ISSN: 2346-3775 Vol. 8 No. 2 May 2020 Published by: Centro Internacional de Agricultura Tropical (CIAT), Cali, Colombia In association with: Chinese Academy of Tropical Agricultural Sciences (CATAS), Haikou, Hainan, P.R. China This issue is dedicated to the memory of Myles J. Fisher (10 August 1935 – 27 May 2020), Australian ecophysiologist who after working at CSIRO in the Northern Territory and Queensland, Australia, joined the CIAT Tropical Pastures Program in 1985. Upon his retirement he was appointed CIAT Emeritus Scientist and was a member of the Editorial Board of Tropical Grasslands-Forrajes Tropicales since the journal´s inception. His friends and colleagues in the tropical forages scientific community will not forget him for his visionary pioneer role in the area of carbon sequestration by tropical pastures, his contribution to our understanding of factors affecting persistence of legumes in pastures and his thorough scientific thinking and pleasant personality. International Center for Tropical Agriculture (CIAT) retains copyright of articles with the work simultaneously licensed under the Creative Commons Attribution 4.0 International License (to view a copy of this license, visit creativecommons.org/licenses/by/4.0/). Accordingly, users/readers are free to share (to copy, distribute and transmit) and to remix (to adapt) the work under the condition of giving the proper attribution. i Editors Rainer Schultze-Kraft, Lyle Winks, The Alliance of Bioversity International and CIAT, Former editor of “Tropical Grasslands”, Colombia Australia Management Committee Robert J. Clements, Agricultural Consultant, Rainer Schultze-Kraft, Australia The Alliance of Bioversity International and CIAT, Colombia Huan Hengfu, Chinese Academy of Tropical Agricultural Sciences Cacilda B. do Valle, (CATAS), Empresa Brasileira de Pesquisa Agropecuária (Embrapa), P.R. China Brazil Asamoah Larbi, Lyle Winks, Agricultural Consultant, Former editor of “Tropical Grasslands”, Ghana Australia Michael Peters, The Alliance of Bioversity International and CIAT, Kenya Editorial Board Caterina Batello, Orlando Guenni, Food and Agriculture Organization of the United Nations Universidad Central de Venezuela (UCV), (FAO), Venezuela Italy Jean Hanson, Michael Blümmel, International Livestock Research Institute (ILRI), International Livestock Research Institute (ILRI), Ethiopia India Michael David Hare, Robert J. Clements, Ubon Ratchathani University, Agricultural Consultant, Thailand Australia Huan Hengfu, Myles Fisher, Chinese Academy of Tropical Agricultural Sciences The Alliance of Bioversity International and CIAT, (CATAS), Colombia P.R. China Albrecht Glatzle, Mario Herrero, Iniciativa para la Investigación y Transferencia de Commonwealth Scientific and Industrial Research Tecnología Agraria Sostenible (INTTAS), Organisation (CSIRO), Paraguay Australia ii Masahiko Hirata, Bruce Pengelly, University of Miyazaki, Agricultural Consultant, Japan Australia Peter Horne, T. Reginald Preston, Australian Centre for International Agricultural Research University of Tropical Agriculture Foundation (UTA), (ACIAR), Colombia Australia Johann Huguenin, Kenneth Quesenberry, Centre de Coopération Internationale en Recherche University of Florida, Agronomique pour le Développement (CIRAD), USA France H. Max Shelton, Muhammad Ibrahim, The University of Queensland, Australia Centro Agronómico Tropical de Investigación y Enseñanza (CATIE), Werner Stür, Costa Rica Australian Centre for International Agricultural Research (ACIAR), Asamoah Larbi, Australia Agricultural Consultant, Ghana Cacilda B. do Valle, Carlos E. Lascano, Empresa Brasileira de Pesquisa Agropecuária (Embrapa), Universidad Nacional de Colombia - Sede Bogotá, Brazil Colombia Robert Paterson, Agricultural Consultant, Spain Principal Contacts Rainer Schultze-Kraft The Alliance of Bioversity International and CIAT Colombia Phone: +57 2 4450100 Ext. 3036 Email: CIAT-TGFT-Journal@cgiar.org Technical Support José Luis Urrea Benítez The Alliance of Bioversity International and CIAT Colombia Phone: +57 2 4450100 Ext. 3354 Email: CIAT-TGFT-Journal@cgiar.org iii Table of Contents Research Papers Seasonal herbage accumulation, plant-part composition and nutritive value of signal grass (Urochloa 48-59 decumbens) pastures under simulated continuous stocking Gustavo José Braga, Carlos Guilherme Silveira Pedreira, Aliedson Sampaio Ferreira, Eliara Anaí de Oliveira, Valdinei Tadeu Paulino Spectral sensors prove beneficial in determining nitrogen fertilizer needs of Urochloa brizantha cv. Xaraés 60-71 grass in Brazil Helizani C. Bazame, Francisco A.C. Pinto, Domingos S. Queiroz, Daniel M. de Queiroz, Daniel Althoff Evaluation of forage quantity and quality in the semi-arid Borana Lowlands, Southern Oromia, Ethiopia 72-85 Gemedo Dalle Ammonium sulfate enhances the effectiveness of reactive natural phosphate for fertilizing tropical grasses 86-92 Carlos E.A. Cabral, Carla H.A. Cabral, Alyce R.M. Santos, Kassio S. Carvalho, Edna M. Bonfim-Silva, Jenifer S. Mattos, Letícia B. Alves, Ana P. Bays Vertical distribution, nutrient concentration and seasonal changes of fine root mass in a semi-deciduous 93-104 tropical dry forest and in two adjacent pastures in the Western Llanos of Venezuela Ana Francisca González-Pedraza, Nelda Dezzeo The effects of bovine urine application on two soil nitrogen compounds and growth of three forage grasses 105-114 in the Colombian Piedmont plains Jaime E. Garzón, Oscar Pardo, Edgar A. Cárdenas Effects of swine manure application and row spacing on growth of pearl millet (Cenchrus americanus) 115-124 during the establishment period and quality of silage produced in Southwest Nigeria V.O.A. Ojo, F.T. Adeshina, G.A. Adetokunbo, S.O. Jimoh, T.A. Adeyemi, J.L. Njie, O.S. Onifade Effect of seed storage on seed germination and seedling quality of Festulolium in comparison with related 125-132 forage grasses Rade Stanisavljevic, Dobrivoj Poštić, Ratibor Štrbanović, Marijenka Tabaković, Snežana Jovanović, Jasmina Milenković, Dragoslav Đokić, Dragan Terzić Selection based on meiotic behavior in Urochloa decumbens hybrids from non-shattered seed 133-140 Joana Neres da Cruz Baldissera, Andréa Beatriz Diverio Mendes, Marlon Mathias Dacal Coan, Claudete Aparecida Mangolin, Cacilda Borges do Valle, Maria Suely Pagliarini Short Communications The effect of stage of regrowth on the physical composition and nutritive value of the various vertical strata 141-146 of kikuyu (Cenchrus clandestinus) pastures Marcelo A. Benvenutti, Craig Findsen, Jean V. Savian, David G. Mayer, David G. Barber Continued iv In vitro digestion characteristics of various combinations of elephant grass hay, gliricidia hay or silage, 147-152 soybean meal and corn meal in rations for sheep Juliana Caroline Santos Santana, Jucileia Aparecida da Silva Morais, Gelson dos Santos Difante, Luís Carlos Vinhas Ítavo, Antonio Leandro Chaves Gurgel, Vinicius da Silva Oliveira, Maria Juciara Silva Teles Rodrigues Evaluation of Asystasia gangetica as a potential forage in terms of growth, yield and nutrient concentration 153-157 at different harvest ages N.R. Kumalasari, L. Abdullah, L. Khotijah, L. Wahyuni, Indriyani Indriyani, N. Ilman, F. Janato Pest insects in natural and sown pastures of Paraguay 158-161 Humberto J. Sarubbi, María B. Ramírez Pasto Certo® version 2.0 - An application about Brazilian tropical forage cultivars for mobile and desktop 162-166 devices Sanzio Carvalho Lima Barrios, Camilo Carromeu, Márcio Aparecido Inácio da Silva, Edson Takashi Matsubara, Cacilda Borges do Valle, Liana Jank, Mateus Figueiredo Santos, Giselle Mariano Lessa de Assis, Leonardo Lazarino Crivellaro, Thallyson Danchen Teixeira Gonçalves, José Marcos Queiroz Júnior, Anderson Ramires Candido, Wyverson Kim Rocha Machado, Beatriz Tomé Gouveia, Alana Aparecida Amarilha Nobre, Ayhan Liell Zanella v Tropical Grasslands-Forrajes Tropicales (2020) Vol. 8(2):48–59 48 doi: 10.17138/TGFT(8)48-59 Research Paper Seasonal herbage accumulation, plant-part composition and nutritive value of signal grass (Urochloa decumbens) pastures under simulated continuous stocking Acumulación estacional de forraje, composición morfológica y valor nutritivo de Urochloa decumbens bajo pastoreo continuo simulado GUSTAVO JOSÉ BRAGA1, CARLOS GUILHERME SILVEIRA PEDREIRA2, ALIEDSON SAMPAIO FERREIRA2, ELIARA ANAÍ DE OLIVEIRA3 AND VALDINEI TADEU PAULINO3 1Embrapa Cerrados, Planaltina, DF, Brazil. embrapa.br/cerrados 2Departamento Zootecnia, ESALQ, Universidade de São Paulo, Piracicaba, SP, Brazil. zootecnia.esalq.usp.br 3Instituto de Zootecnia, APTA, Nova Odessa, SP, Brazil. iz.sp.gov.br Abstract In order to optimize the regrowth and harvest of signal grass (Urochloa decumbens) cv. Basilisk pastures it is necessary to establish more precise grazing management guidelines. The objective of this study was to evaluate herbage accumulation, plant-part composition and nutritive value of signal grass managed under contrasting levels of steady- state canopy heights. Treatments included 3 canopy height targets, i.e. 10 (S-short), 17.5 (M-medium) and 25 cm (T-tall), in a completely randomized design with 4 replications. Experimental units were 144-m2 plots which were grazed by groups of steers for short periods in an endeavor to keep canopy heights at the 3 desired targets. On average, herbage accumulation rate (HAR) in T pastures was greater than in M and S pastures, including the dry-wet season transition period in spring (September‒November). The S pastures had higher crude protein and lower acid detergent fiber concentrations than M and T pastures, especially in the first half of the calendar year. However, in vitro organic matter digestibility was similar for all treatments (612 g/kg). As S and M pastures had lower HARs than T pastures in the spring, it appears advantageous to maintain the signal grass canopy at ~25 cm in order to ensure quick regrowth with the return of the wet season. However, longer-term studies are needed with recording of animal performance before these initial findings can be promoted widely. Keywords: Crude protein, digestibility, grazing management, light interception, pasture height, tropical pastures. Resumen Para optimizar la producción de pasturas de Urochloa spp. es necesario establecer pautas de manejo del pastoreo más precisas. En Brotas, Estado de São Paulo, Brasil, se evaluaron la acumulación de forraje, la morfología de planta y el valor nutritivo de Urochloa decumbens cv. Basilisk en función de diferentes alturas de planta. Los tratamientos incluyeron 3 alturas del pasto: 10 cm (S - baja), 17.5 cm (M - mediana) y 25 cm (T - alta), en un diseño completamente al azar con 4 repeticiones en parcelas de 144 m2 que fueron utilizadas por grupos de novillos durante períodos cortos con el fin de mantener las alturas de planta de acuerdo con los tratamientos. En promedio, la tasa de acumulación de forraje (TAF) en las pasturas del tratamiento T fue mayor que en las pasturas en los tratamientos M y S, incluyendo el período de transición de estación seca a lluviosa en la primavera (septiembre‒noviembre). Las pasturas en el tratamiento S presentaron mayores concentraciones de proteína cruda y menores concentraciones de fibra detergente ácido que las pasturas en M y T, especialmente en el primer semestre del año calendario. Sin embargo, la digestibilidad in vitro de la materia orgánica fue similar para todos los tratamientos (612 g/kg). Las pasturas en los tratamientos S y M presentaron ___________ Correspondence: Gu.Js.t aBvroa gJao,s éE Bmrbargaap,a E Cmebrrapdao sC, eBrrRa d0o2s0, ,B kRm 0 1280, kPmla n1a8lt, iPnala, nCaEltPin 7a,3 C31E0P- 977303,1 D0-F9,7 B0r, aDziFl., Brazil. Email: gustavo.braga@embrapa.br Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) Managing signal grass under continuous stocking 49 TAF más bajas en la primavera que las pasturas en T, lo que sugiere que mantener una altura de pastura de ~25 cm sobre el nivel del suelo permite un rápido crecimiento al comienzo de la época de lluvias. Sin embargo, se necesitan estudios de producción animal a largo plazo antes de que estos resultados iniciales puedan promoverse en forma amplia. Palabras clave: Altura de pastura, digestibilidad, intercepción de luz, manejo de pastoreo, pastos tropicales, proteína cruda. Introduction Although some research-based recommendations regarding defoliation strategies for signal grass are Signal grass [Urochloa decumbens (Stapf) R.D. Webster, available (Braga et al. 2009; Santos et al. 2010; Portela et syn. Brachiaria decumbens Stapf] cv. Basilisk is a al. 2011; Pedreira et al. 2017), a better comprehension of decumbent perennial tropical grass, that despite being the effects of year-round grazing at steady-state (constant) susceptible to spittlebug (Deois spp. and Zulia spp.) is canopy heights is still necessary rather than just average widely used in forage-livestock systems in central Brazil, wet-season estimates. Forage growth in central Brazil is owing to its great tolerance to acidic and infertile soils markedly seasonal, concentrated in the warm, wet months (Rao et al. 1996). With the increasing competition for land (November‒April), and overgrazing during the dry associated with expansion of cropping areas, i.e. maize season is common due to limited forage accumulation (Zea mays), soybean (Glycine max) and sugarcane during this period. This raises the question of how canopy (Saccharum officinarum), pastures must be more structure in the dry season affects plant regrowth at the productive and sustainable; improvement of grazing onset of the following wet season. The objective of this management is required to achieve this objective. study was to evaluate herbage accumulation, plant-part Plant growth and harvest efficiency in grazed composition (leaf blade, stem and dead material) and pastures are closely related to canopy structure (Bircham nutritive value on a monthly basis in signal grass pastures and Hodgson 1983). The presence of photosynthetically managed under various steady-state canopy heights active leaves supports fast regrowth, eventually mimicking continuous stocking management. followed by stem elongation, a process triggered by the complete or near-complete interception of light by the Materials and Methods forage canopy (maximum leaf area index, LAI) (Korte et al. 1982). For tropical grasses, excessive elongation of Experimental site stem has a negative effect on pasture utilization (Hodgson and Silva 2002) due to increasing proportion The research was carried out at APTA (Agência Paulista of rejected patches and plant lodging. Grazing de Tecnologia dos Agronegócios) in Brotas, State of São management, based on the maintenance of specific Paulo, Brazil (21°59ʹ S, 47°26ʹ W; 650 masl). The climate canopy structure by controlling LAI, canopy height or at the site is a subtropical Cwa, according to the Köppen- herbage mass, may avoid excessive stem growth and Geiger classification (Peel et al. 2007). The experimental allow more predictable levels of herbage accumulation area was a 25-years-old pasture of signal grass cv. and animal performance (Silva et al. 2013). In addition, Basilisk. The soil at the site is a Quartzipsamment with plant-part composition is an important variable for cattle 9% clay, 33% fine sand, 57% coarse sand and 1% silt. nutrition, as animals can select a higher quality diet from Chemical analysis in the 0–20 cm layer showed 5 mg a leafy pasture than from one with lower leaf proportion. P/dm3 (Presin), 3 mmolc Ca/dm3, 3 mmol 3c Mg/dm , 2 mmolc Canopy height ranges recommended for grazing K/dm3, 42 mmolc H+Al/dm3, 22 g OM/dm3, 16% base management are associated with the canopy architecture, saturation and pH(CaCl2) 4.2. Rainfall data were recorded as well as the plant-part (leaf, stem and dead material) by the Department of Environment of the City of Brotas, accumulation dynamics under grazing. For example, and the maximum and minimum monthly average keeping canopy height of Marandu palisade grass temperatures were recorded at a weather station 45 km (Urochloa brizantha) at 10 cm by heavy grazing from the experimental site (Figure 1). intensity negatively affected herbage accumulation (Silva et al. 2013). For signal grass pastures, optimum Treatments, experimental design and grazing management canopy height seems to be around 15‒25 cm under continuous stocking (Santos et al. 2013), with a pre- Treatments included 3 canopy height targets, 10 (S-short), grazing target under rotational stocking of 18‒30 cm 17.5 (M-medium) and 25 cm (T-tall), set in a completely (Pedreira et al. 2017). randomized design with 4 replications. On 24 October Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) 50 G.J. Braga, C.G.S. Pedreira, A.S. Ferreira, E.A. de Oliveira and V.T. Paulino 800 35 700 30 600 25 500 20 400 15 300 10 200 100 5 0 0 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Rainfall 2009 Rainfall average Tmax Tmin Figure 1. Monthly rainfall and temperatures during the experimental period (2009) and average long-term rainfall (1983‒2006). 2008, the experimental area was mechanically clipped to Fertilizers were split-applied in equal amounts on 24 reduce the canopy height to approximately 5 cm. In December 2008, 7 February 2009, 10 March 2009, 4 May November 2008, dolomitic lime (0.98 t/ha) and single 2009 and 24 September 2009. superphosphate (44 kg P/ha) were surface-applied to increase the base saturation to 40% and soil P to Measurements >15 mg/dm (Presin), respectively. The area was divided by electric fence into 12 experimental plots measuring Canopy height and herbage mass (HM) were evaluated 144 m2 (12 × 12 m) each. The experimental period was using a rising plate meter (RPM). Periodic calibrations were January 2009‒January 2010. Mob stocking was used to necessary to correlate the RPM reading with canopy height impose defoliation on the experimental plots according to and HM. The calibrations were made 6 times during the their respective treatments. Two crossbred steers, with experimental period by selecting 2 sites (0.30 m2) per plot, mean live weight of 650 kg were used to graze each plot representing the extremes of height, i.e. the tallest and the at each grazing event, always after overnight fast for shortest canopy areas. To measure canopy height a light- solids. Defoliation mimicked continuous stocking, with transparent acetate sheet (21 × 30 × 0.02 cm) was placed on animals assigned to pastures whenever canopy height was top of the canopy and an RPM reading was taken. Herbage approximately 10% above target height. During the wet inside the quadrat (0.30 m2) was then clipped at soil level season, animals grazed pastures approximately once a (double sampling). The samples were dried in a forced-air week, while during the dry season the period between oven for 72 h at 60 °C to estimate HM on a dry matter basis. grazing events was 1‒2 weeks. As paddocks were small, Linear regression curves were established to estimate grazing events lasted for only 15 min to 2 h, and steers canopy height and HM as a function of the RPM readings. grazed non-experimental pastures when they were not RPM readings were taken every 5 days in the wet season, grazing treatment pastures. Aiming not to decrease the and every 7 days in the dry season at 42 sites per plot in a height of pastures by more than 10% below the target, systematic way following a grid-like pattern. All HM values several visual observations and measurements of canopy were expressed on a dry matter basis and estimated from the height were made during grazing. Ammonium sulfate and RPM calibration derived from the double sampling sites. potassium chloride were used to provide a total of 250 kg Light interception (LI) was evaluated monthly (except N + 210 kg K/ha during the course of the experiment. August) with an LAI-2000 canopy analyser (Li-COR, Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) Rainfall (mm) Temperature (°C ) Managing signal grass under continuous stocking 51 Lincoln, NE, USA) at 20 sites per plot, with a single reading Statistical analysis above the canopy for every 5 readings near the base of the canopy. Data were analyzed using the MIXED procedure of SAS Herbage accumulation rate (HAR) was estimated at 3 (Littell et al. 2006) using an auto-regressive first order sites per plot which were protected from grazing using covariance model. Canopy height, period and their cylindrical exclusion cages (0.9 m diameter × 1.5 m in interactions were considered fixed effects, and period was height) in 21-day cycles on average. Herbage analyzed as a repeated measure (Littell et al. 2006). accumulation inside the cages was estimated from RPM Treatment means were estimated using LS means and readings inside and outside the cages, and cages were compared using the probability of the difference (PDIFF) repositioned at new sites in each plot for the following by t-test (5%). Rising plate meter calibration curves were accumulation cycle. Herbage accumulation rate was analyzed for their need for sorting by date or canopy estimated by the difference, as follows: HAR = (HMlast day height treatments using the covariance analysis of PROC - HMfirst day)/d, where HMlast day = herbage mass inside the GLM in SAS (SAS Institute 2002). cages on the last day of exclusion, HMfirst day = herbage mass on the pasture on the first day of exclusion and d = Results number of days of the accumulation cycle. Tiller population density was measured every month at 3 Canopy height and herbage mass random 0.2 × 0.5 m sites per plot inside a metal frame. Tillers were classified as basal, aerial or reproductive, The calibration curves between RPM and canopy height did allowing for the calculation of their participation on each not differ across treatments and dates of calibration evaluation date. Tillers were considered basal when (P>0.05). Consequently, canopy height was monitored with originated from basal tissue buds, and aerial when RPM readings using a single calibration curve (Figure 2). originated from axillary buds of the main tiller. Tillers For HM prediction there was no effect of treatment with visible inflorescence were classified as reproductive. (P>0.05), but there was an effect of calibration date Forage samples were clipped monthly, except in August (P<0.05), so results for HM (including HAR estimates) used and September, at 3 sites per plot inside a 0.3 m2 quadrat at 2 calibration curves covering 2 periods (January‒February soil level to evaluate plant-part composition. Sites that had and March‒December) (Figure 2). The response was been previously sampled were avoided in subsequent positive and linear, and models were adjusted satisfactorily, samplings. A subsample (~0.25 kg) was taken from each both for canopy height (R2 = 0.96) and HM, for the first 2 sample and separated into green leaf blades, green stems calibration dates (R2 = 0.88) and for the last 4 dates (R2 = (true stems plus leaf sheaths) and dead material. Dead 0.92). Monitoring canopy height throughout the year material was visually defined as senescent leaves and stems revealed some variation around the target heights, especially with >50% area of yellow or dry tissue. The samples were for M and T treatments (Figure 3). Canopy heights of S dried in a forced-air oven for 72 h at 60 °C to calculate the pastures were maintained near the target of 10 cm proportion of each plant-part component on a dry matter throughout the year, whereas M and T pastures tended to basis and then the amount of each based on HM estimates. exceed the target heights (17.5 and 25 cm, respectively) Forage nutritive value was estimated from hand-plucked during the months of greater plant growth such as January‒ samples taken monthly, except in September. The samples March and November‒December. The opposite happened were collected from the top of the canopy after observation during the dry season (May‒September), when canopy of the grazing behavior of the animals, and then dried in a height decreased to 20‒25 cm in T pastures. forced-air oven for 72 h at 55 °C. They were then ground in There was a height × month interaction effect on HM a Wiley mill to pass a 1 mm screen and taken to the (P = 0.0024), although T canopies always had greater HM laboratory for chemical analyses. Ash concentration was throughout the experimental period (2,590‒4,010 kg/ha) determined by incineration at 600 °C. Crude protein (CP) than M (1,810‒3,040 kg/ha) and S pastures (1,070‒1,660 concentration was calculated as N × 6.25, with N concentra- kg/ha) with smaller differences between M and T pastures tion determined using the Micro Kjeldahl method (AOAC (~700 kg/ha) in the dry season (May‒September) (Figure 1990). Neutral detergent fiber (NDF) and acid detergent 4A), similar to canopy height variation. Leaf mass also fiber (ADF) concentrations were determined according to showed a height × month interaction (P<0.0001). Early in Van Soest et al. (1991). In vitro organic matter digestibility the year (January‒April), leaf mass followed the order (IVOMD) was estimated according to the two-stage T>M>S pastures (Figure 4B) but there was no canopy procedure of Tilley and Terry (1963) modified by Moore height effect between May and October (P>0.05), and Mott (1974). followed by a greater leaf mass on M pastures, especially Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) 52 G.J. Braga, C.G.S. Pedreira, A.S. Ferreira, E.A. de Oliveira and V.T. Paulino A 8,000 B 8,000 y (Mar-Dec) = 90.0x + 418.4 y = 81.468x + 390.82 7,000 7,000 R² = 0.92 R² = 0.86 6,000 6,000 5,000 5,000 12-Jan 4,000 4,000 11-Feb 26-Mar 3,000 Short 3,000 1-Jun 2,000 Medium 2,000 3-Aug Tall y(Jan-Feb) = 66.1x + 358.91,000 1,000 23-Dec R² = 0.88 0 0 0 20 40 60 80 100 0 20 40 60 80 100 RPM RPM C D 60 60 y = 0.5991x + 3.1055 y = 0.5991x + 3.1055 50 R² = 0.96 50 R² = 0.96 40 40 12-Jan 11-Feb 30 30 Short 26-Mar 20 Medium 20 1-Jun 3-Aug 10 Tall 10 23-Dec 0 0 0 20 40 60 80 100 0 20 40 60 80 100 RPM RPM Figure 2. Prediction of herbage mass and canopy height as a function of rising plate meter (RPM) readings considering the effects of treatment (A and C) and date of calibration (B and D) in signal grass (Urochloa decumbens) cv. Basilisk pastures at 3 canopy heights in Brotas, SP, Brazil. Figure 3. Actual canopy heights in signal grass (Urochloa decumbens) cv. Basilisk pastures while aiming at 3 target canopy heights (10 – short, 17.5 – medium and 25 – tall cm) in Brotas, SP, Brazil. Bars correspond to ± standard deviation. Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) Canopy height (cm) Herbage mass (kg/ha) Herbage mass (kg/ha) Canopy height (cm) Managing signal grass under continuous stocking 53 A B 5,000 Short Medium Tall 1,400 Short Medium Tall 1,200 4,000 1,000 3,000 800 2,000 600 400 1,000 200 0 0 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec C 1,800 D 3,000 Short Medium Tall Short Medium Tall 1,500 2,500 1,200 2,000 900 1,500 600 1,000 300 500 0 0 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Figure 4. A) Herbage mass; B) leaf; C) stem; and D) dead material in signal grass (Urochloa decumbens) cv. Basilisk pastures maintained at 3 canopy heights in Brotas, SP, Brazil. Bars correspond to ± standard error of mean. in December. A height × month interaction effect on stem and S pastures, the advantage being most evident in mass (P<0.0001) was also observed. Tall pastures had the Periods 2 and 3 (February‒April) and Periods 8, 9 and 10 greatest stem mass, followed by M and S pastures (Figure (September‒December). During the dry season (May‒ 4C), with stem mass being greatest during January‒April August; Periods 4‒7), there was no difference among and October‒December, especially in T and M pastures, treatments (P>0.05). but displaying much less variation in S pastures over the course of the year. Dead material mass displayed a Tiller population density and light interception height × month interaction (P<0.0001) but was greater in T pastures, followed by M and S pastures (Figure Time of year was the only factor affecting total tiller 4D). Mass of dead material increased during the first population density (P<0.0001), and the increase in tiller half of the year peaking in June, regardless of canopy numbers in February and March was largely an increase height. in aerial tillers (Figure 6). Aerial tiller numbers were affected by a height × month interaction (P<0.0045) as S Herbage accumulation rate pastures had fewer aerial tillers only in February and March (440 and 145 tillers/m2, respectively) than M and Overall, HAR mirrored rainfall and temperature levels T pastures (626 and 317 tillers/m2, respectively). There being high initially before dropping to near zero in May‒ were effects of month (P<0.0001) and height (P = 0.0474) June (Periods 4 and 5), then increasing to peak in on basal tiller population density as M pastures had more December (Period 10) (Figure 5). Herbage accumulation than T pastures (1,340 vs. 1,250 tillers/m2, respectively). rate was affected by a height × period interaction (P = Reproductive tiller numbers displayed a height × month 0.0005). In general, T pastures had greater HAR than M interaction (P<0.0001) and were present mainly between Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) Stem (kg/ha) Herbage mass (kg/ha) Dead (kg/ha) Leaf (kg/ha) 54 G.J. Braga, C.G.S. Pedreira, A.S. Ferreira, E.A. de Oliveira and V.T. Paulino Figure 5. Daily herbage accumulation rates (HAR) during Periods 1‒11 in signal grass (Urochloa decumbens) cv. Basilisk pastures maintained at 3 canopy heights in Brotas, SP, Brazil. Bars correspond to ± standard error of mean. 2,500 Aerial Reproductive Basal 2,000 1,500 1,000 500 0 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Figure 6. Population density of various tiller categories in signal grass (Urochloa decumbens) cv. Basilisk pastures maintained at 3 canopy heights in Brotas, SP, Brazil. Bars correspond to ± standard error of mean. February and May, with S pastures having fewer of them Forage nutritive value in this period (38 tillers/m2) followed by M (67 tillers/m2) and T (74 tillers/m2) pastures. Crude protein concentration was affected by a height × There was a height × month interaction effect (P<0.0001) month interaction (P<0.0001). CP% in S pastures was on LI. Light interception was more variable in S pastures, greater than in M and T pastures in January, February, peaking at 90% in June and decreasing to 50% in September May, June and October (Figure 8A), with no differences (Figure 7). LI in M and T pastures was relatively stable in the other months. After peaking in October (130‒170 throughout, fluctuating between 90 and 100%. g/kg), mean CP concentration decreased to approximately Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) Population density (tiller/m²) Managing signal grass under continuous stocking 55 100 80 60 40 20 Short Medium Tall 0 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Figure 7. Light interception in signal grass (Urochloa decumbens) cv. Basilisk pastures maintained at 3 canopy heights in Brotas, SP, Brazil. Bars correspond to ± standard error of mean. A B 750 180 Short Medium Tall 160 700 140 650 120 600 100 550 80 60 500 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec C 800 D 400 Short Medium Tall Short Medium Tall 750 360 700 320 650 280 600 240 550 200 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Figure 8. (A) Crude protein (CP) concentration; (B) In vitro organic matter digestibility (IVOMD); (C) Neutral detergent fiber (NDF) concentration; and (D) Acid detergent fiber (ADF) concentration of signal grass (Urochloa decumbens) cv. Basilisk maintained at 3 canopy heights in Brotas, SP, Brazil. Bars correspond to ± standard error of mean. Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) NDF (g/kg) CP (g/kg) Light interception (%) ADF (g/kg) IVOMD (g/kg) 56 G.J. Braga, C.G.S. Pedreira, A.S. Ferreira, E.A. de Oliveira and V.T. Paulino 110 and 90 g/kg in November and December, range of canopy conditions, and sometimes the contrasting respectively. canopy heights do not result in contrasting HAR values Time of year had the greatest effect on IVOMD (Bircham and Hodgson 1983). Since short canopies have (P<0.0001), which peaked in January-February and again small tillers with greater population density (Sbrissia and in October, before declining sharply in November and Silva 2008), the photosynthetic apparatus, i.e. leaf area December (Figure 8B). Both neutral detergent fiber and index, could partially compensate. In the current study, acid detergent fiber concentrations were affected by a however, differences in tiller population density were small height × month interaction (P<0.0001). Both tended to and this compensation was not evident. peak in February before declining slowly until August‒ Considering the low proportion of reproductive tillers, October before increasing to December (Figures 8C and all treatments inhibited flowering, a positive effect in terms 8D). of livestock performance considering the negative impact of reproductive tillers on stem proportion, food intake and Discussion diet quality (Benvenutti et al. 2008). Under our management system, production of reproductive tillers was Steady-state canopy heights did not ensure a constant concentrated between February and May, although signal plant-part component mass within treatments throughout grass typically concentrates its flowering between the year, regardless of canopy height. There was a December and January at this latitude. There was a strong decrease in leaf and stem mass from January to June, seasonal variation in total tiller population density, which especially in M and T pastures, followed by an increase reached a maximum of 2,300 tillers/m2 in February and in dead material, even for the S pastures. This process decreased to ~1,100 tillers/m2 between June and peaked at the end of the wet season likely due to increased September, although basal tiller numbers were less variable tissue senescence triggered by the imminent soil water (1,000‒1,700 tillers/m2). According to Portela et al. (2011) deficit during this period (Figure 1). In the second half of in signal grass pastures managed under rotational stocking, the year, dead material mass remained relatively constant there was less variation than in the current study in tiller showing differences among treatments and greater density throughout the year and the range was 1,100‒1,500 senescence in T pastures, followed by M and S pastures. basal tillers/m2, with pastures managed at heavier grazing As expected, S pastures had less HM (leaf + stem + dead intensity (5 cm post-grazing canopy height) having 10‒ material) than M and T pastures and less stem and dead 20% more basal tillers. material, as also observed by Santos et al. (2010) in 4 Plants in T pastures were larger and probably invested areas of signal grass grazed at 10, 20, 30 and 40 cm more energy in maintenance respiration. Under canopy height during the wet season. environmental restrictions, this could result in negative As expected, mean HAR varied seasonally and greater HAR, as observed in Period 4 of the current study (Figure values were recorded during the warm and wet periods 5). This was reported by Silva et al. (2013) in palisade (January‒April and September‒December). During the grass pastures grazed at >30 cm canopy height during the cooler months (May‒August; Periods 4‒7), HAR was near dry season. Santos et al. (2013) also reported negative zero because the daily minimum temperatures, i.e. under HAR, i.e. death of plant parts was greater than the growth 15 °C (Figure 1), during this period are probably close to of new plant tissue, in signal grass pastures maintained at the threshold values that restrict the growth of C4 species 25 cm canopy height in the dry season. In the current (Silva et al. 2012). The partially greater HAR in T pastures study, the onset of the wet season in spring resulted in can be attributed to the additional amount of leaves, i.e. greater HAR in T pastures (Periods 8‒10; Figure 5), LAI, and plant reserves normally associated with the similar to the findings of Silva et al. (2013) at the same greater shoot and root plant structures (Donaghy and time of year. This greater herbage accumulation is Fulkerson 1998). The advantage of T pastures was important in forage-based systems in central Brazil particularly significant in the transition between the dry and because feed supply at the end of the dry season can wet seasons (September‒November). The positive effect of become critically low and a rapid growth response by the greater canopy height on HAR was similar to that reported pasture with the return of wet season conditions helps for Xaraés palisade grass (Urochloa brizantha) pastures buffer the roughage demand on the farm. Using an managed at 15, 30 and 45 cm (Euclides et al. 2010). On the alternative grazing scheme, Santos et al. (2013) other hand, Euclides et al. (2010) reported a negative effect temporarily lowered canopy height to 15 cm in the early of height on HAR of Marandu palisade grass, even though dry season, which boosted HAR in the following spring in both trials HAR differences were minimal for different compared with a pasture kept at 25 cm, in contrast with canopy heights. This similarity is expected for a given results of the current study. Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) Managing signal grass under continuous stocking 57 There have been many studies on the effects of LI on components such as lignin, minimizing potential negative herbage accumulation dynamics of tropical grasses, consequences for diet nutritive value. As observed by especially in rotationally stocked pastures, e.g. Pedreira et Hernández Garay et al. (2004) and Sollenberger and al. (2017) and Moura et al. (2017). In general, maximum Vanzant (2011), CP% and digestibility increase with canopy light interception (~95‒100% LI) modifies plant- heavier grazing management as a consequence of forage part growth, shifting the primary leaf accumulation to a less utilization increase and its effect on forage maturity and desirable stem elongation, especially in well-fertilized leaf proportion. This greater forage nutritive value in S pastures. For this reason the 95% LI criterion has been pastures would normally result in better animal proposed as the moment when the rest/regrowth period performance. However, this might not occur, because should be terminated in intermittent (rotational and its performance depends on forage intake, also regulated by variations) grazing schemes (Carnevalli et al. 2006). By herbage allowance (Herling et al. 2011; Sollenberger and associating LI with canopy height it may be possible to Vanzant 2011). Short canopies may be associated with recommend specific grazing management targets for smaller herbage allowance and, if taken to extreme levels, different forage species and cultivars. However, the may lead to overgrazing, limiting forage intake by bite size phenotypic plasticity of species such as signal grass can restrictions and/or insufficient grazing time to satisfy lead over time to a more prostrate plant architecture, which appetite (Silva et al. 2013). At the end of the year (October‒ in turn can modify the established association between December) forage nutritive value declined, regardless of canopy LI and height (Braga et al. 2006; Pedreira et al. treatment. This may be associated with N fertilizer 2017). In the current study, LI values measured in M and T application, which in the second half of the year happened pastures were close to the ceiling (95‒100%) in the first only in September, reducing grazing frequency and plant half of the year, whereas in the second half, LI approached tissue renewal at the end of the experimental period, and 95% for T pastures and 90% for M pastures. These values consequently lowering overall forage nutritive value. fall within the range of canopy LIs and canopy heights Regardless of treatment, however, the nutritive value of observed by Pedreira et al. (2017) for signal grass pastures. signal grass can be considered satisfactory since CP The large LI values were probably related to the presence concentrations in hand-plucked samples which mimicked of dead leaves near the bottom of the canopy as observed animal selection, for example, remained above 100 g/kg for by Braga et al. (2006), even for the M pastures kept at most of the year, and IVOMD above 600 g/kg, similar to shorter canopy heights. Although T pastures displayed levels observed by Silva et al. (2013) in palisade grass greater HAR than shorter pastures (M and S), the plant-part pastures grazed at 4 canopy heights (10, 20, 30 and 40 cm). composition profile changed at the end of the experimental The steady-state canopy allows not only better harvest period (November and December) and M pastures showed efficiency, but also results in forage of greater nutritive more leaf than S and T pastures. At the same time, T value compared with pastures that are managed without a pastures showed increasing stem mass and less leaf mass. canopy-based criterion (Nave et al. 2010). Conversely, As signal grass usually starts flowering at this time of the adhering to a canopy condition and the intensification of year, more lenient grazing could favor the development of harvest efficiency during the warm wet season does not stems as opposed to leaves. If excessive stem elongation allow for a build-up of forage of lower nutritive value to occurs, additional effort may be needed to maintain canopy be consumed in the cool and dry season, when forage height, something we did not achieve in the current study, mass usually does not meet the livestock demand even in the taller canopies. The control of canopy structure (stockpiling or deferred grazing). Producers often use lax is important because the presence of mature stems may lead grazing intensity in the wet season, especially in low- to decreased harvest efficiency by livestock associated with input forage-livestock systems in order to accomplish rejected patches and lodging, a process that becomes more this. In contrast, in intensive, high-input grazing systems, important as the intensity of use of the pasture, i.e. fertilizer such as the one represented in the current study, it may be application, irrigation, etc., increases. advantageous to efficiently harvest forage of greater Forage nutritive value in S pastures was greater than in nutritive value, maximizing the animal output in terms of M and T pastures, although no differences in IVOMD were both performance, i.e. daily weight gain, milk production, recorded. On the other hand, higher CP concentration and etc., and pasture carrying capacity, to justify the high lower ADF concentration, especially in the first half of the production costs, mainly where land is expensive. At the year, were recorded in S pastures. The higher grazing same time, to deal with a shortage of forage on offer intensity in S pastures combined with greater defoliation during the dry season in central Brazil, feeding options frequency led to greater tissue renewal and predominance such as stockpiling, protein and/or energy supplements or of younger plant tissues with lower proportion of cell wall mixed grass-legume pastures may be required. Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) 58 G.J. Braga, C.G.S. Pedreira, A.S. Ferreira, E.A. de Oliveira and V.T. Paulino Despite expectations to the contrary, the short canopy allowance. Scientia Agricola 63:121–129. doi: 10.1590/ management (~10 cm) in signal grass pastures was not S0103-90162006000200003 detrimental to forage productive vigor (in terms of HAR) Braga GJ; Portela JN; Pedreira CGS; Leite VBO; Oliveira EA. during the wet season. In addition, leaf mass was almost 2009. Herbage yield in Signalgrass pastures as affected by grazing management. South African Journal of Animal the same as in M and T pastures throughout the Science 39:130–132. doi: 10.4314/sajas.v39i1.61168 experimental period. However, this may not be true in the Carnevalli RA; Silva SC da; Bueno AAO; Uebele MC; Bueno long term, especially without fertilizer application. The S FO; Hodgson J; Silva GN; Morais JPG. 2006. Herbage and M pastures produced forage with greater nutritive production and grazing losses in Panicum maximum cv. value, i.e. higher CP and lower fiber concentrations, than Mombaça under four grazing managements. Tropical T pastures. However, with the onset of the wet season, Grasslands 40:165–176. goo.gl/EilPlN HAR of T pastures exceeded those of S and M pastures, Donaghy DJ; Fulkerson WJ. 1998. Priority for allocation of suggesting an advantage in maintaining signal grass at water-soluble carbohydrate reserves during regrowth of ~25 cm in order to ensure rapid growth response in spring Lolium perenne. Grass and Forage Science 53:211–218. doi: 10.1046/j.1365-2494.1998.00129.x if pastures were continuously stocked. Euclides VPB; Valle CB do; Macedo MCM; Almeida RG de; The results of the present study have shown the Montagner DB; Barbosa RA. 2010. Brazilian scientific phenotypic plasticity of signal grass under a mimicked progress in pasture research during the first decade of XXI continuous stocking condition with no clear evidence of century. Revista Brasileira de Zootecnia 39:151–168. doi: stand decline or loss of vigor, i.e. severe reduction of yield 10.1590/S1516-35982010001300018 and/or tiller density, in the S pastures. How this situation Herling VR: Pedreira CGS; Luz PHC de; Braga GJ; Marchesin will apply under long-term grazing remains to be WA; Macedo FB; Lima CG de. 2011. Performance and answered. Grazing target recommendations for optimal productivity of Nellore steers on rotationally stocked animal performance still need to be developed, but it palisadegrass (Brachiaria brizantha) pastures in response to herbage allowance. The Journal of Agricultural Science is expected that keeping canopy height in signal 149:761–768. doi: 10.1017/S0021859611000116 grass pastures between 15 and 25 cm should ensure Hernández Garay A; Sollenberger LE; McDonald DC; maximum animal production, as a result of quality Ruegsegger GJ; Kalmbacher RS; Mislevy P. 2004. Nitrogen of available forage being maintained at a high level. fertilization and stocking rate affect stargrass pasture and Testing of this hypothesis commercially and recording cattle performance. Crop Science 44:1348–1354. doi: animal performance is warranted. 10.2135/cropsci2004.1348 Hodgson J; Silva SC da. 2002. Options in tropical pasture Acknowledgments management. 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This work is licensed under the Creative Commons Attribution 4.0 International (CC BY 4.0) license. Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) Tropical Grasslands-Forrajes Tropicales (2020) Vol. 8(2):60–71 60 doi: 10.17138/TGFT(8)60-71 Research Paper Spectral sensors prove beneficial in determining nitrogen fertilizer needs of Urochloa brizantha cv. Xaraés grass in Brazil Evaluación del beneficio de los sensores espectrales para determinar los requerimientos de nitrógeno en pasturas de Urochloa brizantha cv. Xaraés en Brasil HELIZANI C. BAZAME1, FRANCISCO A.C. PINTO2, DOMINGOS S. QUEIROZ3, DANIEL M. DE QUEIROZ2 AND DANIEL ALTHOFF2 1Departamento de Engenharia de Biossistemas, Escola Superior de Agricultura "Luiz de Queiroz", Universidade de São Paulo, Piracicaba, SP, Brazil. leb.esalq.usp.br 2Departamento de Engenharia Agrícola, Universidade Federal de Viçosa, Viçosa, MG, Brazil. dea.ufv.br 3Empresa de Pesquisa Agropecuária de Minas Gerais, Viçosa, MG, Brazil. epamig.br Abstract The objective of the present work was to evaluate the use of spectral sensors to determine nitrogen fertilizer requirements for pastures of Urochloa brizantha cv. Xaraés in Brazil. The experimental design was a randomized block design with 4 replications of 4 treatments: a control treatment (TT) without application of N; a reference treatment (TR) with N applied at a standard predetermined fixed rate (150 kg urea/ha/cycle); a treatment using GreenSeekerTM (TG) to determine N requirement by the canopy normalized difference vegetation index (NDVI); and a treatment using SPAD 502 (TS) to determine N requirement by foliar chlorophyll assessment. For treatments involving spectral sensors, N fertilizer was applied at half the rate of that in the reference treatment at the beginning of each cycle and further N was applied only when the nitrogen sufficiency index dropped below 0.85. The sensors used in the work indicated that no additional N fertilizer was required by these pastures above the half rates applied. Applying N at the reduced rates to the pastures was more efficient than the pre-determined fixed rate, as both sensor treatments and the fixed rate treatment produced similar total forage yields, with similar crude protein concentrations. All fertilized pastures supported similar stocking rates, while the sensor treatments used less N fertilizer, i.e. 75 kg urea/ha/cycle less than the reference plot. Longer-term studies to verify these findings are warranted followed by promotion of the technology to farmers to possibly reduce fertilizer application rates, improve profitability and provide environmental benefits. Keywords: GreenSeekerTM, pasture support capacity, precision agriculture, Spad 502, tropical grasses. Resumen El objetivo del trabajo fue evaluar el uso de sensores espectrales para determinar los requerimientos de fertilizantes nitrogenados en pasturas de Urochloa brizantha cv. Xaraés, Para el efecto en Leopoldina, Minas Gerais, Brasil, en un diseño experimental de bloques al azar con 4 repeticiones se evaluaron, durante 3 ciclos de rebrote del pasto y subsiguiente pastoreo, los tratamientos: (1) control (TT) sin aplicación de N; (2) de referencia (TR) con aplicación de N al inicio de cada ciclo en dosis fija predeterminada estándar (150 kg de urea/ha/ciclo); (3) uso del sensor GreenSeekerTM (TG) para determinar el requerimiento de N por el índice de vegetación de diferencia normalizada (NDVI); y (4) uso del sensor SPAD 502 (TS) para determinar el requerimiento de N por evaluación foliar de clorofila. Para los tratamientos con sensores espectrales, el ___________ Correspondence: Helizani Couto Bazame, Departamento de Engenharia de Biossistemas, Escola Superior de Agricultura "Luiz de Queiroz", Universidade de São Paulo, Av. Pádua Dias, 11 – Piracicaba, CEP 13418-900, SP, Brazil. E-mail: helizanicouto@gmail.com Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) Spectral sensors for determination of N needs in Xaraés grass 61 fertilizante nitrogenado se aplicó usando la mitad de la tasa del tratamiento de referencia al comienzo de cada ciclo, con aplicación de N adicional solo cuando el índice de suficiencia de nitrógeno era menor que 0.85. Los sensores utilizados mostraron que las pasturas no requerían fertilizante adicional por encima de las tasas medias de N aplicadas. La aplicación de N a las pasturas usando las tasas reducidas fue más eficiente que la tasa fija predeterminada, ya que los tratamientos con sensores y el tratamiento de tasa fija produjeron rendimientos totales de forraje similares, con concentraciones de proteína cruda similares. Todas las pasturas fertilizadas soportaron cargas animal similares, mientras que los tratamientos con sensores demandaron menos fertilizante de N, i.e. 75 kg de urea/ha/ciclo menos que la parcela de referencia. Se justifican estudios a más largo plazo para verificar estos resultados, seguidos de la promoción de la tecnología a los agricultores para posiblemente reducir las tasas de aplicación de fertilizantes, mejorar la rentabilidad y proporcionar beneficios ambientales. Palabras clave: Agricultura de precisión, capacidad de carga, GreenSeekerTM, pastos tropicales, Spad 502. Introduction of the level of N taken up by the plant. Another example of use of sensors for site-specific N management is the One important characteristic of Brazilian livestock systems GreenSeekerTM. This sensor uses radiation emission diodes is the raising of pasture-fed cattle (Ferraz and Felício 2010), at the red (650 nm) and near infrared (770 nm) wavelengths which is regarded as one of the most economical ways to over the vegetation canopy, providing the normalized produce beef and milk (Carvalho et al. 2009; Deblitz 2009). difference vegetation index (NDVI) (Bredemeier et al. In this scenario, factors such as climatic conditions and 2013). availability of nutrients in the soil must be considered for the The GreenSeekerTM seems to present some advantages adequate development of the pastures (Fernandes et al. when compared with the SPAD 502. Since the 2015). Farmers must be aware that low availability of GreenSeekerTM is a canopy sensor, it has a wider field of nutrients in soils can result in low production and quality of vision and is able to cover a larger area of study, integrating tropical forage (Hare et al. 2015). Determining optimal information about the vegetation as a whole (Chapman and levels of fertilizer to apply to pastures is critical for Barreto 1997). maintaining a sustainable business both financially and The use of spectral sensors in the management of biologically. nitrogen fertilizer application has become a promising Among the essential nutrients, nitrogen (N) is considered technique for farmers seeking practical, easily applied and the most important for plant growth and increasing crop reliable methods for pasture management. Research has yields (Subbarao et al. 2013; Cecato et al. 2014). Nitrogen is shown that measurements with SPAD and the NDVI index present in the amino acids that act in the synthesis of correlate with plant nitrogen concentration in tissue and/or structural and functional proteins (Barbieri et al. 2017), and yield of various crops, including the osier (Daniel et al. directly involved within the photosynthetic process due to its 2016), Japanese cucumber (Pôrto et al. 2014), irrigated rice participation in the chlorophyll molecule. It also increases (Pocojeski et al. 2015), wheat (Theago et al. 2014), potato tillering and improves the nutritional value of pastures (Giletto and Echeverría 2016), cotton (Lee et al. 2009), (Marques et al. 2016). forest species (Ribeiro et al. 2009) and forage (Bravin and A number of precision farming techniques have been Oliveira 2014; Villar et al. 2015; Corrêdo et al. 2019). We developed to address the spatial variability of nutrients in designed this study to evaluate the use of spectral sensors for crop fields more effectively (Hedley 2015). These determining desirable levels of nitrogen fertilizer application techniques consist of site-specific management of to Urochloa brizantha cv. Xaraés pastures. agricultural crops based on information from each location. Among the techniques of precision agriculture, spectral Materials and Methods sensors have been used widely to obtain data which may be related to agronomic characteristics of the crops (Handcock The study was carried out in an experimental field, located et al. 2016; Wachendorf et al. 2018; Viana et al. 2019). in Leopoldina, Minas Gerais, Brazil (21º28'25" S, 42º43'15" Minolta's indirect chlorophyll meter, SPAD (Soil-Plant W; 187 masl), during the period April‒August 2017. Analyses Development) 502, is an example of such sensors. According to the climatic classification of Köppen, the The SPAD 502 quantitatively evaluates the intensity of the climate type of the region is Aw, tropical humid with dry green color in leaves by measuring light transmitted at 650 winters and rainy summers, with average temperature of the nm, where light absorption by the chlorophyll molecule coldest month being above 18 ºC. Soil chemical properties occurs, and at 940 nm, where absorption ceases (Nogueira et in the 0‒20 cm layer of the experimental area are described al. 2018). Intensity of green color in leaves is an indication in Table 1. Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) 62 H.C. Bazame, F.A.C. Pinto, D.S. Queiroz, D.M. de Queiroz and D. Althoff Table 1. Soil chemical properties and nutrients before mineral fertilizer application. Property Unit Block 1 Block 2 Block 3 Block 4 Acidity (pH) H2O 6.1 5.8 5.8 6.0 P mg/dm3 4.6 5.3 6.8 7.3 K mg/dm3 64 37 75 53 Ca2+ cmol /dm3c 2.3 2.3 2.4 2.1 Mg2+ cmolc/dm3 1.0 1.0 0.9 0.8 H+Al cmol /dm3c 2.6 3.1 3.1 2.5 SB cmol /dm3c 3.5 3.4 3.5 3.0 CEC(t) cmolc/dm3 3.5 3.4 3.5 3.0 CEC(T) cmol 3c/dm 6.1 6.5 6.6 5.5 BS % 57.0 52.0 53.0 55.0 SB = sum of basic cations; CEC(t) = effective cation-exchange capacity; CEC(T) = potential cation-exchange capacity; BS = percent base saturation. With the exception of N, soil nutrient levels were Phosphate Institute (PPI). The Potash & Phosphate corrected prior to the implementation of the experiment, Institute published a bulletin (Francis and Piekielek 1999) based on the recommendation of the 5th approximation with guidelines and recommendations for site-specific (Ribeiro et al. 1999), where each plot received 80 kg management of N fertilizer rates. The principle of the PPI P2O5/ha (35 kg P/ha) and 100 kg K2O/ha (83 kg K/ha). methodology is that the plants in the reference plot point to the N absorption potential for a given edaphoclimatic Experimental design and management condition (Villar et al. 2015). The NSI is determined based on the readings of given spectral variables, The experimental area consisted of 16 plots of 0.175 ha according to Equation 1 (Francis and Piekielek 1999): each, where Urochloa brizantha cv. Xaraés grass has been VS psNSI = (1) cultivated since its establishment in December 2008. The VSpr experimental design was a randomized block design, with where: 4 blocks, each containing 4 plots, i.e. a replicate for each of NSI is nitrogen sufficiency index; the 4 treatments: TT – control plot, without application of VSps is spectral variable in the sensor plots; and nitrogen fertilizer; TR – reference plot, with application at VSpr is spectral variable in the reference plot. a fixed rate of 150 kg urea/ha/cycle (~46% N); TG and TS – Whenever sensor plots presented NSI below 0.85, 25% experimental plots, with 75 kg urea/ha applied at the of the N fertilizer dose of the reference plot, equivalent to beginning of each cycle and further applications based on 37.5 kg urea/ha, was applied. The spectral variables were measurement of spectral variables by the sensors, i.e. the determined based on readings when plants in the reference normalized difference vegetation index (NDVI) plot reached 20 and 25 cm in height. To determine the determined by the GreenSeekerTM (TG) and the readings of spectral variable for each plot by the chlorophyll meter the portable chlorophyll meter SPAD 502 (TS). SPAD 502, readings were performed in the middle third of Before the beginning of the experiment on 3 April the the newest fully expanded leaf blade of 30 randomly grass was cut with a brushcutter coupled to a tractor to a identified plants. The average of readings represented the stubble height of 10 cm above soil level. The study SPAD value of the plot. The determination of the NDVI continued for 3 cycles with each cycle including the spectral variable was performed by GreenSeekerTM regrowth period following the exit of animals (cows) from (Trimble), where 40 readings were performed randomly the pasture plus the subsequent grazing period until cows throughout the plot with the apparatus positioned at a height were again removed. As a result of limitations in labor of 1.0 m above the canopy. The average of the readings availability, commencement of studies in the 4 blocks was represented the NDVI value of the plot. staggered with a delay of 1 week between Blocks 1 and 2, Blocks 2 and 3 etc. Production parameters The full nitrogen fertilizer dose (150 kg urea/ha) was applied at the beginning of each cycle for reference plots, Forage yields were determined before grazing when pastures i.e. when animals were removed, while sensor treatments in each plot reached 30 cm in height and after grazing in each received only 75 kg urea/ha. Further applications of N grazing cycle. Representative areas of 3 m2 were selected fertilizer to sensor treatments were based on the nitrogen and sampled using an iron frame of 1 m² at 3 different sufficiency index (NSI) as proposed by the Potash & locations in the plot and weight of fresh forage recorded. Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) Spectral sensors for determination of N needs in Xaraés grass 63 From the cut forage, 300 g subsamples were taken and treatments than in Control treatments as animals took longer separated into leaf blade, culm+leaf sheath and dead forage, to consume the available forage above 15 cm. before being weighed and heated to 65 °C for 72 hours to The temperature and rainfall observed during the study determine dry matter (DM) percentage for the calculation of are presented in Figure 1. Total rainfall during the period forage DM. The growth rate was determined by the differ- was 74 mm, with the highest rainfall concentration ence between amount of forage available when animals were occurring in late May. No rain fell in July-August and removed from a block and that when they were reintroduced mean minimum temperatures fell below 15 °C on within each cycle divided by the length of the regrowth numerous occasions during this period. period, i.e. the amount of forage produced during the regrowth period divided by the length of the regrowth Spectral index during growth cycles of Xaraés grass period. The crude protein (CP) concentration in pasture was determined by the Kjeldahl method (Rodrigues et al. 2017) Data for the NDVI and SPAD variables and the mean of on samples collected prior to the commencement of grazing the NSI variation are shown in Figure 2. The 3 cycles of in each cycle. crop management presented NSI values constantly above After pasture sampling was complete, lactating cows 0.85 for treatments where N fertilizer was applied and (crossbred Holstein × Zebu) were introduced according to below 0.85 for the Control, which received no N fertilizer. pasture availability. The plots were grazed until the plants During each cycle, none of the sensor treatment plots reached a mean of 15 cm in height, at which point the received additional fertilizer after the application of 75 kg cows were removed and the average stocking rate urea/ha at the beginning of the cycle, i.e. they received 75 (AU/ha) during each grazing cycle was calculated kg urea/ha/cycle less than the reference plots. according to the methodology of Delevatti et al. (2019). The yield response to fertilizer (Hare at al. 2015), also Pasture growth and quality known as yield efficiency (Abassi et al. 2005), was then calculated, according to Equation 2, for each treatment at The average presentation yields of the different the end of each management cycle: components of Xaraés grass for each treatment and for the DMfertilized – DMcontrol 3 management cycles when the cows entered the pasture Yield response = (2) Napp are presented in Table 3. During the experimental period, where: the availability of total forage and leaf blades in both DMfertilized is dry matter yield of the N fertilized plots (TS, reference plots and sensor plots were similar (P>0.05). TG or TR); Total forage available for the Control treatment was DMcontrol is dry matter yield of the control plots (TT); and inferior to that of N-fertilized treatments during all cycles Napp is the amount of N applied during the management but differences were significant (P<0.05) only in Cycles cycle. 2 and 3. The amounts of leaf blade available were higher Data on total forage yield, crude protein concentration, in fertilized treatments than in Controls in all cycles but growth rate, stocking rate and leaf blade consumption differences were significant only in Cycles 1 and 2. were subjected to analysis of variance. The comparison of These differences understate the value of fertilizer as the means was performed by the Tukey test (P<0.05), using Control treatment took longer to reach the necessary height the programming language and environment, R. for grazing so data for Controls represent a longer period than those for the N-fertilized treatments. To obtain a more Results valid comparison between treatments, their respective daily pasture growth rates, plus stocking rates and leaf blade Rest periods and weather details consumptions are presented in Table 4. Growth rates of total forage during Cycle 2 and leaf blades during both Cycles 2 The rest periods, i.e. regrowth periods, for each treatment for and 3 were significantly higher (P<0.05) for the fertilized the 3 management cycles plus the duration of grazings are than for the Control treatments. Only during Cycle 2 did the shown in Table 2. The length of each growth cycle of Xaraés TG treatment present growth rate of leaf blades significantly grass for the different treatments varied according to inferior to the growth rate of TS. However, TG growth rate prevailing weather conditions and whether or not N fertilizer was still similar to the TR treatment and superior to the was applied. Fertilized treatments presented similar rest Control. As yield data were not collected when pastures periods in each cycle, while the Control plots required much reached 15 cm height and the study officially commenced longer rest periods for forage to reach the desired height. In following the initial cut on 3 April, growth rates for Cycle 1 Cycles 2 and 3, grazing periods were longer in fertilized are not presented in Table 4. Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) 64 H.C. Bazame, F.A.C. Pinto, D.S. Queiroz, D.M. de Queiroz and D. Althoff Table 2. Average length (days) of the 3 cycles (regrowth and grazing) of Xaraés grass. Treatment Cycle 1 Cycle 2 Cycle 3 Regrowth Grazing Regrowth Grazing Regrowth Grazing TT 38 3 38 2.5 46 2.5 TR 30 3 23 4 30 4 TG 31 3 23 4 30 4 TS 30 3 23 4 30 4 TT = Control; TR = Reference; TG = GreenSeekerTM sensor; TS = SPAD 502 sensor. Figure 1. Length of regrowth and grazing periods and the climatic variables observed during the 3 management cycles. The average stocking rates for treatments that received treatments receiving N fertilizer had higher CP N fertilizer in the 3 crop management cycles (Table 4) percentages in forage than the Control treatment, but were not significantly different (P>0.05), while stocking differences were significant only for Cycles 2 and 3. rates on the Control plots were significantly lower than on Despite differences in amounts of N applied to the various fertilized treatments in all management cycles (P<0.05). fertilized treatments, CP% did not differ between The difference in favor of the fertilized treatments fertilized treatments (P>0.05), although the reference increased in Cycles 2 and 3. treatment always presented higher absolute values. While The estimated crude protein (CP) concentrations in CP% in N treatments remained constant over the 3 cycles, available forage pre-grazing for each treatment in the 3 CP% in the Control treatment declined from Cycle 1 to crop management cycles are presented in Table 5. All Cycle 3. Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) Spectral sensors for determination of N needs in Xaraés grass 65 Figure 2. Spectral variables (NDVI and SPAD) and the mean of the Nitrogen Sufficiency Index (NSI) variation based on the GreenSeekerTM and SPAD 502 measurements for each treatment during the regrowth period for the 3 management cycles of Xaraés grass. TT = Control; T TMR = Reference; TG = GreenSeeker sensor; and TS = SPAD 502 sensor. Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) 66 H.C. Bazame, F.A.C. Pinto, D.S. Queiroz, D.M. de Queiroz and D. Althoff Table 3. Presentation yields of the different components of Xaraés grass for each treatment and cycle when the cows entered the pasture – mean per evaluation. Parameter TT TR TG TS P-value¹ CV (%) Cycle 1 Total forage (kg DM/ha) 1,982 2,405 2,352 2,451 0.15 12.3 Leaf blade (kg DM/ha) 1,279b2 1,661a 1,596ab 1,737a 0.01 9.5 Culm (kg DM/ha) 578 604 618 544 0.84 20.6 Dead forage (kg DM/ha) 133 140 131 173 0.90 61.6 Cycle 2 Total forage (kg DM/ha) 1,993b 2,725ab 2,635ab 3,069a 0.01 13.0 Leaf blade (kg DM/ha) 1,038b 1,708a 1,595a 1,918a 0.00 11.5 Culm (kg DM/ha) 771 767 821 902 0.74 29.2 Dead forage (kg DM/ha) 166 194 116 243 0.34 58.9 Cycle 3 Total forage (kg DM/ha) 2,045b 2,759ab 2,969a 2,793ab 0.04 14.9 Leaf blade (kg DM/ha) 1,427 1,759 1,854 1,714 0.15 14.4 Culm (kg DM/ha) 491 899 969 979 0.09 32.2 Dead forage (kg DM/ha) 122 85 91 71 0.29 38,9 1Probability values by the F test of the analysis of variance. 2Means followed by the same letter on the same line do not differ by the Tukey test (P>0.05), while means with different letters are significantly different (P<0.05). TT = Control; TR = Reference; TG = GreenSeekerTM sensor; TS = SPAD 502 sensor. Table 4. Growth rates of the various components of Xaraés grass, stocking rate and leaf blade consumption for each treatment at the end of the 3 management cycles (mean per evaluation). Parameter TT TR TG TS P-value¹ CV (%) Cycle 1 Stocking rate (AU/ha) 2.3b 3.5a 3.4a 3.3a 0.00 8.7 Leaf blade consumption (kg DM/ha) 792 949 922 1179 0.09 19.5 Cycle 2 Total forage (kg DM/ha/d) 20.7b2 51.9a 56.6a 78.0a 0.00 25.9 Leaf blade (kg DM/ha/d) 15.0c 44.6ab 40.8b 61.1a 0.00 21.3 Culm (kg DM/ha/d) 7.0 6.2 17.3 16.8 0.26 80.8 Stocking rate (AU/ha) 2.1b 6.1a 5.7a 5.6a 0.00 14.1 Leaf blade consumption (kg DM/ha) 329b 931a 825ab 1,159a 0.00 28.0 Cycle 3 Total forage (kg DM/ha/d) 9.7 23.6 29.0 26.8 0.10 46.7 Leaf blade (kg DM/ha/d) 12.9b 32.7a 33.9a 32.7a 0.00 23.9 Culm (kg DM/ha/d) ~0 ~0 1.6 3.3 0.44 >100.0 Stocking rate (AU/ha) 1.5b 4.3a 4.1a 4.1a 0.00 7.3 Leaf blade consumption (kg DM/ha) 908 703 944 742 0.48 30.5 1Probability values by the F test of the analysis of variance. 2Means followed by the same letter on the same line do not differ from each other by the Tukey test (P>0.05), while means followed by different letters are significantly different (P<0.05). TT = Control; TR = Reference; TG = GreenSeekerTM sensor; TS = SPAD 502 sensor. Table 5. Crude protein concentration (% DM) in forage pre-grazing for each treatment in the 3 crop management cycles. Management cycle TT TR TG TS P-value¹ CV (%) Cycle 1 9.2 13.4 11.4 11.9 0.09 19.0 Cycle 2 8.0b2 13.6a 12.4a 12.0a 0.00 11.6 Cycle 3 6.5b 13.4a 11.6a 11.7a 0.00 11.5 1Probability values by the F test of the analysis of variance. 2Means followed by the same letter on the same line do not differ by the Tukey test (P>0.05), while means followed by different letters are different (P<0.05). TT = Control; TR = Reference; TG = GreenSeekerTM sensor; TS = SPAD 502 sensor. Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) Spectral sensors for determination of N needs in Xaraés grass 67 The yield responses to nitrogen fertilizer (kg DM/kg N productive potential is high and plants are well supplied applied) for all management cycles are presented in Table with N. In those cases, there would be no response to 6. As growth data were not available for Cycle 1, we higher doses of nitrogen, and forage production would not based our calculations for Cycle 1 on the differences in suffer (Bredemeier et al. 2013). presentation yields of forage when cows entered the The positive results of using the NSI methodology pastures for grazing and the amounts of N applied (Table were made possible by the spectral responses evaluated in 3). We considered this approach was valid because the this experiment. For instance, the NDVI values presented pastures received equal treatment prior to the a similar response in the 3 management cycles, tending to commencement of the study and any differences between increase as the canopy height increased. Increases in treatments at commencement of grazing were a function NDVI values were expected, since the biomass of the of the N applied. Yield response values were higher for plots increased with the development of the pasture. In the Sensor treatments (TG and TS) than for the reference addition, there was little difference in the amplitude of treatment (TR), although they were not statistically NDVI curves between treatments receiving nitrogen different for Cycles 1 and 3. fertilizer. This explains why, in situations where available nitrogen levels are high, the maximum potential of the Table 6. Yield response to fertilizer (kg DM above Control/kg photosynthetic system is reached and the surplus of total N applied) for each treatment during the regrowth period nitrogen is stored as other reserve compounds (Argenta et for each management cycle. al. 2001; Amaral and Molin 2014). In this context, the value of using sensors is enhanced because, besides Cycle TR TG TS detecting plots which are deficient in nutrients, it is also 1 6.1 10.7 13.6 1 possible to infer when fertilizer application exceeds the 2 5.5c 14.2b 27.9a needs of the pasture, i.e. over-fertilizing. Over-fertilizing 3 6.7 13.4 10.6 1 leads to an increase in nitrogen losses and a decrease in Means followed by the same letter on the same line do not differ from each other by the Tukey test (P>0.05), while means the efficiency of nutrient use by the plants. with different letters are different (P<0.05). T = Reference; T Saturation in sensors that work with NDVI may occur R G = GreenSeekerTM sensor; TS = SPAD 502 sensor. when a high leaf area index is reached, where the linear relationship will no longer apply between sensor Discussion measurements and parameters such as biomass increase (Tremblay et al. 2009; Tian et al. 2016). NDVI saturation Despite its short duration, this study has shown the did not occur during the first 2 cycles since grazing began benefits of using sensors to determine the need for N when height of the pasture reached 30 cm, corresponding fertilizer applications to U. brizantha cv. Xaraés pastures to 95% light interception by the leaf canopy of Xaraés during the April‒August period in the Southeast region of grass. This light interception condition prevents the crop Brazil. Application of N according to readings made with canopy from reaching very dense levels, avoiding auto- the sensors produced as much forage with similar CP% as shading and senescence of the lower leaves, which applying N at a set rate at pre-determined intervals, and represents forage loss. There was only a small tendency resulted in a saving of 75 kg urea/ha/cycle. Since fertilizer for NDVI saturation during Cycle 3. This may be a costs are substantial this would result in significant response to cattle grazing and trampling on pasture, which savings to a farmer. helped cover small regions were soil had been exposed The absence of statistical differences between the 3 N after the drastic cut at the beginning of the experiment. fertilizer treatments confirms the benefits to be gained We also observed a tendency for the SPAD index to from the use of precision tools for determining N fertilizer increase with growth of the pasture. Supporting evidence requirements for Xaraés grass pastures rather than for this increase was provided by Cancellier et al. (2013), applying fertilizer at fixed rates at set times. The sensor who evaluated the dynamics of chlorophyll indices treatments used half the amount of fertilizer applied to the resulting from the application of N fertilizer to upland rice fixed rate treatment, but produced similar amounts of total genotypes in the municipality of Gurupi, Tocantins, forage and leaf blade, allowing similar stocking rates on Brazil. The authors concluded that the younger leaves at all fertilized treatments. Therefore, using this technology the top caused an increase in the readings, which were to apply N at variable rates in areas with spatial variability carried out on the last fully expanded leaf of the plant. could contribute to higher efficiency of nitrogen use. As a consequence of the drastic initial brushcut, the Additionally, using this methodology would lead to a rest period for the first growth cycle (30 days for the reduction in application of N in locations where the treatments with N applied) was the same as for the third Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) 68 H.C. Bazame, F.A.C. Pinto, D.S. Queiroz, D.M. de Queiroz and D. Althoff cycle, when not only the days were shorter but also total Since Urochloa brizantha is a tropical species, precipitation and average temperatures were lower. There variation in climatic factors could have contributed to the are limited studies on the benefits or setbacks from cutting drop in productivity with time in the Control treatment. tropical pastures too close to ground level, but Santos and Tropical forage species have optimal growth within a Fonseca (2016) suggest that cutting pasture short to temperature range of 25‒35 °C and their growth is eliminate material of low acceptance to animals could reduced at lower temperatures, ceasing at temperatures compromise the speed of regrowth. One must remember between 10 and 15 °C (Dantas et al. 2016). Minimum that pasture had to regrow from 10 cm after the initial cut temperatures were below 15 ºC on only a few occasions, to the 30 cm target for Cycle 1, while regrowth in Cycles but more frequently during the third management cycle of 2 and 3 was from 15 cm to 30 cm. the experiment. This could result in thermal limitation to The higher forage yields in the second crop management the growth of pastures. cycle may have been a response to the timing of the rainfall, Total forage yield depends on factors including genetic which occurred mostly at the beginning of the cycle (Figure composition of the species, availability of soil nutrients 1). Another contributing factor could be the fact that and climatic factors such as temperature, luminosity, soil defoliation of pasture during the study was carried out by moisture, etc. In the work carried out by Galzerano et al. animals and was not as severe or as rapid as defoliation by (2011), the authors evaluated Xaraés grass during the wet the brushcutter, so the residual pasture was in a more season in the region of Jaboticabal, São Paulo, Brazil, favorable condition to regenerate. using 95% interception of photosynthetically active Only in Cycle 1 has the Control treatment yielded radiation by the sward as a management criterion and total forage values approaching those of the fertilized applying 100 kg N/ha. The total forage yield obtained was treatments. Before the implementation of the 149 kg/ha/d, which was similar to the 118‒138 kg/ha/d experiment, the area was fertilized every 60 days with found in Cycle 2 for treatments that received N fertilizer, N, according to the farmer’s own criteria, for the even though the current study was conducted in the dry maintenance of forage supply for cattle so there may season, when the climate was much less favorable for the have been residual N in all plots when the study growth of tropical forages. commenced. However, the lack of N fertilizer Yields of leaf blade tended to exceed those of culm and application on the Control treatment during the study dead forage in all 3 management cycles, which is a would have resulted in the depletion of N stocks in the function of pasture being managed at lower heights and soil and caused a reduction in green leaf color (Figure 2) high grazing intensity, favoring greater control of stem and a decrease in forage yield when compared with the elongation (Euclides et al. 2009; Carloto et al. 2011). The fertilized treatments (Table 3). Leaf blade production on higher presence of leaf blades relative to culm and dead the Control treatment was also statistically similar to forage positively affects animal performance, as leaf has those of fertilized treatments during Cycle 3. This may higher nutritional value than the other structures (Castro be only a reflection of significantly lower leaf blade et al. 2013). consumption in the Control plots during Cycle 2, Euclides et al. (2009) evaluated animal production and resulting in high leaf availability at the commencement its relationship with the characteristics of Urochloa of grazing in Cycle 3 (Table 4). In Cycle 3, there were brizantha cvv. Marandu, Xaraés and Piatã, in Campo clear signs of N deficiency in the Control pasture, Grande, Mato Grosso do Sul, Brazil. Mean stocking rates including leaf chlorosis, the appearance of smaller for Xaraés were 3.8 AU/ha in the wet season and 1.4 leaves and growth restrictions that reflected the extended AU/ha during the dry season, which were generally lower duration of the crop cycle (Table 2). than stocking rates obtained in our study. While many Consideration of individual parameters in isolation factors impact on stocking rates of pastures, e.g. age of represents only part of the true differences between the stand, soil fertility, climatic conditions, etc., those treatments. An important factor was the difference in the authors applied only 50 kg N/ha in November-December lengths of time to complete each cycle by the various of each year, so there was probably insufficient N treatments, i.e. fertilized versus Control. Table 3 and available to meet demands of the sward to produce higher Figure 1 show clearly that not only did fertilized forage yields. treatments produce more forage than the Control in each In pastures of Marandu grass fertilized with 1,000 cycle, but also they did it in a much shorter time. A kg/ha/yr (20:05:20, N:P:K) and managed with 30 days nitrogen deficiency was the most likely reason for the rest in Valença, Rio de Janeiro, Brazil, Fukumoto et al. increase in length of regrowth periods and lower DM (2010) obtained an average stocking rate of 5 AU/ha in yields in the Control treatment. the period from January to June 2005. Similar stocking Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) Spectral sensors for determination of N needs in Xaraés grass 69 rate data were obtained in Cycle 2 in our study for the grazing cycles, applying N fertilizer at a lower rate treatments that received N fertilizer, confirming that the determined by readings taken with sensors was more application rates determined by the sensors were efficient than applying N at a fixed rate at preset intervals. consistent with the actual needs of the crop. Longer-term studies to confirm these findings seem It is interesting that crude protein concentration in warranted followed by promotion of this technology to available forage during Cycle 1 did not differ significantly make pasture production more cost-efficient with between treatments, although absolute values were higher associated environmental benefits. in fertilized treatments. One possible explanation for the lack of differences is the history of fertilizer application Acknowledgments to the area. Before the experiment commenced, the area received nitrogen fertilizer every 60 days in an endeavor This study was supported by the Agricultural Research to maintain the forage supply for cattle. Residual N in the Corporation of Minas Gerais (EPAMIG) and the Federal soil may have boosted CP% in the forage produced in the University of Viçosa (UFV), and was funded by the Control treatment. However, as the study advanced, CP% Minas Gerais State Agency for Research and declined in the Control while it was maintained in the N Development (FAPEMIG). treatments despite much higher forage yields in these treatments, which should have produced a dilution effect. References The lack of any significant difference in CP values (Note of the editors: All hyperlinks were verified 7 April 2020.) between reference and sensor treatments in Cycles 2 and 3 suggests that there was a possible N loss in the reference Abassi MK; Kazmi M; Hussan Fu. 2005. Nitrogen use treatment that received N fertilizer at a higher fixed rate. The efficiency and herbage production of an established grass key to optimizing the relationship between crop yield, profit sward in relation to moisture and nitrogen fertilization. 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Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) Tropical Grasslands-Forrajes Tropicales (2020) Vol. 8(2):72–85 72 doi: 10.17138/TGFT(8)72-85 Research Paper Evaluation of forage quantity and quality in the semi-arid Borana Lowlands, Southern Oromia, Ethiopia Evaluación de la cantidad y calidad del forraje natural en la zona semi- árida de Borana Lowlands, Southern Oromia, Etiopía GEMEDO DALLE Center for Environmental Science, Addis Ababa University, Ethiopia. aau.edu.et Abstract This study was conducted with the aim of determining herbaceous biomass during different seasons, plus nutritive value of herbaceous species and forage on selected woody plants and documenting pastoralists’ perceptions of the value of various forage species in Borana Zone, Oromia, Ethiopia. Data were collected from a total of 92 main plots of 500 m2 during rainy and dry seasons located across different functional Land Use Units called Kalo (enclosed areas), Worra (grazed by lactating stock) and Foora (more remote and grazed by dry and non-lactating stock). Total herbage and leaves of woody plants were analyzed for concentrations of crude protein (CP), organic matter (OM), neutral detergent fiber (NDF), acid detergent fiber (ADF), acid detergent lignin (ADL) and ash. Perceptions of farmers were determined through group discussions. Herbage biomass plus chemical composition of both herbaceous and woody forage species varied significantly across seasons and Land Use Units. Mean herbaceous biomass in all Land Use Units was poor (876‒1,469 kg DM/ha). Mean CP, NDF and ADF concentrations of the herbaceous samples were 62, 749 and 444 g/kg DM, respectively. Mean CP% of leaves from woody plants was higher (11%) than from herbage (6%). In both groups, crude protein concentrations were highest during the wet season and lowest during the dry season, whereas fiber concentrations were highest in the dry season. Mean CP% of herbaceous forage species was below the critical level recommended for both beef cattle (7%) and small ruminants (9%) but forage from woody species should provide a reliable supply of supplementary nitrogen. Management of rangelands should be designed to ensure that desirable herbaceous species are preserved, while desirable woody species are also a valuable asset. Determination of management strategies to ensure that the desirable mix of species is maintained is imperative if sustainable production is to continue. Keywords: Borana pastoralists, herbaceous biomass, nutritive value, pasture management, tropical rangelands. Resumen En la zona semi-árida de Borana, Oromia, Etiopía, se midió la biomasa herbácea disponible en pasturas naturales durante diferentes estaciones del año y se determinó el valor nutritivo de las especies herbáceas y del follaje de plantas leñosas seleccionadas. Además se documentó la percepción de los productores pastoriles tradicionales sobre el valor de varias especies forrajeras. Los datos se obtuvieron durante las épocas lluviosas y secas en un total de 92 parcelas de 500 m2 cada una, ubicadas en diferentes unidades funcionales de uso pastoril: Kalo (áreas encerradas), Worra (pasturas utilizadas con hembras lactantes) y Foora (áreas remotas utilizadas con animales no lactantes). De las plantas herbáceas enteras y del follaje de las especies leñosas se analizaron las concentraciones de proteína cruda (PC), materia orgánica (MO), fibra detergente neutra (FDN), fibra detergente ácida (FDA), lignina detergente ácida y cenizas. Las percepciones de los pastores fueron registradas con base en reuniones grupales. La biomasa disponible y la composición química de las especies herbáceas y del follaje de las leñosas variaron significativamente según las estaciones y las unidades de uso pastoril. En promedio de las unidades de uso pastoril, la biomasa herbácea disponible fue baja (876‒1,469 kg MS/ha). ___________ Correspondence: Gemedo Dalle, Center for Environmental Science, Addis Ababa University, P. O. Box 80119, Addis Ababa, Ethiopia. E-mail: gemedo.dalle@aau.edu.et Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) Forage quantity and quality in Borana Lowlands 73 Las concentraciones, promedio, de PC, FDN y FDA de las muestras herbáceas fueron de 62, 749 y 444 g/kg de MS, respectivamente. La concentración, promedio, de PC en el follaje de las plantas leñosas fue mayor (11%) que la las herbáceas (6%). En ambos grupos, las concentraciones de PC fueron más altas durante la estación lluviosa y más bajas durante la estación seca, mientras que las concentraciones de fibra fueron más altas en la estación seca. El porcentaje de PC promedio de las especies herbáceas fue menor que el nivel crítico recomendado tanto para ganado bovino (7%) como para pequeños rumiantes (9%), mientras que se espera que el follaje de las especies leñosas proporcione un suministro confiable de nitrógeno complementario. Los resultados demuestran que el manejo de las pasturas naturales debe ser diseñado para asegurar la conservación de las especies herbáceas deseables, considerando que las especies leñosas deseables son un activo valioso. La identificación y aplicación de estrategias de manejo, tendientes a mantener una combinación deseable de especies en las pasturas, son imprescindibles para asegurar una producción ganadera sostenible en la región. Palabras clave: Biomasa herbácea, manejo pastoril, pasturas naturales tropicales, productores pastoriles, valor nutritivo. Introduction Rangeland management markedly affects botanical composition and, consequently, herbage quantity and The Borana Lowlands occupy 95,000 km2 (Alemayehu quality. In order for the grazing system to be sustainable, Mengistu 2004) in Ethiopia and are populated by better understanding of the characteristics of the forage pastoralists who represent a vital part of Ethiopia’s available is needed. However, little or no data are currently population, contributing significantly to the nation’s available on the quality of plant resources in the study area. GDP. Review of different studies, e.g. Shapiro et al. Therefore, this study was conducted in the Borana (2017), estimated direct contribution of livestock Lowlands to determine both quality and quantity of forage production in lowland pastoral systems of Ethiopia to resources in this semi-arid pastoral production system agricultural GDP and national GDP to be 39 and 17%, throughout the year. The specific objectives were to respectively. The area supports 480,000 families with an determine herbaceous biomass and nutritive value of forage annual population growth rate of 2.5–3% (Homan et al. species and document pastoralists’ perceptions on forage 2004). Livestock production dominated by the Boran species. breed has been the major source of livelihood for Borana pastoralists. According to CSA (2008), in 2007 there were Materials and Methods 1,771,589 cattle, 1,991,196 goats, 699,887 camels and 52,578 donkeys in the Borana zone. The Boran breed Study area remains one of the most productive breeds as it is fast- growing and fertile with good milk production compared The study was conducted in Arero and Yaballo Districts of with other indigenous cattle breeds in Ethiopia (Aynalem Borana Zone, Oromia, Ethiopia (Figure 1). This study was Haile et al. 2011). part of a larger project of the Borana Lowland Development Livestock play a crucial role in the subsistence Program (BLPDP)/Deutsche Gesellschaft für Technische economy, culture and religion of pastoralists in Ethiopia, Zusammenarbeit (GTZ) aimed at developing a pastoral- and represent both social capital and an insurance against oriented self-help concept for sustainable natural resource disaster (Herlocker 1999). Borana pastoralists are known management under changing ecological and socio-economic for their strong tradition of livestock production through conditions. Field data were collected from 2001 to 2003 in using their indigenous rangeland and water management different seasons. strategies. The herbage on offer in the rangelands, The main study sites were Dida Hara Pastoral however, is highly variable, both in quantity and quality. Association (PA) in Yaballo and Web PA in Arero. In Vázquez-de-Aldana et al. (2000) reported that the addition, a government ranch called Dida Tuyura and Foora botanical composition of available forage was highly (an area used for dry or non-lactating livestock) were variable as was the nutritional quality, which was further selected randomly for the forage resource assessment. The exacerbated by topographic relief, soil characteristics, government ranch was reputed to be in relatively good climate, season and management. The semi-sedentary rangeland condition and was included as a benchmark for Borana pastoralists have developed strategies to exploit comparing the other Land Use Units. Yaballo town is 570 this highly variable resource, and are known for km south of Addis Ababa (9°0'19'' N, 38°45'49'' E; 2,355 sustainably using the Borana land in southern Ethiopia for masl). Dida Hara and Web are located about 30 km northeast livestock production. and 85 km southeast of Yaballo town, respectively. Foora is Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) 74 Gemedo Dalle located in Dida Hara PA, about 48 km southeast of Yaballo rainy season. The difference between the long rains and town. Dida Tuyura Ranch is in Dida Yaballo PA (Yaballo short rains is the amount of rain that the area receives. district), about 25 km northeast of Yaballo town. Livestock populations in the Borana Lowlands are Soil characteristics predominantly cattle, while small ruminants and camels are also important in the production system. Rearing The soils in the study area are granitic and volcanic soils and dromedaries has expanded since the 1990s. Estimates have their mixtures (Coppock 1994). Valley bottomlands of the shown that herd composition in Tropical Livestock Units Borana rangelands are dominated by vertisols. Review of (TLU) was 90% cattle, 5% small ruminants and 4% studies that described upland rangeland soils in the study dromedaries (Homann 2004). Borana cattle are a Bos indicus area showed that the soils vary in color (yellow, brown, grey breed that belong to the Large East African Zebu breed or red) and have almost equal proportions of sand, silt and group (Homann 2004). clay (Alemayehu Mengistu 2004). In general, Dida Hara soils are the lightest, containing the highest proportion of sand, whereas Web has soils with higher levels of available P, Ca, Mg, CEC and pH. Mean available P ranged from 2.0 ppm in Foora to 30 ppm in Web Worra. Concentrations of P and Ca and CEC are highly variable in both Dida Hara and Web (Gemedo Dalle 2004). Sampling strategy Borana pastoralists classify their grazing lands into enclosed grazing lands for calves (Kalo), grazing lands for lactating livestock (Worra) and grazing lands for dry livestock (Foora). Based on suitability for different classes of livestock (i.e. availability of forage and watering points), the pastoralists establish their villages (pastoral camps) locally called Olla. Classification and demarcation of the grazing land into Kalo, Worra and Foora is based on distance from the villages and accessibility of watering points: Kalo is adjacent to the villages, Worra the next removed and Foora Figure 1. Location of Borana Zone, in Oromia, Southern Ethiopia. the most remote. Similarly, Kalo and Worra are located (Source: Google Earth). within walking distance (distance from water covered by grazing livestock in a single day, which is about 12 km) from Climatic characteristics watering points, whereas Foora are remote from the watering points (having no permanent watering point within The elevation of the study area ranges from 750 to about the grazing area) and dry livestock utilizing this area depend 2,000 m above sea level. Rainfall is bimodal, with the long on surface rainwater or must walk long distances to access rains during March‒May and short rains during October‒ watering points. Kalo was fenced and protected from November (Haugen 1992; Coppock 1994). Mean annual grazing from early wet season to hot dry season, and was rainfall is 412 mm in Web (Web weather station; data from accessible for grazing only during the hot dry season. Worra Southern Range Development Unit) and 566 mm in Dida and Foora were open to livestock throughout the year. A Hara (Yaballo town as the nearest station; data from the stratification sampling technique was used to collect samples National Meteorological Services Agency of Ethiopia). from these functional Land Use Units. Within each Land While mean annual temperature varies from 19 to 24 °C Use Unit, the initial sampling point was established (Alemayehu Mengistu 1998), the mean maximum randomly, but subsequent units were established at 200 m temperatures for Yaballo stations ranged from 24.4 to 26.4 intervals on a linear transect. °C and minimums from 13.8 to 14.8 °C (1989‒2001 raw Samples of both herbaceous and woody forage species data from the National Meteorological Services Agency of were gathered from different Land Use Units. Herbaceous Ethiopia). In general, December‒February is the hot dry samples were collected during cool dry (June‒August), short season, March‒May is the long rainy season, June‒August rains (September‒November) and long rains (March‒May) is the cool dry season and September‒November is the short seasons, whereas woody samples were taken during short Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) Forage quantity and quality in Borana Lowlands 75 rains, hot dry (December‒February) and long rains seasons. sample of each browse species was collected and only from Within the various districts, an effort was made to sample a single tree/shrub. from different sites in all seasons, in an attempt to ensure that Nomenclature of plant species follows published the samples were representative for the specific study sites. volumes of Flora of Ethiopia and Eritrea (Hedberg and Edwards 1995; Edwards et al. 1995, 1997) and was updated Forage sampling according to the taxonomy of the Genetic Resources Information Network GRIN (npgsweb.ars-grin.gov/ Forage samples were collected from a total of 92 main plots gringlobal/taxon/taxonomysearch.aspx). of 50 × 10 m (500 m2) each (Table 1). Within each 500 m2 plot, 5 subplots of 0.5 × 0.5 m (0.25 m2), 4 at the corners and Chemical analyses 1 in the center, were established and herbaceous samples were collected for both biomass and forage nutritive value After oven-drying of samples at 105 °C, dry matter (DM), determination. Samples from the 5 subplots were pooled and organic matter (OM), crude protein (CP), neutral detergent assumed to represent the main plot. To demarcate these fiber (NDF), acid detergent fiber (ADF), acid detergent subplots, a 3-sided frame of welded metal (0.5 × 0.5 m), left lignin (ADL), ash, ADF-Ash and in vitro digestibility of dry open at one side as recommended by Whalley and Hardy matter (IVDMD) were determined in the laboratory of the (2000), was used. All grasses, herbaceous forbs and sedges International Livestock Research Institute (ILRI), Addis rooted within the marked area of 0.25 m2 were cut at 2 cm Ababa, Ethiopia. IVDMD was analyzed only during the cool above ground following the method of Vázquez-de-Aldana dry season. et al. (2000). Immediately after harvesting, the material CP was determined using the Kjeldahl method (N × sampled in each plot was sorted manually into species and 6.25), IVDMD by the in vitro rumen digestibility procedure weighed in the field using a portable digital balance to (Van Soest and Robertson 1985) and NDF, ADF and ADL determine contributions of individual species to total fresh by the detergent system of analysis (Van Soest and biomass. Because of logistical issues sorted samples could Robertson 1985). Ash was determined by igniting samples not be dried individually and were pooled again, dried at 60 at 500 °C (AOAC 1990). °C for about 48 hours in a well-ventilated oven (Adesogan et al. 2000) and weighed to determine both total herbaceous Pastoralists’ perceptions biomass and the contribution of individual species to total dry biomass. Eight community-level group discussions were held in 4 For woody plants, samples of green leaves (including places: Dikale (DIK), Dambala Abba Chana (DAC) (both in young and old) of each plant and each species were collected Dida Hara), Tesso Qallo (TSQ) and Dhibu Kolocho (DBK) at random. For Vachellia tortilis (syn. Acacia tortilis), fruits (both in Web PA). One hundred and eight pastoralists (52 (pods) were also collected as they were preferred by animals. men and 56 women) participated in the group discussion. A total of 75 samples (25 samples for each of the 3 seasons) Pair-wise preference ranking was used to identify the most were collected and analyzed. An effort was made to sample preferred forage species. According to their palatability to from the same species in all 3 seasons. However, during the livestock, grass species were classified as highly desirable hot dry season, some species had shed leaves, so samples (decreasers), intermediate (increasers) and least desirable were taken from other drought-resistant forage species as (pioneers), based on pastoralists’ perceptions and field indicated by the pastoralists. In each season, only 1 sample observations. Decreasers were defined as desirable grass per species was taken from the first site where the identified species that are likely to decrease with heavy grazing woody plant was encountered. In other words, only a single pressure (Baars et al. 1996). Table 1. Descriptions of Land Use Units and sampling details in the Borana Lowlands, Ethiopia. Land Use Unit Explanation Sampling intensity No. of samples No. of seasons Total Dida Hara Kalo (DHK) Dida Hara grazing land for calves 21 3 63 Dida Hara Worra (DHW) Dida Hara grazing land for lactating livestock 14 3 42 Web Kalo (WBK) Web grazing land for calves 19 3 57 Web Worra (WBW) Web grazing land for lactating livestock 14 3 42 Dida Tuyura Ranch (DTR) Government ranch used for conserving Borana breeds 10 2 20 Foora (FOR) Grazing land for dry livestock between Dida Hara and 14 3 42 Web Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) 76 Gemedo Dalle Data analysis Tuyura Ranch, which were relatively protected sites, were higher than those in other Land Use Units, which were Descriptive statistics were used in organizing, communally grazed, during the main rainy season. summarizing and describing the data. Comparison of Mean herbage biomass over all Land Use Units ranged mean values was performed using two-tailed t-test from 921 kg DM/ha in the early wet season to 1,241 kg following Fowler and Cohen (1996). ANOVA was DM/ha in the cool dry season following the long rains. applied to investigate variability across Land Use Units Available herbage biomass during the cool dry season and seasons. Effects of season × land use interactions was higher than in the other seasons. were determined using GLM (General Linear Model). Biomass contribution of herbaceous species Results The contribution to available biomass by various Rainfall herbaceous species varied among sites and seasons. After short rains, Chrysopogon aucheri, Digitaria milanjiana Rainfall data for Dida Hara were taken from Yaballo town, and Eragrostis papposa were the main contributors in which was the nearest station to Dida Hara and that of Web Dida Hara, C. aucheri alone contributing almost half of was from Web station, which was collected by Southern the herbaceous biomass. In Web, most of the contribution Range Development Unit. The mean annual rainfall in Dida Hara (Yaballo) and Web is presented in Table 2. Table 2. Mean monthly rainfall in mm (1988‒2001) and the While mean monthly rainfall ranged from 5.9 to 144.1 coefficient of variation at the main study sites in the Borana mm in Dida Hara and 2.0 to 113.9 mm in Web (Table 2), lowlands, Ethiopia (raw data for Yaballo were taken from the variation within individual months was great as observed National Meteorological Services Agency of Ethiopia). from the CV%. Month Dida Hara Web Two rainfall peaks are conspicuous, demonstrating the Mean CV (%) Mean CV (%) bimodal nature of rainfall in the Borana Lowlands. Annual totals were highly variable, ranging from 188 to January 24.5 125.7 17.1 193.6 803 mm with mean of 545 mm in Dida Hara, and from February 33.6 129.5 9.2 208.7 211 to 638 mm with mean of 412 mm in Web. This March 56.4 76.4 68.6 66.9 difference in annual rainfall between the two sites was April 144.1 49.2 113.9 60.9 statistically significant (t = 2.196, df = 22 at P=0.05), May 77.1 69.8 57.9 68.6 indicating that Web is more arid than Dida Hara. June 14.5 65.5 3.2 146.9 July 12.0 106.7 3.1 151.6 Herbaceous biomass August 5.9 105.1 2.0 170 September 35.2 98.3 15.7 135.0 The above-ground herbaceous presentation yields October 87.4 59.3 58.6 93.7 (standing crop) were highly variable both spatially and November 37.1 83.6 51.7 98.1 temporally (Tables 3 and 4), with significant differences December 17.4 69.5 11.6 171.5 across seasons (P = 0.026) and Land Use Units (P = Overall total 545 413 0.000). Presentation yields for Dida Hara Kalo and Dida SD 40.2 35.3 Table 3. Effects of season on mean herbaceous presentation yields (kg/ha) on different Land Use Units in the Borana Lowlands, Ethiopia. Land Use Unit Cool dry (Jun-Jul 2001) Short rains (Nov-Dec 2001) Long rains (Mar-Apr 2002) Mean SD Mean SD Mean SD Dida Hara Kalo 1,285 119 1,841 676 1,093 540 Dida Hara Worra 1,220 68 680 297 850 320 Web Kalo 1,269 93 1,393 662 712 210 Web Worra 1,162 174 983 496 576 658 Dida Tuyura Ranch -1 - 1,542 251 1,396 740 Foora 1,270 12 9 458 15 2 901 63 5 Mean 1,241 1,150 921 1Logistical issues prevented data collection at Dida Tuyura Ranch in June-July 2001. Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) Forage quantity and quality in Borana Lowlands 77 Table 4. Herbaceous biomass contribution by each species (% DM basis) on different Land Use Units in the short and long rainy seasons, and their forage value (FV) as perceived by pastoralists in the Borana Lowlands, Ethiopia. Plant family Species Land Use Unit1 (%) FV2 DHK DHW WBK WBW FOR DTR Short rains (November) Acanthaceae Barleria spinisepala 0.8 0.2 0.0 0.0 0.1 0.5 H Asparagaceae Chlorophytum gallabatense 6.0 0.5 0.1 0.0 0.0 1.8 H Commelinaceae Commelina africana 1.8 1.0 0.1 0.0 0.2 0.3 H Cyperaceae Cyperus bulbosus 0.0 0.3 0.0 0.0 1.9 0.0 I Cyperus sp. 2.7 1.6 0.2 0.4 0.4 0.4 I Fabaceae Indigofera volkensii 0.3 0.0 0.0 1.4 0.0 0.0 I Poaceae Cenchrus ciliaris 0.0 0.0 6.2 2.4 0.0 0.0 H Tetrapogon roxburghiana (syn. Chloris roxburghiana) 0.0 1.4 5.5 4.4 13.4 0.0 I Chrysopogon aucheri 41.3 50.2 4.5 2.5 57.1 37.5 I Cynodon dactylon 0.0 0.0 10.9 0.0 0.0 0.0 H Digitaria milanjiana 12.5 10.7 0.0 0.0 16.1 1.7 H Eleusine intermedia 3.1 0.0 0.0 0.0 0.0 0.9 L Eragrostis papposa 13.1 21.1 0.3 1.0 10.2 2.1 I Harpachne schimperi 4.4 1.0 0.0 0.0 0.6 0.7 I Heteropogon contortus 2.4 7.3 0.0 0.0 0.0 33.0 I Ischaemum afrum 0.0 0.0 5.3 0.0 0.0 0.0 L Leptothrium senegalense 0.0 0.0 0.1 0.7 0.0 0.0 I Megathyrsus maximus (syn. Panicum maximum) 1.5 0.0 0.0 5.9 0.0 0.0 H Cenchrus mezianus (syn. Pennisetum mezianum) 4.7 0.0 66.9 81.6 0.0 0.0 L Themeda triandra 4.1 3.2 0.0 0.0 0.0 21.0 I Velloziaceae Xerophyta humilis 0.8 1.2 0.0 0.0 0.0 0.2 I Long rains (April) Acanthaceae Barleria spinisepala 0.0 0.0 0.0 0.0 0.7 0.0 H Asparagaceae Chlorophytum gallabatense 3.4 0.8 0.0 0.0 0.3 0.1 H Commelinaceae Commelina africana 3.1 1.2 0.4 3.9 1.5 0.0 H Cyperaceae Cyperus bulbosus 0.0 0.0 2.7 0.4 0.0 0.0 I Cyperus sp. 5.9 0.8 4.9 1.8 0.0 0.4 I Poaceae Andropogon chinensis 0.0 0.0 0.0 0.0 0.0 29.7 L Bothriochloa radicans 0.0 0.0 0.0 0.0 0.0 3.2 L Cenchrus ciliaris 0.0 2.4 42.8 26.1 0.0 0.0 H Tetrapogon roxburghiana (syn. Chloris roxburghiana) 1.3 0.0 0.4 8.2 0.0 0.4 I Chrysopogon aucheri 40.4 52.1 8.5 13.4 57.8 14.2 I Digitaria milanjiana 11.7 14.3 19.5 7.6 7.5 0.0 H Digitaria neghellensis 0.0 0.0 0.0 0.8 0.0 0.0 H Eleusine intermedia 3.3 0.0 0.0 0.0 0.0 0.0 L Eragrostis papposa 11.3 12.1 0.0 0.0 1.6 0.0 I Harpachne schimperi 0.0 0.0 0.0 0.0 0.0 0.4 I Heteropogon contortus 10.4 5.6 0.0 0.0 0.0 34.9 I Leptothrium senegalense 0.0 3.4 0.0 0.0 0.0 0.0 I Megathyrsus maximus (syn. Panicum maximum) 0.0 0.0 0.0 6.2 0.0 0.0 H Cenchrus mezianus (syn. Pennisetum mezianum) 3.2 1.6 0.0 18.9 30.6 0.0 L Setaria verticillata 0.0 0.0 0.2 0.1 0.0 0.0 H Sporobolus pellucidus 0.0 0.0 20.3 12.7 0.0 0.0 I Themeda triandra 6.1 0.8 0.0 0.0 0.0 16.7 I Velloziaceae Xerophyta humilis 0.0 4.7 0.0 0.0 0.0 0.0 I 1Land Use Unit: DHK = Dida Hara Kalo; DHW = Dida Hara Worra; WBK = Web Kalo; WBW = Web Worra; FOR = Foora; and DTR = Dida Tuyura Ranch; 2Forage value: H = highly palatable; I = intermediate; and L = least palatable, as assessed by pastoralists from the main study sites, Dida Hara and Web. Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) 78 Gemedo Dalle to biomass (74%) was from the least palatable grass, D. microphylla, F. sycomorus and Ziziphus sp. were Cenchrus mezianus (syn. Pennisetum mezianum). In Foora, sampled only during the hot dry season when other highly most of the biomass (57%) was from a single species ranked species had shed their leaves. Salvadora persica, C. aucheri, although Tetrapogon roxburghiana (syn. Chloris Kirkia burgeri, F. sycomorus, Commiphora kataf (syn. roxburghiana) (13%), D. milanjiana (16%) and E. papposa Commiphora erythraea) and Terminalia prunioides had (10%) made marked contributions. On Dida Tuyura Ranch, high amounts of ADF, NDF and ADL. C. aucheri (37%), Heteropogon contortus (33%) and CP% in woody plants was highest in the wet season Themeda triandra (21%) were major contributors (Table 4). (169 g/kg DM) and lowest in the dry season (139 g/kg In the early wet season (Long rains; Table 4), C. aucheri DM; Table 8). contributed nearly half (46%) to available herbaceous biomass at Dida Hara, and about 58% at Foora sites. Table 5. Mean contribution (% DM basis) to available biomass by C. mezianus made a large contribution (31%) at Foora site. highly desirable, intermediate and least desirable species in the In Web, Cenchrus ciliaris contributed the highest proportion Borana Lowlands, Ethiopia for the various Land Use Units. (34%), while at Dida Tuyura Ranch, the dominant 1 contributor was H. contortus (35%). Overall, C. aucheri was Land Use Unit the main contributor to herbaceous biomass at most sites DHK DHW WBK WBW DTR FOR during both seasons. At Web sites, the dominant species Short rains (November) were C. mezianus in November (short rains) and C. ciliaris Grasses in April (long rains). Highly desirable 14.0 10.7 17.1 8.3 1.7 16.1 Estimation of contributions to available biomass by Intermediate 65.3 84.2 10.4 8.6 94.3 81.3 highly desirable, intermediate and least desirable forage Least desirable 7.8 0.0 72.2 81.6 0.9 0.0 grasses showed that intermediate grasses were most Sedges 2.7 2.0 0.2 0.4 0.4 2.3 Forbs 10.2 3.0 0.2 1.4 2.8 0.3 prominent in Dida Hara, Dida Tuyura and Foora areas but Long rains (April) not in Web zones (Table 5). Grasses Highly desirable 11.7 16.9 62.5 40.8 0.0 7.5 Nutritive quality of herbaceous species Intermediate 69.5 74.1 29.2 34.3 66.6 59.5 Least desirable 6.5 1.6 0.0 18.9 32.9 30.6 Chemical composition of herbaceous samples was Sedges 5.9 0.8 7.6 2.2 0.4 0.0 compared across both Land Use Units and seasons (Table Forbs 6.5 6.7 0.5 3.9 0.1 2.5 6). In general, there was a strong trend for an increase in Mean of the two seasons CP% from the cool dry season to the long rainy season. Grasses Mean CP% was 48 g/kg DM in cool dry season, 62 g/kg DM Highly desirable 12.8 13.8 39.8 24.5 0.8 11.8 after short rains and 76 g/kg DM during the long rainy Intermediate 67.4 79.1 19.8 21.4 80.4 70.4 season. There was variation in CP% across the seasons (P = Least desirable 7.2 0.8 36.1 50.2 16.9 15.3 0.000) and Land Use Units (P = 0.013). Sedges 4.3 1.4 3.9 1.3 0.4 1.1 However, there were no differences in CP levels across Forbs 8.3 4.9 0.4 2.6 1.5 1.4 various Land Use Units during the cool dry season. During 1Land Use Units: DHK = Dida Hara Kalo; DHW = Dida Hara the short rains, Dida Hara and Foora sites had higher CP than Worra; WBK = Web Kalo; WBW = Web Worra; FOR = Foora; Web sites and Dida Tuyura Ranch. During the long rains, and DTR = Dida Tuyura Ranch. Web sites showed higher CP levels than Dida Hara sites with Dida Tuyura Ranch showing lowest levels (Table 6). Pastoralists’ perceptions Nutritive value of woody plants Ranking of the forage value of plant species was performed through group discussions with pastoralists. Mean chemical composition of leaves from woody plants is According to the perceptions of Borana pastoralists, summarized in Table 7. CP concentration ranged from 47 to Cenchrus ciliaris and Digitaria milanjiana were grasses 168 g/kg DM. Senegalia brevispica (syn. Acacia with the highest nutritive value (Tables 4 and 9). brevispica), Balanites aegyptiaca, Chionothrix latifolia, Similarly, Senegalia brevispica, Grewia tembensis and Combretum hereroense, Cordia sinensis (syn. Cordia Maeroa triphylla were the most highly appreciated gharaf), Dalbergia microphylla, Ficus sycomorus, Maerua woody species (Table 8). Some forbs, such as Commelina triphylla and Ziziphus sp. were the top species with higher africana, have high nutritional quality. All sedges CP%. CP% for most species reported in Table 6 was an (Cyperus spp.) were perceived to have intermediate average for 3 seasons. However, C. hereroense, C. sinensis, nutritional value (Table 4). Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) Forage quantity and quality in Borana Lowlands 79 Table 6. Mean chemical composition of the herbaceous biomass sampled in three seasons across different Land Use Units (LUU) in the Borana Lowlands, Ethiopia. LUU1 Ash OM NDF ADF ADL ADF-Ash CP IVDMD (%) (%) (g/kg) (g/kg) (g/kg) (g/kg) (g/kg) (%) Cool dry season (June-July) DHK 11 89 761 429 91 68 45 38 DHW 13 87 742 434 89 82 45 32 WBK 11 90 754 439 144 59 52 37 WBW 9 91 780 491 277 42 47 33 DTR2 - - - - - - - - FOR 10 90 768 468 95 67 49 36 Short rains (November-December) DHK 10 90 768 481 101 51 67 DHW 11 89 763 418 87 69 79 WBK 9 91 798 504 111 52 49 WBW 9 91 803 508 126 52 52 DTR 9 91 768 479 89 51 50 FOR 11 89 730 407 80 68 76 Long rains (March-April) DHK 12 88 717 413 91 57 78 DHW 15 86 696 383 78 77 84 WBK 21 79 638 368 78 134 96 WBW 23 77 653 375 112 143 101 DTR 9 91 779 468 90 46 40 FOR 11 89 778 450 114 65 58 1Land Use Units: DHK = Dida Hara Kalo; DHW = Dida Hara Worra; WBK = Web Kalo; WBW = Web Worra; FOR = Foora; and DTR = Dida Tuyura Ranch. 2During the cool dry season, samples were not collected from DTR. OM = organic matter; NDF = neutral detergent fiber; ADF = acid detergent fiber; ADL = acid detergent lignin; CP = crude protein; IVDMD = in vitro dry matter digestibility. Table 7. Mean nutritive values of leaves from woody plants across 3 seasons and frequency of appreciation (FAP) of the forage species by pastoralists in the Borana Lowlands, Ethiopia. Family Species DM Ash NDF ADF ADL CP FAP (%) (%) (g/kg) (g/kg) (g/kg) (g/kg) (%) Amaranthaceae Chionothrix latifolia1 91 19 378 251 36 136 383 Anacardiaceae Rhus natalensis1 91 10 538 422 246 90 75 Asteraceae Vernonia phillipsiae 91 12 480 328 119 76 50 Apocynaceae Cynanchum viminale (syn. Sarcostemma viminale)1 89 11 498 482 93 47 38 Burseraceae Commiphora kataf (syn. Commiphora erythraea) 88 10 307 296 127 93 13 Commiphora habessinica 89 14 473 484 291 114 25 Capparaceae Boscia mossambicensis1,3 92 8 616 414 143 100 633 Cadaba farinosa1 91 24 379 290 156 113 25 Maerua triphylla1 92 14 451 294 107 141 63 Combretaceae Combretum hereroense2 91 11 314 268 72 140 13 Terminalia prunioides1 89 9 290 286 68 100 25 Convolvulaceae Cladostigma hildebrandtioides 92 9 546 390 79 117 253 Cordiaceae Cordia sinensis (syn. Cordia gharaf)1,2 91 18 365 407 89 141 13 Ebenaceae Euclea divinorum1,2 92 8 245 329 151 80 383 Fabaceae Senegalia brevispica (syn. Acacia brevispica)1 91 7 421 278 132 154 88 Senegalia goetzei (syn. Acacia goetzei)1 91 7 511 458 250 109 133 Vachellia tortilis (syn. Acacia tortilis)1 91 7 490 440 208 113 633 Dalbergia microphylla1,2 92 8 293 217 65 150 25 Continued Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) 80 Gemedo Dalle Family Species DM Ash NDF ADF ADL CP FAP (%) (%) (g/kg) (g/kg) (g/kg) (g/kg) (%) Kirkiaceae Kirkia burger 88 8 280 217 68 109 13 Lamiaceae Plectranthus igniarius 90 18 502 540 250 116 38 Malvaceae Grewia damine (syn. Grewia bicolor)1,3 91 9 484 367 128 104 1003 Grewia tembensis1 90 13 407 303 67 130 88 Moraceae Ficus sycomorus1,2 89 15 250 234 54 134 133 Oleaceae Olea europaea subsp. cuspidata1 93 6 384 297 127 73 383 Schrebera alata1,2 92 6 306 301 79 97 - Phyllanthaceae Phyllanthus sepialis 91 12 432 298 97 99 383 Rhamnaceae Ziziphus sp.2 92 5 177 153 41 168 - Rutaceae Vepris glomerata1 93 10 465 380 173 144 38 Salvadoraceae Salvadora persica1 88 36 252 162 39 89 13 Sapindaceae Haplocoelum foliolosum1 93 7 473 359 153 77 253 Pappea capensis1,3 92 6 526 423 163 83 753 Zygophyllaceae Balanites aegyptiaca1,3 92 11 408 287 132 120 38 1Drought-resistant forage species (according to the pastoralists’ perceptions). 2Species sampled only once during hot dry season (December‒February) but were not appreciated as highly desirable forage species by the pastoralists (Gemedo Dalle 2004). 3Woody forage species appreciated by the pastoralists for grazers (cattle). DM = dry matter; NDF = neutral detergent fiber; ADF = acid detergent fiber; ADL = acid detergent lignin; CP = crude protein; FAP = frequency of appreciation. Table 8. Mean seasonal chemical composition of forage from woody plants in the Borana Lowlands, Ethiopia. Season Ash (%) NDF (g/kg) ADF (g/kg) ADL (g/kg) CP (g/kg) Hot dry (Jan-Feb) 10.9 326 300 96 139 Short rains (Nov-Dec) 12.5 506 391 161 167 Long rains (Mar-Apr) 11.6 444 332 138 169 NDF = neutral detergent fiber; ADF = acid detergent fiber; ADL = acid detergent lignin; CP = crude protein. Table 9. Preference ranking (1 = highest rank) of the top 5 grass and woody forage species by men (M) and women (W) pastoralists (number of participants in parenthesis) during group discussions in the Borana Lowlands, Ethiopia. Site1 and gender group DIK DAC TSQ DBK M (11) W(13) M (13) W (10) M (13) W(21) M (15) W (12) Forage grasses Cenchrus ciliaris 1 2 1 2 1 2 1 2 Chrysopogon aucheri 4 4 3 3 - 4 3 5 Digitaria milanjiana 2 1 2 1 3 1 2 4 Digitaria neghellensis 3 -2 - - 2 - - - Eleusine intermedia - - 5 - - - 5 - Halchisoo (botanical name not determined) - - - - 4 - - 1 Megathyrsus maximus (syn. Panicum maximum) - - 4 5 - - 4 - Cenchrus mezianus (syn. Pennisetum mezianum) 5 3 3 3 Themeda triandra - 5 - 4 5 5 - - Woody plants for browsers Senegalia brevispica (syn. Acacia brevispica) 1 - 1 1 2 - 2 1 Vachellia nilotica subsp. nilotica (syn. Acacia nilotica) - 5 - - - - - - Vachellia tortilis (syn. Acacia tortilis) 4 - - - - - - - Balanites aegyptiaca - 4 2 - - - - - Blepharispermum pubescens3 - - - - - 2 - - Cadaba farinosa - - - - 4 1 - - Continued Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) Forage quantity and quality in Borana Lowlands 81 Site1 and gender group DIK DAC TSQ DBK M (11) W(13) M (13) W (10) M (13) W(21) M (15) W (12) Commiphora kataf (syn. Commiphora erythraea) - - - - - - 3 - Dalbergia microphylla - - - - - - - 5 Grewia damine (syn. Grewia bicolor) - - - - - 5 - - Grewia tembensis 5 2 3 3 1 - 1 4 Kirkia burgeri - - - - - - 4 - Maerua triphylla 3 - 5 4 - 4 - 3 Phyllanthus sepialis - - 4 - - - - - Plectranthus barbatus (syn. Plectranthus comosus)3 - - - - 5 - - - Rhus natalensis 3 2 Cynanchum viminale (syn. Sarcostemma viminale) 2 1 - 2 - - - - Vepris glomerata - - - - 3 - 5 - Vernonia phillipsiae 3 5 1DIK = Dikale, DAC = Dambala Abba Chana, TSQ = Tesso Qallo and DBK = Dhibu Kolocho. 2Empty cell (-) means the species was ranked below rank 5. 3Species not listed in Table 7 (Table 7 contains species that were sampled, whereas this table contains species mentioned by local communities during free listing). Discussion Although the author of this manuscript agrees with protection of the ranch area, proper management Herbaceous standing crop following standard range management techniques should be followed. It should serve as a demonstration farm and The greater availability of forage on the government learning laboratory for surrounding pastoralists. Ayana ranch and Kalos (enclosures) than on open-grazed areas Angassa et al. (2010) also reported that there was such as the Worra and Foora showed the importance of variation as a result of rangeland management that affect- exclosures for conserving and sustainably using forage ed biomass of most herbaceous species, plus grass basal resources during the dry season. This was in agreement cover and herbaceous species richness and diversity. with previous reports (Oba et al. 2001; Ayana Angassa et Quantifying the contributions of various species to al. 2010). The pattern for highest mean herbaceous forage mass allows useful comparisons of the productivity biomass to occur in the cool dry season (Jun-Jul) and of different species and different management practices, lowest in the main rainy season (Mar-Apr), after the hot and provides a basis for appropriate stocking rates to be dry season, was according to expectations. In the cool dry developed (Sollenberger and Cherney 1995). Rainfall season growth of vegetation has been stimulated by the (precipitation), soil moisture, radiation, temperature, soil long rains and herbage is mature. It is traditional for nitrogen and phosphorus are important environmental Borana pastoralists to protect Kalos from grazing during factors that affect herbage production (Gutman et al. 1990). the wet season and open them for general grazing in the The maximum presentation yield of herbage recorded in hot dry season (Dec-Jan), when there is relatively high this study was 1,840 kg DM/ha during the short rains, while accumulation of herbage mass. Grazing land management studies in similar semi-arid ecosystems reported much practices may be the main reasons for the significant higher values. In similar arid areas of northern Kenya, differences in presentation yields of herbaceous biomass mean herbaceous biomass inside exclosures was 4,180 kg across the Land Use Units in this study rather than the DM/ha and that of continuously grazed open rangelands actual productive ability of these areas. The different 1,802 kg DM/ha (Oba et al. 2001). Our results for presentation yields of forage on the government ranch herbaceous biomass yields from all sites in the Borana from those on the communal grazing lands may reflect Lowlands fall within the category of poor, and such low grazing management imposed. There were only few herbaceous yields would directly affect livestock Borana cattle on the government ranch during the study production and ecosystem stability. years and it was therefore subjected to only low stocking Unlike the study of Ayana Angassa and Baars (2000), pressure during all seasons. Old growth accumulated and very low percentages of highly palatable grasses were rank grass dominated the new growth of desirable species. found. Grasses considered of intermediate nutritive value Furthermore, the ranch was highly encroached by woody by pastoralists were dominant at most sites, while highly species that might have contributed to low forage quality. palatable species were present at much lower levels, in Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) 82 Gemedo Dalle agreement with the rangeland condition assessment report aucheri and Digitaria milanjiana as drought-resistant by Gemedo Dalle et al. (2006). This aspect is discussed forage grasses, concurring with the report by Skerman further in a subsequent section. and Riveros (1989). Among the woody species Vachellia tortilis, Boscia mossambicensis, Chionothrix latifolia, Seasonal changes in forage value Grewia damine and Pappea capensis for grazers (cattle) and Senegalia brevispica, Grewia tembensis and Maerua Both herbage biomass and chemical composition of the triphylla for browsers, e.g. camels and goats, were ranked herbaceous forage samples varied significantly across the as the top forage species. Le Houérou and Corra (1980) seasons. Physiological age of forage species, time of also reported that most of the woody plants identified grazing, species and botanical fraction are some of the during this study were considered palatable for animals, factors that cause variability in chemical composition of being preferred over other species. forages (Adesogan et al. 2000). The decline in CP% in Comparing pastoralists’ indigenous knowledge with herbaceous forage from 7.6% in the wet season to 4.8% laboratory results showed that Borana pastoralists have an in the dry season, and that of woody plants from 16.9 to accurate perception of the nutritive value of the various 13.9%, respectively, is in agreement with previous forage species. In general, the pastoralists’ knowledge of findings (Pérez Corona et al. 1998; Hussain and Durrani forage species growth and quality indicated that they know 2009; Habtamu Teka et al. 2013; Zhai et al. 2018). which species should be retained in the pasture and which According to the report by Habtamu Teka et al. (2013), were least useful to ensure sustainable animal production chemical composition of all grass species showed in the area. significant (P<0.05) variation between sites, seasons and species in agreement with our results. Comparison of the Land Use Units In contrast, nutrient concentrations of browse from woody plants is subject to relatively less seasonal variation The high fluctuation of species composition on Web sites (Crowder and Chheda 1982) and this particularly enhances might be due to presence of permanent watering points their value as dry season feeds for livestock (Dicko and (Web deep wells or Ela) in the area, which allowed high Sikena 1992). About three-quarters of the woody forage numbers of livestock to remain in the area resulting in plants studied were perceived as drought-resistant species overgrazing and consumption of desirable species. by pastoralists. Several studies from arid ecosystems, e.g. During the long rains, most livestock are taken away from Dicko and Sikena (1992), have shown that perennial shrubs the deep wells as water is more readily available and retained high CP% for a longer period than herbaceous highly desirable species get the opportunity to regrow species, with a range of 7% in winter to 14% in spring. during this time. During the hot dry season when herbaceous species were When comparing Land Use Units, CP% varied across almost dry and in limited supply for livestock, mean CP% the Land Use Units as reported by Habtamu Teka et al. in leaf material on woody plants was 13.9%, which would (2013). Overall, forage on the Dida Tuyura Ranch had the provide a valuable N supply to rumen microflora provided lowest CP levels and the highest concentrations of NDF the nitrogenous components were digestible. Mean in vitro and ADF reflecting the under-utilization of this area and dry matter digestibility of the herbaceous forage was very accumulation of a bulk of mature fibrous material. This low (35%) with a range from 32 to 38% indicating its suggests that ‘over-protection’ of rangelands is not limited potential to contribute energy. Conservation of necessarily a desirable management strategy and these drought-resistant species is an important strategy for significant defoliation by grazing animals at certain times sustaining livestock production, especially during dry might be needed to stimulate pastures and ensure a periods. sustainable system. Comparing pastoralists’ perceptions and scientific Status of forage nutritive value in relation to livestock knowledge on forage nutritive value production The pastoralists identified Cenchrus ciliaris, Digitaria Herbaceous species had lower forage quality than the milanjiana, Megathyrsus maximus and Themeda triandra woody browse species in agreement with previous reports as highly palatable grasses. Similar perceptions of Borana (Hussain and Durrani 2009; Gete Zewudu and Gemedo pastoralists were reported by Habtamu Teka et al. (2013) Dalle 2019). CP concentrations in standing forage and in an earlier report by Skerman and Riveros (1989). exceeded the threshold level of 7% (Humphreys 1991; Furthermore, the pastoralists identified Chrysopogon Pérez Corona et al. 1998; Ganskopp and Bohnert 2001) Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) Forage quantity and quality in Borana Lowlands 83 only during the long rains. This and other studies, e.g. condition where desirable species are more prevalent. Habtamu Teka et al. (2013), showed that quality of Surprisingly, in Web areas desirable species were standing herbaceous forage in the Borana Lowlands was dominant during the long rains. Further studies are largely below the threshold level for good livestock warranted to determine why these differences occurred production. The minimum recommended CP concentra- between the Land Use Units and whether strategies can be tion in diets for small ruminants is even higher (9%) developed to improve the situation in the Dida Hara area. (Araújo Filho et al. 1998). While animals would select a It seems that ‘over-protection’ as has been practiced on the higher quality diet from herbaceous forage than the whole Dida Tuyura Farm is not the solution and more intensive plant data indicate, as the seasons progressed CP study of the Web sites may yield possible solutions for concentrations in the selected diet would decline. Access testing. Furthermore, herd diversification for effective to some browse from woody species would alleviate this utilization of browse species was recommended as a result N deficiency as time progressed, while feeding of N of this study. supplements would also increase intake of the low quality forage. The study highlighted the importance of restoring Acknowledgments degraded rangelands and also the need to improve forage quality through focused interventions aimed at increasing I thank the Borana pastoralists for their hospitality, CP concentration in the herbaceous forage. patience and unreserved willingness to share their Tree leaves are nutritionally desirable, mainly as a knowledge, and am grateful for the support from local source of CP (Forwood and Owensby 1985). The mean CP administration. concentration in foliage of woody plants determined by this I sincerely acknowledge the in-depth and professional study was 11%. This was significantly higher than the CP review by anonymous reviewers. Their contributions concentration in herbaceous forage in agreement with have improved the quality of this manuscript. reports from other areas, e.g. Musco et al. (2016). Further- more, NDF, ADF and ADL concentrations in forage from References woody plants were lower than those of herbaceous species. (Note of the editors: All hyperlinks were verified 15 April 2020.) Trees and shrubs represent an integral part of diets for domestic ruminants in Africa and may constitute an Adesogan AT; Givens DI; Owen E. 2000. Measuring chemical composition and nutritive value in forages. In: Mannetje L’t; important source of proteins, minerals and vitamins, Jones RM, eds. Field and laboratory methods for grassland and especially during the dry season (Dicko and Sikena 1992). animal production research. CAB International, Wallingford, Borana pastoralists recognized the importance of woody UK. p. 263–278. doi: 10.1079/9780851993515.0263 species with higher CP concentration in this semi-arid Alemayehu Mengistu. 1998. The Borana and the 1991–92 environment. For long-term sustainability of the system, drought. A rangeland and livestock resource study. 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Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) Tropical Grasslands-Forrajes Tropicales (2020) Vol. 8(2):86–92 86 doi: 10.17138/TGFT(8)86-92 Research Paper Ammonium sulfate enhances the effectiveness of reactive natural phosphate for fertilizing tropical grasses Sulfato de amonio como mejorador de la efectividad del fosfato natural reactivo en la fertilización de gramíneas tropicales CARLOS E.A. CABRAL1, CARLA H.A. CABRAL1, ALYCE R.M. SANTOS2 , KÁSSIO S. CARVALHO3, EDNA M. BONFIM-SILVA1, LUIZ J.M. MOTTA1, JENIFER S. MATTOS1, LETÍCIA B. ALVES1 AND ANA P. BAYS2 1Instituto de Ciências Agrárias e Tecnológicas (ICAT), Universidade Federal de Mato Grosso, Rondonópolis, MT, Brazil. ufmt.br 2Faculdade de Agronomia e Zootecnia (FAAZ), Universidade Federal de Mato Grosso, Cuiabá, MT, Brazil. ufmt.br 3Instituto Federal de Educação, Ciência e Tecnologia de Mato Grosso, Sorriso, MT, Brazil. srs.ifmt.edu.br Abstract Reactive natural phosphate is a slow and gradual solubilizing fertilizer, which makes it difficult to use in neutral to alkaline soils. Nitrogen fertilizers which acidify the soil may increase the possibility of using this phosphate fertilizer commercially. Two greenhouse experiments were conducted to compare responses of Xaraés palisadegrass (Urochloa brizantha syn. Brachiaria brizantha cv. Xaraés) and Mombasa guineagrass (Megathyrsus maximus syn. Panicum maximum cv. Mombasa), when different combinations of P and N fertilizers were applied during the establishment phase in non-acidic soils or with corrected acidity. The experiments were carried out in a completely randomized design with 3 fertilizer combinations (simple superphosphate plus urea, SSU; natural reactive phosphate plus urea, RPU; and natural reactive phosphate plus ammonium sulfate, RPAS). There was no difference in tiller density, leaf numbers, forage mass, leaf mass and stem mass for either forage on SSU and RPAS treatments but they exceeded those on RPU. Soil pH was lower in soil fertilized with ammonium sulfate than in soil fertilized with urea. Applying natural reactive phosphate plus ammonium sulfate seems as effective as simple superphosphate plus urea in promoting increased growth in tropical grasses on low-P soils. Longer-term and more extensive field studies are needed to determine if these results can be reproduced in the long term, and the level of soil acidification over time. Keywords: Ammonium sulfate, establishment fertilization, Megathyrsus maximus, phosphorus, soil acidification, urea, Urochloa brizantha. Resumen El fosfato natural reactivo es un fertilizante que se solubiliza en forma lenta y gradual, lo que dificulta su uso en suelos de reacción neutra a alcalina. Los fertilizantes nitrogenados que acidifican el suelo pueden favorecer el uso del fosfato natural reactivo. Por tanto, el objetivo de este trabajo fue verificar cuál fertilizante nitrogenado favorece el uso de fosfato natural reactivo durante la fase de establecimiento de 2 gramíneas tropicales, en suelo no ácido o con acidez corregida. Para el efecto en condiciones de invernadero se realizaron sendos experimentos con un diseño completamente al azar, con los pastos Urochloa brizantha (syn. Brachiaria brizantha) cv. Xaraés y Megathyrsus maximus (syn. Panicum maximum) cv. Mombasa. Los tratamientos consistieron en fertilización con superfosfato simple y urea (SSU), fosfato reactivo natural y urea (RPU) y fosfato reactivo natural y sulfato de amonio (RPAS). No se encontraron diferencias en la densidad de rebrotes, número de hojas, biomasa forrajera, biomasa de hoja y tallo en ambos pastos entre los ___________ Correspondence: Carlos Eduardo Avelino Cabral, Instituto de Ciências Agrárias e Tecnológicas (ICAT), Universidade Federal de Mato Grosso, Rondonópolis, CEP 78735-901, MT, Brazil. Email: carlos.eduardocabral@hotmail.com Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) Fertilizing tropical grass with reactive natural phosphate 87 tratamientos de SSU y RPAS, aunque estos parámetros presentaron valores más bajos en el tratamiento RPU. El pH fue más bajo en el suelo fertilizado con sulfato de amonio, en comparación con la urea. El sulfato de amonio, al acidificar el suelo más que la urea, favorece el uso de fosfato natural reactivo en la fertilización de pastos en suelos bajos en fósforo. Se necesitan estudios de campo a plazo más largo y más extensivos para determinar el nivel de acidificación del suelo a lo largo del tiempo y si los resultados de este estudio pueden ser reproducidos a largo plazo. Palabras clave: Acidificación del suelo, fertilización de establecimiento, fósforo, Megathyrsus maximus, sulfato de amonio, urea, Urochloa brizantha. Introduction Urea is the most popular choice of nitrogen fertilizer, due to its high nitrogen concentration and relatively low Phosphorus plays an important role in development of root cost. However, high losses of N can occur through systems of plants, and concentration of this nutrient in volatilization. Ammonium sulfate is an alternative source tropical soils is often low (Marcante et al. 2016; Zambrosi of N and is less susceptible to volatilization, but is more et al. 2017). Deficiency of P during pasture establishment expensive than urea per unit of N and has a lower reduces forage photosynthetic activity (Ghannoum et al. concentration of this nutrient which increases freight 2008), which has an impact on leaf elongation (Kavanova costs (Werneck et al. 2012). Both urea and ammonium et al. 2006) plus forage mass and root production (Rezende sulfate can be applied to enhance the viability of applying et al. 2011; Waddell et al. 2017; Ros et al. 2018). In P- natural phosphate to forages (Nascimento et al. 2002). deficient soils applying P fertilizer is essential to get This work was designed to determine which nitrogen satisfactory pasture growth and animal performance, which fertilizer promoted better responses in tropical grasses represents a significant cost for cattle farmers. grown in non-acid or corrected acid soils when applied While water-soluble phosphate fertilizers, such as with reactive natural phosphate. triple and single superphosphate, are most commonly used, a lower-cost alternative is reactive natural Materials and Methods phosphate. Reactive natural phosphates come from sedimentary rocks and differ from those from igneous and The experiments were performed in a greenhouse at metamorphic rocks, which have low reactivity and are Federal University of Mato Grosso, Cuiabá, MT, in a commonly called ‘rock’ phosphate (Corrêa et al. 2005). completely randomized design with 3 treatments. The lower cost of reactive natural phosphate results from Treatments consisted of different combinations of physical processing being all that is involved in nitrogen and phosphorus fertilizers, i.e. simple super- manufacture, as opposed to water-soluble fertilizers, phosphate plus urea (SSU), reactive natural phosphate which are both milled and chemically (Ivanova et al. plus urea (RPU) and reactive natural phosphate plus 2006) or thermally solubilized. ammonium sulfate (RPAS). The SSU treatment was Despite its low cost, an obstacle to the use of natural considered the Control treatment, because it combined a reactive phosphate is that it requires low soil pH, which readily available source of P with the N fertilizer most enables phosphorus in the fertilizer to be converted to a commonly used. soluble form and enter the soil solution (Guedes et al. 2009). However, soil acidity also has a negative effect on Experiment 1 availability of P in tropical soils, as it increases the adsorption of P by oxides and promotes the precipitation Experiment 1 was conducted with Xaraés palisadegrass of this nutrient with free aluminum and cationic micro- [Urochloa brizantha (Hochst. ex A. Rich.) R.D. Webster nutrients (Souza et al. 2006). Therefore, for productive cv. Xaraés] with 6 replicates of the fertilizer treatments. pastures, correction of soil acidity is an important The soil used was the top 20 cm of an Oxisol, collected in practice. native Cerrado areas, with texture characterized by 57.5% Most grass pastures will respond to N fertilizer sand, 5.0% silt and 37.5% clay. Chemical composition of application and P fertilizer is rarely applied without added the soil was: P = 1.1 mg/dm3; potassium (K) = 47 mg/dm3; N fertilizer. During the nitrification process release of calcium (Ca) = 0.2 cmol 3c/dm ; magnesium (Mg) = 0.1 ammonia from nitrogen fertilizers also releases hydrogen cmol /dm3c ; hydrogen and aluminum (H+Al) = 5.7 in the soil solution, which acidifies the soil by lowering cmol 3c/dm ; cation exchange capacity = 6.1 cmol 3c/dm ; pH (Schroeder et al. 2011; Bortoluzzi et al. 2017; Cabral base saturation = 6.9%; aluminum saturation = 70.4%; et al. 2018). and pH (in calcium chloride) = 4.1. After collection, the Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) 88 C.E.A. Cabral, C.H.A. Cabral, A.R.M. Santos, K.S. Carvalho, E.M. Bonfim-Silva, L.J.M. Motta, J.S. Mattos, L.B. Alves and A.P. Bays soil was sieved through a 4.0 mm mesh and transferred to regrowth cycles were evaluated for Xaraés palisadegrass and pots with 3.5 dm³ volume. Dolomitic limestone was 2 regrowth cycles for Mombasa guineagrass. applied to raise base saturation to 50%. After the Plant height was measured with a graduated rule and incorporation, the soil was left for 30 days with soil the numbers of tillers per pot were recorded at the end of moisture kept at 80% field capacity for limestone each regrowth cycle, before harvest. All leaves present in reaction. Thirty days after limestone incorporation, P each pot above the residue height were counted, to obtain fertilizer was applied at the rate of 300 mg P O /dm3 2 5 leaf numbers (LN). Leaf appearance rate (LAR) was (131 mg P/dm3). Seed was sown after P fertilizer estimated by the ratio of number of leaves per tiller and application, and after seedling emergence seedlings were the interval between cuts. Phyllochron (PHY) was thinned to leave 3 plants per pot. The criteria for thinning calculated as the inverse of LAR. were based on seedling vigor and uniformity. After At the end of each regrowth period the harvested seedling emergence, soil moisture was maintained near material was separated into morphological components, field capacity, estimated according to the methodology i.e. leaf and stem (stem + sheath). These fractions were described by Cabral et al. (2016). conditioned in paper bags, then subjected to drying in an air circulation oven at 65 ºC for 72 hours and weighed. Experiment 2 Forage accumulation rate (FAR) was calculated by dividing forage mass (FM) at each cut by the interval Experiment 2 was conducted with Mombasa guineagrass between cuts. [Megathyrsus maximus (Jacq.) B.K. Simon & S.W.L. At the final evaluation, plants were harvested at ground Jacobs cv. Mombasa] and involved 5 replicates of the level and the stem residue and roots were collected. The fertilizer treatments. The soil used was the top 20 cm of root system was washed with running water using a 4 mm an Inceptisol from a degraded pasture with texture mesh sieve. Afterwards, residue and roots were dried in a characterized by 80% sand, 12% silt and 8% clay. forced air circulation oven under the same conditions Chemical composition of the soil was: P = 22 mg/dm3; mentioned for FM to obtain dry matter data. Soil pH (in potassium (K) = 152 mg/dm3; calcium (Ca) = 7.4 calcium chloride) was determined at this time. cmolc/dm3; magnesium (Mg) = 2.0 cmolc/dm3; hydrogen and aluminum (H+Al) = 1.4 cmol 3c/dm ; cation exchange Statistical analysis capacity = 11.2 cmolc/dm3; base saturation = 87%; aluminum saturation = 0%; and pH (in calcium chloride) Data were submitted to analysis of variance using the = 5.9. After collection, the soil was sieved through a 4.0 general linear mixed model method, using the PROC mm mesh and transferred to 5.0 dm³ pots. Phosphorus MIXED command (SAS® Institute Inc., Cary, NC, USA). fertilizer was applied on the day of sowing at rates of 300 Least squares means of treatments were compared by mg P2O5/dm3 (131 mg P/dm3). After seedling emergence Tukey test (P<0.05). excess seedlings were removed leaving 3 plants per pot. The model was as follows: Criteria for thinning and maintenance of soil moisture yijk = μ + Ti + eij + Ck + ɛijk; were as described in Experiment 1. where: For both experiments, fertilizer application after yijk = expected response; thinning consisted of N fertilizer application (200 mg µ = average/constant, associated with the experiment; N/dm3) as urea or ammonium sulfate and potassium Ti = treatment effect (different nitrogen fertilizers) i; fertilizer application (100 mg K2O/dm3 or 83 mg K/dm3) eij = treatment error i, in repetition j, normally and as potassium chloride. independently distributed; Ck = random effect associated with regrowth cycle k, Measurements normally distributed; and ɛijk = experimental error associated with treatment i, in Measurements commenced 30 days after seedling repetition j, in cycle k, normally distributed. emergence. Estimation of the chlorophyll index of the youngest adult leaf of a representative tiller was performed Results with a non-destructive method using a Clorofilog (CLF 1030 Falker, Brazil) for Experiment 1 only. Experiment 1 Aerial parts were then cut at 10 and 25 cm above the soil for Xaraés and Mombasa, respectively. Forages were Fertilizer type had no significant effect (P>0.05) on height, allowed to regrow and evaluated at intervals of 20 days. Four leaf appearance rate, phyllochron or root mass of Xaraés Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) Fertilizing tropical grass with reactive natural phosphate 89 (Tables 1 and 2). However, forage mass, leaf mass, stem Experiment 2 mass, leaf number, tiller density, forage accumulation rate and stem residue for RPAS and SSU treatments were As for Experiment 1, fertilizer combination had no greater (P<0.05; Table 1) than for RPU. significant effect (P>0.05) on leaf appearance rate, phyllochron, stem residue or root mass of Mombasa Table 1. Effects of N and P fertilizer combinations on productive guineagrass (Tables 3 and 2). However, plant height, and structural characteristics and chlorophyll index of Urochloa forage mass, leaf mass, stem mass and forage brizantha cv. Xaraés plus soil pH during establishment. accumulation rate were greater (P<0.05) for RPAS and SSU than for RPU. Leaf number followed the pattern Variable SSU RPU RPAS s.e.m. P- SSU>RPAS>RPU, while tiller density for SSU exceeded value that for RPAS and RPU (P<0.05). Height (cm) 48 50 49 1.14 0.53 FM (g DM/pot) 19.5a 11.1b 19.1a 0.59 <0.01 Table 3. Effects of N and P fertilizer combinations on FAR (g/d) 0.89a 0.52b 0.89a 0.02 <0.01 productive and structural characteristics of Megathyrsus LM (g DM/pot) 15.9a 9.0b 15.6a 0.49 <0.01 maximus (syn. Panicum maximum) cv. Mombasa plus soil pH SM (g DM/pot) 3.6a 2.1b 3.5a 0.19 <0.01 during establishment. LN (No./pot) 83a 50b 80a 2.66 <0.01 TD (No./pot) 32a 19b 33a 0.84 <0.01 Variable SSU RPU RPAS s.e.m. P- LAR (No./tiller/d) 0.12 0.12 0.11 0.003 0.38 value PHY (No. of days/leaf) 8.6 8.8 9.0 0.27 0.50 Height (cm) 62a 56b 64a 1.04 <0.01 Soil pH 4.14b 4.66a 3.56c 0.04 <0.01 FM (g DM/pot) 9.00a 5.23b 8.44a 0.62 <0.01 Chlorophyll index 52.7a 45.7b 53.0a 0.82 <0.01 FAR (g/d) 0.30a 0.17b 0.28a 0.02 <0.01 Means within rows with different letters differ (P<0.05) by LM (g DM/pot) 8.68a 5.13b 8.14a 0.59 <0.01 Tukey’s test. SM (g DM/pot) 0.32a 0.09b 0.29a 0.04 <0.01 SSU = simple superphosphate plus urea; RPU = reactive natural LN (No./pot) 35a 22c 28b 5.26 <0.01 phosphate plus urea; RPAS = reactive natural phosphate plus TD (No./pot) 11a 7b 9b 0.57 <0.01 ammonium sulfate. LAR (No./tiller/d) 0.10 0.11 0.11 0.005 0.80 FM = forage mass; FAR = forage accumulation rate; LM = leaf PHY (No. of days/leaf) 9.7 10.0 9.5 0.44 0.71 mass; SM = stem mass; LN = leaf number; TD = tiller density; Soil pH 7.89a 7.91a 6.12b 0.10 <0.01 LAR = leaf appearance rate; PHY = phyllochron. Means within rows with different letters differ (P<0.05) by Tukey’s test. Table 2. Effects of N and P fertilizer combinations on stem SSU = simple superphosphate plus urea; RPU = reactive natural residue and root mass (g DM/pot) of Urochloa brizantha cv. phosphate plus urea; RPAS = reactive natural phosphate plus Xaraés (Experiment 1) and Megathyrsus maximus (syn. Panicum ammonium sulfate. maximum) cv. Mombasa (Experiment 2) during establishment. FM = forage mass; FAR = forage accumulation rate; LM = leaf mass; SM = stem mass; LN = leaf number; TD = tiller density; Treatment Xaraés Mombasa LAR = leaf appearance rate; PHY = phyllochron. Residue Root Residue Root mass mass mass mass Final pH for SSU and RPU exceeded that for RPAS SSU 17.6a 67.1 13.0 4.8 (P<0.05) with that for RPAS being similar to the original RPU 9.8b 49.7 7.1 2.9 pH while those for SSU and RPU were higher than the RPAS 18.2a 58.6 9.6 4.5 P-value <0.01 0.15 0.05 0.43 original. s.e.m. 0.83 5.88 1.68 1.21 Means within columns with different letters differ (P<0.05) by Discussion Tukey’s test. SSU = simple superphosphate plus urea; RPU = reactive natural The similar results found for the productive and structural phosphate plus urea; RPAS = reactive natural phosphate plus variables for the RPAS and SSU treatments suggest that ammonium sulfate. reactions between natural phosphate and ammonium sulfate produced sufficient P in a soluble form to meet the Chlorophyll index for RPAS and SSU was greater growth requirements of a high nutrient extraction grass (P<0.05) than for RPU (Table 1). (Galindo et al. 2018). This synergistic effect between Final soil pH had the pattern RPU>SSU>RPAS natural reactive phosphate and ammonium sulfate is due (P<0.05) with RPU being greater than the original pH and to the reduction in soil pH, which promotes the RPAS lower than the original. conversion of phosphorus present in natural phosphate Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) 90 C.E.A. Cabral, C.H.A. Cabral, A.R.M. Santos, K.S. Carvalho, E.M. Bonfim-Silva, L.J.M. Motta, J.S. Mattos, L.B. Alves and A.P. Bays into a soluble form (Degryse et al. 2017). Costa et al. was lower than in the other treatments, differences were (2008) and Vitti et al. (2002) showed reduction in soil pH not large enough to be considered significant. On the of up to 1.1 units when ammonium sulfate was applied as other hand residue weights (residual stem weights) were fertilizer. An additional factor which could have higher in the SSU and RPAS treatments than in RPU. The contributed to forage growth was the sulfur content of combination of lower root development in RPU combined ammonium sulfate, which can increase growth of tropical with lower levels of available N and P in the soil solution forages (Miranda et al. 2017; Santos et al. 2019), when plus lower residual stem weight from which to regrow available sulfur levels in soil are limiting. According to would have contributed to reduced growth of forage on Artur and Monteiro (2014) sulfur has a greater impact on this treatment. Benot et al. (2019) indicated that the stem regrowth than on establishment. base, which is present in the residue mass, accumulates In the case of natural phosphate plus urea, growth of non-structural total carbohydrates, which are important forage was restricted relative to that with SSU and RPAS for regrowth of forages under conditions of lower (Tables 1 and 3). This is probably a function of the lesser photosynthetic activity, such as water deficit, shading and effect of urea in lowering soil pH as observed in Tables 1 after defoliation. and 3 to promote solubilization of natural reactive Commonly, chlorophyll index is a variable used to phosphate. Lower acidification of the soil by urea verify the nitrogen fertilizer efficiency of crops (Cardoso compared with ammonium sulfate is due to hydrolysis et al. 2011). However, in this study, a phosphate fertilizer that occurs soon after the application of urea to the soil, effect was observed, as chlorophyll index for Xaraés grass which consumes protons from the soil (Fageria et al. fertilized with RPAS and SSU exceeded that for RPU. 2010). In the hydrolysis of urea, hydrogen ions in soil are combined with N to produce ammonia, which temporarily Conclusions increases soil pH around the urea granule. This increase in pH promotes ammonia volatilization, which reduces Applying ammonium sulfate as N fertilizer in conjunction the amount of ammonia to be nitrified and/or supplied to with natural reactive phosphate on low-P soils should give plants (Lara Cabezas and Souza 2008). In addition to this similar growth responses in grass pastures as simple increase in pH around the urea granule, when part of the superphosphate and urea. Field studies are needed to nitrogen is lost by volatilization, less nitrogen remains in verify these greenhouse results and determine rate of soil to be oxidized to nitrate in the nitrification process, reactive natural phosphate solubilization, as well as cost- which is one of the factors that contributes to soil of-production, since ammonium sulfate is more expensive acidification (Isobe et al. 2011), the main factor that per unit of N than urea, and requires correction of soil promotes the solubilization of the phosphorus present in acidity, which may increase pasture fertilization costs. natural phosphate. Plants on this treatment would have less available N and P for growth. With SSU, it is References important to note that most of the phosphorus in simple (Note of the editors: All hyperlinks were verified 21 April 2020.) superphosphate is water soluble, and is readily available to be absorbed by the plant as well as to be adsorbed on Artur AG; Monteiro FA. 2014. 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Root morphology and its contribution S0100-204X2012000300020 to a large root system for phosphorus uptake by Zambrosi FCB; Ribeiro RV; Machado EC; Garcia JC. 2017. Rytidosperma species (wallaby grass). Plant and Soil 412: Phosphorus deficiency impairs shoot regrowth of sugarcane 7–19. doi: 10.1007/s11104-016-2933-y varieties. Experimental Agriculture 53:1–11. doi: 10.1017/ Werneck CG; Breda FA; Zonta E; Lima E; Polidoro JC; S0014479715000290 (Received for publication 22 November 2019; accepted 17 April 2020; published 31 May 2020) © 2020 Tropical Grasslands-Forrajes Tropicales is an open-access journal published by International Center for Tropical Agriculture (CIAT), in association with Chinese Academy of Tropical Agricultural Sciences (CATAS). This work is licensed under the Creative Commons Attribution 4.0 International (CC BY 4.0) license. Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) Tropical Grasslands-Forrajes Tropicales (2020) Vol. 8(2):93–104 93 doi: 10.17138/TGFT(8)93-104 Research Paper Vertical distribution, nutrient concentration and seasonal changes of fine root mass in a semi-deciduous tropical dry forest and in two adjacent pastures in the Western Llanos of Venezuela Distribución vertical, concentración de nutrientes y cambios estacionales en la masa de raíces finas en un bosque seco tropical semicaducifolio y dos pastizales adyacentes en los Llanos Occidentales de Venezuela ANA FRANCISCA GONZÁLEZ-PEDRAZA1,2,3 AND NELDA DEZZEO3 1Universidad de Pamplona, Facultad de Ciencias Agrarias, Pamplona, Colombia. unipamplona.edu.co/fagrarias 2Universidad Nacional Experimental Sur del Lago “Jesús María Semprum”, Santa Bárbara, Venezuela. unesur.edu.ve 3Instituto Venezolano de Investigaciones Científicas (IVIC), Altos de Pipe, Venezuela. ivic.gob.ve Abstract With the objective to contribute to a better understanding of ecological consequences of deforestation on the below-ground system in the Western Llanos of Venezuela, we evaluated the vertical distribution, nutrient concentration and seasonal changes of total fine root mass (FRM) (<2 mm diameter) in a semi-deciduous tropical dry forest and in 2 adjacent pastures of Cynodon nlemfuensis: a young pasture (YP, 5 years old) and an old pasture (OP, 18 years old) in the Obispo municipality, Barinas State. This evaluation included measurements at the end of the rainy season, during the dry season and during the subsequent early rainy season in 2005/2006. Highest FRM was recorded during the dry season, which probably indicates a plant water-stress response mechanism. The highest proportion (63‒88%) of FRM was concentrated in the 10–20 cm soil layer at all studied sites, probably due to a higher nutrient and moisture content at that depth. Non-significant differences (P>0.05) were observed in the total concentrations of organic carbon, nitrogen, phosphorus, calcium and magnesium in the FRM in soils supporting forest, OP and YP at the evaluated depths. Non-significant changes in the total FRM and nutrient concentrations were observed between the sampling periods and the 3 study sites. YP soils showed a slight increase in FRM that could be associated with the root growth of secondary vegetation, which is considered a weed and is periodically removed. Our results suggest that the land use change from tropical forest to pastures has not significantly affected the mass of fine roots and their carbon and nutrient concentrations. Further studies are needed to determine if these findings apply to other ecosystems. Keywords: Cynodon nlemfuensis, root distribution, soil C, soil N, tropical pastures. Resumen Con el objetivo de contribuir a entender mejor las consecuencias ecológicas de la deforestación en el sistema suelo-raíces en los Llanos Occidentales de Venezuela, evaluamos la distribución vertical, concentración de nutrientes y los cambios estacionales en la masa total de raíces finas (MRF) (<2 mm de diámetro) en un bosque seco tropical semicaducifolio y en dos pasturas adyacentes de Cynodon nlemfuensis: una pastura de 5 años (PJ) y una de 18 años (PV). Esta evaluación se realizó en 2005/2006 en el municipio Obispo, estado Barinas, e incluyó mediciones al final del período de lluvias, durante el período seco y al inicio del período de lluvias subsiguiente. La mayor MRF se registró durante el período seco, lo que probablemente indica un mecanismo de respuesta al estrés hídrico de la planta. La mayor proporción (63‒88%) de MRF se concentró en la capa de suelo de 10‒20 cm en todos los sitios estudiados, probablemente debido a una mayor concentración de nutrientes y humedad a esa profundidad. No se observaron diferencias significativas (P>0.05) en las concentraciones totales de carbono orgánico, nitrógeno, fósforo, calcio y magnesio en la MRF en suelos de bosque, PJ y PV a las profundidades evaluadas. No se observaron cambios significativos en la MRF total y en las concentraciones de nutrientes entre los períodos de muestreo en ___________ Correspondence: Ana Francisca González-Pedraza, Universidad de Pamplona, Facultad de Ciencias Agrarias, Pamplona, Norte de Santander, Colombia. Email: anagonzalez11@gmail.com Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) 94 A.F. González-Pedraza and N. Dezzeo los 3 sitios de estudio. Los suelos en PJ mostraron un ligero aumento en la MRF que podría estar asociado con el crecimiento de raíces de la vegetación secundaria, que se considera una maleza y se elimina periódicamente. Nuestros resultados indican que la conversión del bosque a pasturas no afectó significativamente la masa de raíces finas y sus concentraciones de carbono y nutrientes. Se requieren estudios adicionales para determinar si los resultados son aplicables a otros ecosistemas. Palabras clave: C en el suelo, Cynodon nlemfuensis, distribución de raíces, N en el suelo, pastos tropicales. Introduction Materials and Methods Fine root mass in tropical forests is the most important Study site component of below-ground C dynamics and can contribute significantly to global net primary production (Malhi et al. The study area was in the El Mangón farm, located in the 2011). In tropical dry forests, fine root production is high, Obispo municipality, Hurtado sector of Barinas State in constituting an important source of carbon (C) and nutrients the Western Llanos of Venezuela (40º01'10"–40º59'10" in the soil (Fiala et al. 2017). It has been suggested that the N, 91º57'30"–91º25'18" W; 120 masl) (Figure 1). roots in these forests provide more N, P and K than the Average annual rainfall is 1,244 mm, with a rainy season above-ground biomass (Singh et al. 1989). from April to December and a dry season from January to Several environmental factors affect production of fine March. Average annual temperature is 26.8 °C, and the roots in tropical dry forests, such as the marked climatic relief is flat with a slope between 0 and 2% (Ewel et al. seasonality (Murphy and Lugo 1986; Singh et al. 1989), 1976; SIRA-INIA-CENIAP 2010). soil nutrients (Blair and Perfecto 2001) and forest disturbances and land use history (Castellanos et al. 2001; Jaramillo et al. 2003). The clearing of dry forests for the establishment of pastures can have important effects on the production and distribution of roots within the soil profile and on the contributions of C and nutrients (Jaramillo et al. 2003). In Venezuela, large areas of tropical dry forests have been cleared and replaced by pastures. It is likely that this conversion has had impacts on the mass and distribution of fine roots within the soil profile. Studies on the replacement of tropical deciduous forests with pastures have shown important effects on C and nutrients in the soil profile (Crespo 2015), and pastures have the potential to store significant amounts of C in soils (Rodríguez et al. 2013; Crespo 2015). The dense and deep root system of grasses contributes to the formation of soil aggregates and provides protection to the soil C, making it least susceptible to oxidation and eventual loss to the atmosphere (Cambardella and Elliot 1993). Considering that the rate of deforestation of tropical dry forests is increasing rapidly, it is important to improve the understanding of the ecological consequences of forest disturbance on the below-ground system. Detailed information on the changes that have occurred in fine root production once forests have been cleared and converted into pastures can be very useful in predicting the consequences of deforestation and to design effective Figure 1. Study area in the Western Llanos of Venezuela pasture management strategies. The objective of this study (SIRA-INIA-CENIAP 2010). was to evaluate the fine root mass and its nutrient composition in a tropical dry forest and in 2 adjacent The landscape corresponds to an alluvial overflow pastures (5- and 18-years-old) planted following the logging plain, influenced by the fluvial dynamics of an old bed of and burning of forest in the Western Llanos of Venezuela. the Santo Domingo River (Schargel and Strebin 1970). Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) Fine root mass under forest and pasture 95 Table 1. Soil characteristics of the study sites (mean ± SD) (Source: González-Pedraza and Dezzeo 2011, 2014a, 2014b). Characteristic Depth (cm) Forest 5-years-old pasture (YP) 18-years-old pasture (OP) 0‒5 5.4±0.4Ba 5.0±0.5Cab 4.8±0.5Db 5‒10 5.8±0.4Ba 5.5±0.2BCa 5.5±0.4Ca pH (H2O) 10‒20 6.0±0.4ABa 5.9±0.2Aa 6.1±0.3Ba 20‒30 5.7±0.7Bab 5.3±0.1BCb 5.9±0.4BCa 30‒40 6.6±1.1Aab 5.9±0.2Ab 6.9±0.7Aa 0‒5 1,390±251.9Aa 1,473±400.2Ba 1,517±249.9Ba Total organic 5‒10 1,379±228.5Ab 2,232±287.9Aa 1,904±436.1Aa carbon 10‒20 916±143.5Bc 1,675±196.4Ba 1,364±229.4BCb (g/m2) 20‒30 1,002±133.6Bb 1,018±118.6Cb 1,123±117.3Ca 30‒40 493±59.9Ca 529±145.5Da 494±84.8Da 0‒5 173±59.6Ba 209±99.8Ba 184±55.2Ba 5‒10 184±33.2Ba 173±35.7Ba 205±28.3Ba Total nitrogen 2 10‒20 641±465.5Ab 838±710.2Aab 1,404±811.1Aa (g/m ) 20‒30 204±89.2Ba 147±12.5Ca 215±114.8Ba 30‒40 137±43Ba 104±25.7Ca 126±23.3Ba 0‒5 588.5±206.2Aa 926.3±329.9Aa 656.5±67.2Aa 5‒10 442.8±85.8Bb 776.1±177.3Ba 512.3±70.6Bb Total phosphorus 10‒20 59.0±36.9Db 163.4±76.8Ca 99.0±68.4Dab (µg/g) 20‒30 295.5±115.4Cb 594.4±120.1Ba 309.9±68.5Cb 30‒40 321.4±65.7BCb 487.3±101.4Ba 298.3±15.4Cb 0‒5 11.0±3.9Aa 12.5±4.6Aa 9.1±1.8Aa 5‒10 6.1±3.0Cc 14.9±3.1Aa 11.4±3.0Ab Calcium 10‒20 9.2±1.6ABb 12.7±3.7Aa 10.6±2.1Aab (meq/100 g) 20‒30 9.0±1.3ABab 8.2±1.4Bb 10.3±1.8Aa 30‒40 7.3±0.7BCb 7.3±1.0Bb 9.2±2.7Aa 0‒5 4.1±1.1ABa 5.0±1.2Aa 5.2±1.8Aa 5‒10 3.4±1.1Ba 4.5±0.9ABa 4.5±2.2Aa Magnesium 10‒20 3.9±1.2ABa 3.9±0.9ABa 4.8±2.1Aa (meq/100 g) 20‒30 4.7±1.0Aab 3.4±1.0Bb 5.3±1.8Aa 30‒40 4.8±1.1Aa 3.4±0.8Bb 4.7±1.2Aa 0‒5 0.5±0.2Ab 1.2±0.7Aa 0.6±0.3Ab 5‒10 0.2±0.2Bb 0.8±0.4ABa 0.3±0.2Bb Potassium 10‒20 0.2±0.1Bb 0.5±0.3BCa 0.2±0.1Bb (meq/100 g) 20‒30 0.1±0.0Bb 0.4±0.2BCa 0.2±0.0Bb 30‒40 0.2±0.2Ba 0.3±0.2Ca 0.2±0.1Ba 0‒5 0.1±0.0Ca 0.1±0.0Ca 0.1±0.1Ba 5‒10 0.1±0.1BCa 0.1±0.0Ca 0.1±0.1Ba Sodium 10‒20 0.1±0.1BCa 0.1±0.0Ca 0.2±0.3Ba (meq/100 g) 20‒30 0.3±0.2Ba 0.2±0.1Ba 0.4±0.3Ba 30‒40 1.2±0.4Aa 0.8±0.1Ab 1.1±0.3Aab 0‒5 1.7±0.1Aa 2.0±0.2Aa 1.8±0.6Aa 5‒10 0.5±0.1CDb 0.6±0.0Ba 0.5±0.0Bab Aluminum 10‒20 0.6±0.1Ca 0.6±0.1Ba 0.6±0.1Ba (meq/100 g) 20‒30 0.7±0.1Ba 0.6±0.1Bb 0.6±0.1Bb 30‒40 0.5±0.1Da 0.4±0.1Ca 0.3±0.1Cb 0‒5 17.2±3.8Aa 20.3±5.5Aa 16.8±3.3Aa Cation exchange 5‒10 10.3±2.4Cc 20.8±3.9Aa 16.8±3.1Ab capacity 10‒20 13.9±1.3Bb 17.7±4.3Aa 16.2±3.7Aab (meq/100 g) 20‒30 14.7±1.7ABab 12.7±1.9Bb 16.5±2.9Aa 30‒40 13.5±1.7Bab 12.3±1.1Bb 15.2±2.9Aa 0‒5 90.5±3.8Ba 90.8±5.4Ba 88.9±4.7Ba 5‒10 95.4±2.1Bb 97.1±0.6Aa 96.7±0.7Aa Base saturation 10‒20 96.2±1.4Ba 96.5±1.0Aa 96.2±1.1Aa (%) 20‒30 95.7±1.4Ba 95.7±1.6Aa 96.5±1.2Aa 30‒40 96.9±1.2Aab 96.6±1.3Ab 98.1±0.9Aa Different lower-case letters indicate significant differences between sites for the same depth (P<0.05). Different upper-case letters indicate significant differences between depths for the same site (P<0.05). Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) 96 A.F. González-Pedraza and N. Dezzeo The soil parent material is of alluvial origin, a sandy clay According to González-Pedraza and Dezzeo (2011), soils loam with kaolinite as dominant clay mineral (Ewel et al. at the study site are fine-textured with a predominance of silt. 1976). In general terms, the soils have been described as Forest and OP soils show similar clay and sand contents, deep, moderately fertile, with moderate concentrations of while YP soils have significantly higher clay content (up to organic matter, nitrogen and phosphorus, high cation 18% more than forest and OP soils). In general, the soil exchange capacity and base saturation (Table 1), with clay properties at the studied sites tended to be similar (Table 2). loam and silty clay loam textures (Table 2) (Schargel and The predominant vegetation in the region is semi- Strebin 1970; González-Pedraza and Dezzeo 2011, deciduous, periodically flooded forest (Ewel et al. 1976). 2014a, 2014b). The main soil orders found in this area These forests consist of 2 strata, the upper one consisting of correspond to Inceptisols and Vertisols, among them deciduous and semi-deciduous trees with heights of 15‒20 Tropaquepts, Usterts and Aquerts (Mogollón and m, and the lower one consisting of deciduous and evergreen Comerma 1994). trees with heights less than 15 m (Table 3) (Aymard 2015). Table 2. Particle size distribution (mean ± SD) in the 0‒40 cm of forest and pasture soils (Source: González-Pedraza and Dezzeo (2011). Particle size distribution Depth (cm) Forest 5-years-old pasture 18-years-old pasture 0–5 19.8±5.9Aa 12.5±7.8ABb 22.3±5.6Aa 5–10 20.8±7.6Aa 10.0±7.4ABb 22.7±7.6Aa % Sand 10–20 15.6±4.4Aa 5.6±1.8Bb 18.3±8.0Aa 20–30 15.8±2.6Aa 15.4±6.8Aa 13.5±5.5Aa 30–40 17.1±2.5Aa 12.9±3.3ABb 15.4±4.3Ab 0–5 49.7±6.2Ba 44.8±6.1Ba 47.1±4.5Ca 5–10 53.9±5.6ABa 46.8±5.7Bb 47.1±3.0Cb % Silt 10–20 57.5±4.1Aa 60.0±3.5Aa 52.7±3.6Bb 20–30 57.5±4.2Aa 60.4±5.3Aa 57.0±4.1ABa 30–40 58.3±2.0Ab 66.3±4.4Aa 60.0±2.7Ab 0–5 30.5±9.6Ab 42.7±10.6Aa 30.6±6.4Ab 5–10 25.2±6.9Ab 43.2±8.3Aa 30.2±6.2Ab % Clay 10–20 26.9±1.6 Ab 34.4±3.2ABa 28.9±5.1Ab 20–30 26.7±2.0Aab 24.2±3.4BCb 29.5±3.3Aa 30–40 24.6±1.0Aa 20.8±2.0Cb 24.6±2.5Aa Different lower-case letters indicate significant differences between sites (P<0.05). Different upper-case letters indicate significant differences between depths for the same site (P<0.05). Table 3. Main species of trees present in the semi-deciduous forest. (Source: Adapted from Figueroa 2006). Family Botanical name Common name Phenology Acanthaceae Trichanthera gigantea (Bonpl.) Nees Naranjillo Evergreen Anacardiaceae Spondias mombin L. Jobo Deciduous Annonaceae Rollinia exsucca (DC.) A. DC. Anoncillo Semi-deciduous Annona jahnii Saff. Manidito Semi-deciduous Bixaceae Cochlospermum vitifolium (Willd.) Spreng. Bototo Deciduous Cordiaceae Cordia collococca L. Caujaro Semi-deciduous Euphorbiaceae Sapium glandulosum (L.) Morong Lechero Semi-deciduous Fabaceae Samanea saman (Jacq.) Merr. Samán Deciduous Inga sp. Guamo Evergreen Albizia sp. Carabali Semi-deciduous Pterocarpus acapulcensis Rose Drago Facultatively deciduous Malvaceae Ceiba pentandra (L.) Gaertn. Ceiba Facultatively deciduous Sterculia apetala (Jacq.) H. Karst. Camuruco Deciduous Luehea candida (DC.) Mart. Guácimo cimarrón Facultatively deciduous Guazuma ulmifolia Lam. Guácimo Evergreen Moraceae Maclura tinctoria (L.) D. Don ex Steud. subsp. tinctoria Mora Deciduous Brosimum alicastrum Sw. Charo Evergreen Rubiaceae Genipa americana L. Caruto Deciduous Sapindaceae Sapindus saponaria L. Paraparo Deciduous Urticaceae Cecropia peltata L. Yagrumo Evergreen Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) Fine root mass under forest and pasture 97 In this region in the last 50 years, large areas of natural for determining the vertical distribution of the fine root forest have been converted into pasture using the slash- mass (<2 mm) and their nutrient concentrations were and-burn method. Estrella grass (Cynodon nlemfuensis collected at 12 points arranged along 3 transects with Vanderyst) is grown for grazing by cattle (Truter et al. 4 sampling points per transect. The samples were 2015). collected with a soil core at 0‒5, 5‒10, 10‒20, 20‒30 and Field work was carried out in an area of tropical dry 30‒40 cm depths at the end of the rainy season 2005 forest with dominant deciduous vegetation and in 2 (November). For determining the seasonal changes in adjacent areas, where the original forest had been cut mass of fine roots, soil samples at 0‒5 and 5‒10 cm depths down manually and burnt, and Estrella grass had been were collected at 12 points in each study plot along planted (5- and 18-years-old, YP and OP, respectively). 3 transects on 3 occasions: end of rainy season 2005 The pastures have never been fertilized, but have been (November), during the dry season (March 2006) and mowed annually to control weeds and to promote grass early rainy season (May 2006) (Figure 2). growth. At sampling time, species from the original forest that could not be cut by hand, as well as vegetation of Fine root mass secondary growth, like palms and some species of legumes, were observed in the pasture. Cattle were All collected soil samples were dried and passed through a introduced into YP for grazing during the dry season and 2 mm soil sieve. The fine roots were extracted from the soil early and late in the rainy season. On each occasion the fraction which passed through the 2 mm sieve. From each cattle remained in this pasture until they consumed all sieved soil sample, 2 × 200 g subsamples were taken. In available grass. Every 1‒2 months cattle were introduced these subsamples all fine roots (both living and dead) were to the old pasture (OP) and remained for 3–7 days, while manually extracted and weighed. Mean weight of fine root consuming all available grass. mass from the 2 subsamples was used to calculate the dry matter (DM) of fine root mass from each soil sample. The Fine root sampling data were converted to t DM/m2 considering the area of the core, before being converted to a hectare basis according to At each selected site (forest, YP and OP), soil samples the following equation: were taken from a 600 m2 plot (20 × 30 m). The distance Fine root mass (t DM/ha) = weight of fine root mass (t between the 3 sites was 1‒3 km. At each plot, soil samples DM/m2) × area of one hectare of soil (10,000 m2/ha). Figure 2. Distribution of precipitation and temperature during the sampling period. [Source: Barinas Agrometeorological Station (SIRA-INIA-CENIAP 2010)]. Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) 98 A.F. González-Pedraza and N. Dezzeo Laboratory analyses 0.57 t DM/ha) than in forest (5.27 ± 0.68 t DM/ha) and OP (4.57 ± 0.54 t DM/ha). Distribution of fine root mass was not Total organic carbon in fine roots (TOCr) was determined uniform down the soil profile as root mass in the 10‒20 cm using the Loss on Ignition (LOI) method (Schulte and horizon was generally greater than in the other horizons at Hopkins 1996). It was assumed that 58% of the organic all study sites (P<0.05; Figure 3). matter (OM) was organic carbon (Nelson and Sommers 1996) and %TOCr was calculated according to the following equation: %TOCr = [pre-ignition weight (g) – post-ignition weight (g)] × 0.58 × 100/pre-ignition weight (g). Concentration of total organic carbon in the fine roots was multiplied by the respective root mass to give TOCr and the results were expressed in kg/ha based on dry weight and bulk density of the soil. Total nitrogen (N), phosphorus (P), calcium (Ca) and magnesium (Mg) in the fine root mass was extracted by digestion with concentrated sulfuric acid and oxidation with hydrogen peroxide (Tiessen and Moir 1993). Total nitrogen in the roots (TNr) was determined by the colorimetric method of Keeney and Nelson (1982) on a Figure 3. Vertical distribution of fine root mass in soils Technicon Autoanalyzer II. Total phosphorus (TPr) was supporting dry semi-deciduous forest and 5-years-old (YP) and measured by the molybdenum blue method of Murphy 18-years-old (OP) pasture. All points are mean values with and Riley (1962). Total Ca and total Mg were measured standard error bars across forest and pastures. Different lower- on an atomic absorption spectrophotometer (AA). Total case letters indicate significant differences between sites N, P, Ca and Mg concentrations obtained in the fine root (P<0.05). Different upper-case letters indicate significant mass were multiplied by their respective root masses, and differences between depths (P<0.05). the results were expressed in kg/ha based on dry weight and bulk density of the soil. Seasonal changes in fine root mass Statistical analysis Within each sampling period, no significant differences (P>0.05) between forest, YP and OP were observed in the Statistical analysis of data was carried out using one-way mass of fine roots in both 0‒5 cm and 5‒10 cm horizons. analysis of variance (ANOVA). Means were separated Differences between seasons in mass of fine roots occurred with Tukey’s test when statistical differences (P<0.05) in the 0‒5 cm horizon, with root mass increasing during the were observed. When necessary, the data were dry season to values exceeding those in the early or late transformed to homogenize variances, and when that did rainy season for all sites (P<0.05; Table 4). not meet this assumption (P>0.05) according to Levenne’s test, a non-parametric Mann-Whitney test was Table 4. Seasonal variation in mass of fine roots (mean ± SD) in the top 10 cm of soils supporting forest and pastures of 2 ages. also applied. To determine relationships between variables at sites of interest, a simple linear regression Season Depth Fine root mass (t DM/ha) analysis was used. All statistics were computed using (cm) Forest YP1 OP STATISTICA for Windows 6.0 (Statistica 2001). End rainy 0–5 0.9±0.5ABa 1.0±0.4Ba 1.0±0.5Ba season 5–10 0.7±0.3Aa 0.9±0.4Aa 0.5±0.5Ba Results 0–5 1.4±0.7Aa 2.1±0.8Aa 1.6±0.7Aa Dry season 5–10 0.8±0.6Aa 1.1±0.5Aa 1.0±0.4Aa Vertical distribution of fine roots Early rainy 0–5 0.7±0.4Ba 1.1±0.6Ba 1.1±0.4Ba season 5–10 0.7±0.4Aa 0.9±0.4Aa 0.6±0.3ABb No significant differences (P>0.05) between study sites 1YP: 5-years-old pasture; OP: 18-years-old pasture. (forest, YP and OP) were detected in the vertical distribution Different lower-case letters indicate significant differences of the fine root mass. However, total mass of fine roots in between depths (P<0.05). Different upper-case letters indicate the top 40 cm of soil was somewhat higher in YP (5.95 ± significant differences between seasons (P<0.05). Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) Fine root mass under forest and pasture 99 Total carbon and nutrients in the fine root mass No significant differences (P>0.05) between forest and pasture soils were found in total phosphorus, The total concentrations of carbon and nutrients in fine roots calcium and magnesium in the mass of fine roots in the in the top 40 cm of soil was determined only in soil samples top 40 cm of soil (Table 6). However, there were collected at the end of the rainy season. No significant changes in concentrations down the profile. A differences (P>0.05) in total organic carbon (TOCr) and total significant increase (P<0.05) in total phosphorus in nitrogen (TNr) in fine roots were found between Forest, YP roots in the forest soil in the 10–20 cm horizon was and OP except for the 30‒40 cm soil depth, where YP observed. Total calcium and magnesium in fine roots contained significantly (P<0.05) more TOCr than forest soil tended to decline with depth but differences were (Table 5). TOCr was highest (P<0.05) in the 10‒20 cm inconsistent and rarely significant. horizon in all soils. In contrast, TNr tended to decrease Significant positive relationships existed between fine steadily with increasing soil depth, with levels in the top root mass and nutrient content in 0–40 cm soil horizons in 5 cm exceeding those in the 30‒40 cm horizon (Table 5). forest and pasture (Table 7). Table 5. Total organic carbon and total nitrogen (mean ± SD) in fine roots (<2 mm) in the top 40 cm of soils supporting forest and 5-years-old and 18-years-old pastures. Nutrient (kg/ha) Depth (cm) Forest 5-years-old pasture 18-years-old pasture Total organic carbon in 0–5 455±246.3Ba 466±227.9Ba 381±204.9Ba the fine root mass 5–10 403±234.4Ba 360±177.5Ba 305±196.5Ba (TOCr) 10–20 864±542.8Aa 894±421.9Aa 706±480.9Aa 20–30 424±309.9Ba 390±130.5Ba 430±228.5Ba 30–40 182±148.7Bb 357±160.3Ba 304±147.6Bab Total nitrogen in the 0–5 7.2±3.5Aa 9.0±3.7Aa 8.0±4.4Aa fine root mass (TNr) 5–10 4.8±1.2Aa 5.9±3.2ABa 4.2±2.5Ba 10–20 5.9±4.3Aa 6.8±4.2Aa 4.8±1.9Ba 20–30 3.5±2.5Ba 2.4±0.6BCa 3.2±1.7Ba 30–40 2.6±2.5Ba 1.8±1.4Ca 2.9±2.5Ba Different lower-case letters indicate significant differences between sites (P<0.05). Different upper-case letters indicate significant differences between depths (P<0.05). Table 6. Vertical distribution of total nutrients (mean ± SD) in fine root mass in soils supporting forest and 5-years-old and 18- years-old pastures. Nutrient (kg/ha) Depth (cm) Forest 5-years-old pasture 18-years-old pasture 0–5 0.7±0.4Ba 1.2±0.6Aa 0.8±0.5Aa 5–10 0.4±0.2Ba 0.6±0.4Aa 0.4±0.2Aa Phosphorus 10–20 1.5±1.4Aa 1.4±0.7Aa 0.8±0.6Aa 20–30 0.5±0.4Ba 0.7±0.4Aa 0.6±0.3Aa 30–40 0.5±0.3Ba 0.4±0.1Aa 0.3±0.1Aa 0–5 8.3±4.9Aa 10.0±6.6Aa 7.0±4.9Aa 5–10 6.5±3.1Aa 9.4±6.5Aa 5.6±42.2Aa Calcium 10–20 8.2±6.2Aa 4.2±2.6ABab 1.9±0.8Bb 20–30 7.3±2.6Aa 6.6±4.7Aab 5.7±2.8Aa 30–40 4.2±2.3Aa 3.2±2.8Ba 3.9±2.4ABa 0–5 4.6±1.9Aa 6.1±3.1Aa 5.9±2.6Aa 5–10 4.1±1.8Aa 4.9±3.3ABa 3.9±1.8Aa Magnesium 10–20 4.7±2.5Aa 6.5±3.4Aa 4.4±2.3Aa 20–30 4.7±2.1Aa 3.3±1.3ABa 4.9±3.9Aa 30–40 2.5±1.8Aa 1.8±1.2Ba 2.6±2.0Aa Different lower-case letters indicate significant differences between sites (P<0.05). Different upper-case letters indicate significant differences between depths (P<0.05). Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) 100 A.F. González-Pedraza and N. Dezzeo Table 7. Linear correlation coefficients (Pearson; r) for relation- forest, submontanous evergreen narrow-leaved forest, ships between fine root mass (<2 mm) and nutrient concentrations semi-deciduous narrow-leaved forest, semi-deciduous in fine roots in the top 40 cm of soils supporting forest and 5-years- broad-leaved forest and 2 species of mangroves, old (YP) and 18-years-old (OP) pasture. Rhizophora mangle and Avicennia germinans). Fifty- seven percent of the dry mass of fine live roots was Nutrient Forest YP OP concentrated in the upper 0–5 cm soil layer in the semi- TOCr 0.99 0.96 0.93 deciduous forests. Semi-deciduous narrow-leaved forest TNr 0.94 0.94 0.92 had live (87 g DM/m2) and dead (284 g DM/m2) fine roots P 0.77 0.82 0.76 and semi-deciduous broad-leaved forest had live (200 g Ca 0.80 0.76 0.68 DM/m2) and dead (210 g DM/m2) (range 371–410 g Mg 0.96 0.82 0.88 DM/m2) fine roots in the top 15 cm of soil, which TOCr: total organic carbon; TNr: total nitrogen; P: phosphorus; exceeded those in evergreen forests. The authors Ca: calcium; Mg: magnesium. concluded that fine root biomass is better predicted by nutrients in litterfall. The mangrove stands had 554 g Discussion live fine roots DM/m2 (A. germinans) and 758 g DM/m2 (R. mangle) in the top 15 cm of soil. The total fine root mass in the top 40 cm of soil was higher Variations in root dry mass and percentage distribution than those reported by Jaramillo et al. (2003) for semi- of roots of Florico grass (Cynodon nlemfuensis) in the deciduous dry forests (3.6–4.3 t DM/ha in the top 60 cm) 0–40 cm soil layer under 4 different grazing strategies and and for pastures of Panicum maximum, Cenchrus ciliaris seasons were evaluated by Barros et al. (2017). High and Andropogon gayanus (3.1–3.7 t DM/ha in the top 60 concentrations of roots were observed in the 0–10 cm layer cm). However, the values for YP and OP can be (51.8–65.6%) for all grazing strategies in all seasons. This considered low compared with that reported by Crespo concentration of roots near the soil surface was explained and Lazo (2001) for a pasture of Cynodon nlemfuensis by the branched architecture of the root system, common (10 t DM/ha in the top 15 cm of soil). in forage grasses and the ability of the plant to acquire While no significant differences in total fine root mass water and nutrients. According to Barros et al. (2017) the were found between sites, the top 20 cm of soil accounted root system has less need to go deeper to acquire water and for 72.5, 68.4 and 66.2% of the total in Forest, YP and nutrients when grazing is less severe. OP, respectively. Accordingly the fine root mass declined Similarly, Rodríguez et al. (2013) reported more than with depth as reported by Du et al. (2019) in 4 vegetation 80% of the below-ground root biomass was present in the types (grassland, shrubland, secondary forest and primary 0–5 cm soil layer in different grasslands of Mayabeque forest) in a karst area, Southwest China during vegetation province, Cuba. restoration. In that study the fine roots were concentrated Despite C. nlemfuensis being easy to establish, in the top 10 cm of soil, which accounted for more than persistent, highly productive and adapted to different 57% of the root biomass, and decreased with increasing climate and soil conditions, this species does not withstand soil depth (soil samples from 0 to 30 cm deep). In karst high-intensity grazing for long periods. After defoliation by ecosystems, fine roots contribute to the regulation of grazing the plants consume organic reserves for restoration nutrient cycling in terrestrial ecosystems and the high of tissues lost and then physiological activity is adjusted as density of fine roots within the top few centimeters of soil the stocks of reserves are gradually restored (Sollenberger is crucial for acquiring nutrients. 2008; Truter et al. 2015). According to Du et al. (2019), although fine root The significant increase in root mass detected in the biomass of all vegetation types decreased with soil depth, 10–20 cm soil horizon at all study sites could be the decrease was more rapid in forests (especially in associated with the highest nutrient concentrations in soil secondary forests) than in other vegetation types. They at that depth (González-Pedraza and Dezzeo 2011; 2014a; suggested that the vertical distribution patterns of fine 2014b). A further hypothesis may be that plant roots roots showed a more rapid decline in species-rich explore deeper soil in search of water, particularly during communities than in species-poor communities, which the dry season, in order to survive during periods of water probably reflected changes in soil water content, nutrient stress, as shown by Snyman (2009) in pastures under concentration and bulk density in the soil profile. different drying conditions in semi-arid regions of Fiala et al. (2017) also measured a reduction in fine southern Africa. A high concentration of roots at this root mass (diameter <1 mm) with increasing soil depth in same depth of soil was also reported for savannas in 6 Cuban forests (submontanous evergreen broad-leaved tropical dry forest areas of India (Tripathi et al. 1999). Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) Fine root mass under forest and pasture 101 In this region the soils have been described as deep, The TOCr and TNr results obtained in the forest and moderately fertile, with moderate concentrations of pastures of this study are similar to those reported by organic matter, nitrogen and phosphorus, high capacity Trujillo et al. (2006), Jaramillo et al. (2003) and Crespo and for cation exchange (CEC) and saturation with bases, with Lazo (2001) for other tropical dry forests and pastures. The clay loam and silty clay loam textures (Schargel and similarity between the studied forest and pastures in the C Strebin 1970; González-Pedraza and Dezzeo 2011). and N concentrations in the mass of fine roots is also Schargel and Strebin (1970) described a buried horizon at consistent with the results found by Jaramillo et al. (2003). a depth of close to 50 cm, where increases in the The high values for TOCr and TNr in fine roots within the percentages of clay, CEC, calcium, potassium, carbon and 10–20 cm soil horizon are closely related to the high root nitrogen were observed. mass present at that depth of soil. A positive and significant Fine roots are usually quick to respond to changes in correlation (P<0.05) was found between the mass of fine vegetation type, soil temperature, moisture and nutrient roots and the amounts of TOCr and TNr throughout the soil content (Du et al. 2019). The increase in fine root mass profile (Table 4). during the dry period at 0–5 cm soil depth (Table 4) is While the sites were not contiguous, soils on which the contrary to the results found by Singh et al. (1989), various sites were located were very similar. Our data Kummerow et al. (1990) and Trujillo et al. (2006). suggest that conversion of forests to pasture did not change However, other studies have reported small increases in carbon and nutrient levels in the soil to any significant root biomass with decreased water availability in the soil degree. Several factors can influence these outcomes such (Castellanos et al. 2001; Snyman 2009; Barros et al. 2017; as the land cover, post-conversion land management, Du et al. 2019), and this has been considered as an climate and soil type and texture (Dengiz et al. 2019). adaptive strategy of plants under significant water stress Dengiz et al. (2019) analyzed the spatial variability of (Snyman 2009). soil organic carbon density under different types of land For example in temperate grasslands, Walter et al. cover within different soil types in a subhumid terrestrial (2012) demonstrated that root length was highest when ecosystem and found that soil type and land cover were rainfall variability was intermediate and was 43 and 24% critical factors influencing spatial variation of soil organic shorter with extreme and low rainfall variability. carbon (SOC) density. Land cover was the primary factor According to this author, although enhanced root growth affecting variation in SOC density, while soil properties under drought conditions is viewed as an adaptive feature like texture, genetic horizons and soil depth also had an of many species, sometimes grassland roots may not important influence. The observation that organic carbon respond to dryness with enhanced root growth. concentration decreases with increasing soil depth under all During the dry season, Barros et al. (2017) reported land cover types has been generally observed in most a higher concentration (65.6%) of fine roots of situations. C. nlemfuensis near the surface (0–10 cm) in more severe Soil fertility may have a direct influence on fine root grazing treatments than in less severe ones. This may be mass production. In this study, soil fertility affected the associated with the renewal of the root system, also quantities of fine roots in these soils (high correlation known as “turnover”, during the favorable dry season between fine root mass and nutrient content in the top 40 growth period and with the accumulation of organic cm of forest and pasture soils). Reynolds and D´Antonio reserves. The lower densities of root dry mass in winter, (1996) indicated the possibility that nutrient availability relative to the other seasons, would reflect the fast has direct effects on root mass because fine roots are recovery of the aerial parts of plants in the rainy season generally plastic organs and plants can deploy and redirection of organic reserves to restore the root photosynthate below-ground to gather growth-limiting system reserves. resources. Similarly, Hutchings and de Kroom (1994) said Pastures under grazing tend to accumulate more reserves that root proliferation in fertile soils promotes extensive in their roots, as a mechanism of adaptation to defoliation exploration of the soil and increases root surface area for (Barros et al. 2017). This aspect, combined with the water greater water and nutrient uptake. shortage during the dry season, could be contributing to the Tropical pastures can accumulate large stores of C in increased mass of fine roots during this time of year. On the the soil. Chaplot et al. (2016) evaluated the potential of other hand, the significant reduction in the mass of fine roots grassland rehabilitation through high density-short with increasing soil depth that occurred during the dry duration grazing to sequester atmospheric carbon and season could be related to a higher concentration of nutrients found a significant increase in SOC stocks with increasing in the first 5 cm of soil, as was noted by Castellanos et al. grass biomass and grass cover to rates as high as (2001) for tropical dry forests and pastures. 12%/year. Amongst the proposed explanations were Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) 102 A.F. González-Pedraza and N. Dezzeo increased root biomass production, greater soil aggregate soils. Soil Science Society of America Journal 57:1071‒1076. stability and associated greater organic matter protection doi: 10.2136/sssaj1993.03615995005700040032x from decomposers. Castellanos J; Jaramillo VJ; Sanford RL; Kauffman JB. 2001. Our findings suggest that the quantities of fine roots, soil Slash-and-burn effects on fine root biomass and productivity in a tropical dry forest ecosystem in Mexico. carbon and other nutrients in soils supporting tropical Forest Ecology and Management 148:41‒50. doi: pastures can be equal to or exceed those in soils supporting 10.1016/S0378-1127(00)00523-5 tropical forest trees. However, the values reported here were Chaplot V; Dlamini P: Chivenge P. 2016. Potential of grassland recorded at 3 specific times of the year from 3 relatively rehabilitation through high density-short duration grazing to similar sites but not strictly the same soils. Nevertheless, the sequester atmospheric carbon. Geoderma 271:10‒17. doi: data do add to the increasing database of soil C storage 10.1016/j.geoderma.2016.02.010 information under various types of vegetation. Crespo G. 2015. Factores que influyen en el reciclaje de nutrientes en pastizales permanentes, avances en el Conclusions desarrollo de su modelación. Revista Cubana de Ciencia Agrícola 49:1‒10. bit.ly/2yBUrCY According to the results of this study, the land use change Crespo G; Lazo J. 2001. A study on the root biomass of from forest to pastures has not significantly affected the C. nlemfuensis cv. Panamanian, P. maximum cv. Likoni and D. annulatum and their nutrient contribution. Cuban Journal mass of fine roots (<2 mm) and their carbon and nutrient of Agricultural Science 35(3):259‒263. concentrations in the soil. Additionally, the changes in Dengiz O; Saygın F; İmamoğlu A. 2019. Spatial variability of distribution of fine root mass down the soil profile were soil organic carbon density under different land covers and closely related to the changes in the nutrient content of the soil types in a sub-humid terrestrial ecosystem. Eurasian soil at the considered depths. Journal of Soil Science 8:35‒43. doi: 10.18393/ejss.486582 The moderate increase in the mass of fine roots in the Du H; Liu L; Su L; Zeng F; Wang K; Peng W; Zhang H; Song young pasture (YP) may be associated with additional T. 2019. Seasonal changes and vertical distribution of fine root inputs from the growth of secondary forest root biomass during vegetation restoration in a karst area, vegetation, which is considered a weed and therefore Southwest China. Frontiers in Plant Science 9, article 2001. removed periodically. Further studies in other regions are doi: 10.3389/fpls.2018.02001 needed to determine if these findings apply to other Ewel JJ; Madriz A; Tosi Jr JA. 1976. Zonas de vida de Venezuela: Memoria explicativa sobre el mapa ecológico. ecosystems. Segunda Edn. Ministerio de Agricultura y Cría, Fondo Further studies are needed to clarify the effects of Nacional de Investigaciones Agropecuarias, Caracas, climate seasonality and soil nutrients on magnitude and Venezuela. distribution of fine root mass within the soil profile and Fiala K; Hernández L; Holub P. 2017. Comparison of vertical its contribution to C storage in soil under both forest and distribution of live and dead fine root biomass in six types pastures. of Cuban forests. Journal of Tropical Forest Science 29:275‒281. jstor.org/stable/44272905 References Figueroa C. 2006. Plan de manejo simplificado y de impacto (Note of the editors: All hyperlinks were verified 4 April 2020.) ambiental, aprovechamiento de productos forestales. Fundo Agropecuario El Mangón, C.A., superficie 1821 hectáreas, Aymard G. 2015. Bosques de los Llanos de Venezuela: Parroquia La Luz, municipio Obispos, Estado Barinas. Estructura, composición florística, diversidad y estado Venezuela. Informe técnico. (Unpublished.) actual de conservación. En: López R; Hétier JM; López D; González-Pedraza AF; Dezzeo N. 2011. Efecto del cambio de Schargel R; Zinck A, eds. Tierras Llaneras de Venezuela. bosque a pastizal sobre las características de algunos suelos 2nd Edn. p. 241‒268. IRD-CIDIAT, Mérida, Venezuela. en los Llanos Occidentales de Venezuela. Interciencia Barros ACC de; Almeida JCC; Camargo Filho ST; Carvalho 36:135–141. bit.ly/2xP8Nzp CAB de; Campana LL; Morais LF de. 2017. Root dry matter González-Pedraza AF; Dezzeo N. 2014a. Changes in the labile mass and distribution of Florico grass under different and recalcitrant organic matter fractions due to grazing strategies. Pesquisa Agropecuária Brasileira transformation of semi-deciduous dry tropical forest to 52:1276‒1285. doi: 10.1590/s0100-204x2017001200017 pasture in the Western Llanos, Venezuela. In: Greer FE, ed. Blair BC; Perfecto I. 2001. Nutrient content and substrate effect Dry forests: Ecology, species diversity and sustainable on fine root density and size distribution in a Nicaraguan management. Nova Science Publishers Inc., New York, rain forest. Biotropica 33:697‒701. doi: 10.1111/j.1744- USA. p. 105‒132. 7429.2001.tb00227.x González-Pedraza AF; Dezzeo N. 2014b. Effects of land use Cambardella CA; Elliot ET. 1993. Carbon and nitrogen change and seasonality of precipitation on soil nitrogen in a distribution in aggregates from cultivated and native grassland dry tropical forest area in the Western Llanos of Venezuela. 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Agriculture, agee.2011.11.015 (Received for publication 7 August 2019; accepted 17 January 2020; published 31 May 2020) © 2020 Tropical Grasslands-Forrajes Tropicales is an open-access journal published by International Center for Tropical Agriculture (CIAT), in association with Chinese Academy of Tropical Agricultural Sciences (CATAS). This work is licensed under the Creative Commons Attribution 4.0 International (CC BY 4.0) license. Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) Tropical Grasslands-Forrajes Tropicales (2020) Vol. 8(2):105–114 105 doi: 10.17138/TGFT(8)105-114 Research Paper The effects of bovine urine application on two soil nitrogen compounds and growth of three forage grasses in the Colombian Piedmont plains Efecto de la aplicación de orina bovina en dos compuestos nitrogenados del suelo y el crecimiento de tres pastos en el piedemonte de los Llanos Orientales de Colombia JAIME E. GARZÓN1, OSCAR PARDO2 AND EDGAR A. CÁRDENAS1 1Departamento de Producción Animal, Universidad Nacional de Colombia, Bogotá, Colombia. unal.edu.co 2Corporación Colombiana de Investigación Agropecuaria (Agrosavia), La Libertad, Colombia. agrosavia.co Abstract The effects of application of bovine urine on biomass and nitrogen (N) accumulation in 3 tropical grasses (Urochloa decumbens cv. Basilisk, U. humidicola cv. Humidicola and Megathyrsus maximus cv. Mombasa), and on available N concentrations in soil (NH +-N, NO -4 3 -N) were studied using a randomized complete block design with 3 replicates. There were significant interactions between species and urine application over time in terms of herbage accumulation and N concentration (P<0.01), with significant differences in the concentrations of N available in the soil (P<0.01). Soil temperature and precipitation had important effects on the concentrations of both soil ions. Application of bovine urine increased dry matter accumulation of all grasses in the short term and of U. decumbens over the whole year. Application of urine increased soil N levels, but for U. humidicola and M. maximus the effects were transient. It is necessary to continue with longer-term studies in the Piedmont plains to determine the effects of livestock grazing on the biogeochemical cycles, environmental impacts and natural mitigation options that the ecosystem offers, e.g. CO2 sequestration, biological nitrification inhibitors and organic matter decomposition. Keywords: Ammonium, herbage accumulation, Megathyrsus maximus, nitrates, nitrogen, Urochloa spp. Resumen En un Oxisol del piedemonte llanero colombiano se estudiaron los efectos de la aplicación de orina de bovinos en la acumulación de biomasa aérea y nitrógeno (N) en las gramíneas tropicales Urochloa decumbens cv. Basilisk, U. humidicola cv. Humidicola and Megathyrsus maximus cv. Mombasa, así como en las concentraciones de N disponible en el suelo (N-NH +4 , N-NO -3 ). Para el efecto se dispuso de un diseño de bloques al azar con tres repeticiones. Para la producción de materia seca y concentración de N se observaron, a través del tiempo, interacciones significativas (P<0.01) entre especies y la aplicación de orina, con significancia estadística en las concentraciones de N disponible en el suelo (P<0.01). La temperatura del suelo y la precipitación fueron factores importantes asociados con las concentraciones de ambos iones de N en el suelo. La aplicación de orina bovina incrementó la acumulación de materia seca en los tres pastos a corto término y en U. decumbens a través del año. Igualmente se incrementaron los niveles de N en el suelo, pero para U. humidicola and M. maximus el efecto fue transitorio. Se sugiere continuar con estudios a largo plazo para determinar efectos del pastoreo de bovinos sobre los ciclos biogeoquímicos, su impacto ambiental y opciones de mitigación naturales que el ecosistema ofrece, como son secuestro de CO2, los inhibidores biológicos de la nitrificación y la decomposición de materia orgánica. Palabras clave: Amonio, Megathyrsus maximus, nitratos, nitrógeno, producción de biomasa, Urochloa spp. ___________ Correspondence: Jaime E. Garzón, Universidad Nacional de Colombia, Departamento de Producción Animal, Facultad de Medicina Veterinaria y de Zootecnia, Carrera 30 # 45-03, Bogotá, Colombia. Email: jegarzona@unal.edu.co Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) 106 J.E. Garzón, O. Pardo and E.A. Cárdenas Introduction Materials and Methods The Colombian Piedmont plains are a transition zone Study site located between the slopes of the eastern Andes mountain range and the eastern plains (Llanos Orientales) in The study was performed at AGROSAVIA “La Libertad” Colombia. With an area of 2,010,000 ha (7.6% of the Research Center (4°03'33.2" N, 74°27'27.1" W; 412 Colombian Orinoquia region), they occupy a strip of land masl). The average temperature is 26.5 °C and mean parallel to the mountain range. In general, the plains are annual precipitation is 2,800 mm (Rincón et al. 2010). known for their low soil fertility, aluminum toxicity and The climate classification corresponds to Tropical Humid predominant acidity with high clay content, aluminum Forest (Holdridge 1978; IGAC 2004). saturation and low concentrations of available phosphorus and interchangeable bases (IGAC 2004). These properties Environmental variables limit plant growth and only species with physiological adaptations can survive (Rao 2001). The soil within the studied area was classified as an oxisol The warm temperatures and humid environment (Mejia 1996) with the following physico-chemical enable the whole territory to be used for cropping and characteristics: bulk density (cylinder method) – 1.23 3 livestock production, mainly under grazing with Zebu g/cm ; pH (suspension soil:water, 1:1 v/v) – 4.4; organic (Bos indicus) cattle. Pastures are composed of native and carbon (Walkley-Black) – 1.95%; total nitrogen (organic increasingly planted species, such as Urochloa carbon × 0.0862) – 0.14%; phosphorus (Bray II) – 8.2 mg/kg; potassium, calcium and magnesium (extraction decumbens (Stapf) R.D. Webster (syn. Brachiaria with NH +4 1 M acetate, pH 7) – 0.08, 1.43 and 0.29 decumbens Stapf), U. humidicola (Rendle) Morrone & meq/100 g, respectively; exchangeable acidity (extraction Zuloaga (syn. B. humidicola (Rendle) Schweick., with 1 M KCl) – 2 meq/100 g; effective cation exchange including cv. Llanero, previously considered as capacity (exchangeable bases + exchangeable acidity) – B. dictyoneura) and Megathyrsus maximus (Jacq.) B.K. 3.83 meq/100 g; and 32, 25 and 43% sand, silt and clay, Simon & S.W.L. Jacobs (syn. Panicum maximum Jacq.) respectively (McKean 1993). plus some legumes, all of which are adapted to the Temperature and water volume in the soil were edaphoclimatic conditions of the area (Bernal 1994). measured daily with a Decagon® datalogger, to calculate Organic matter concentration in soils is low and producers the water-filled pore space (WFPS), using the formula do not usually apply chemical fertilizers to their recommended by USDA (2014). Particle density was grasslands, mainly due to costs. Cattle are relied upon to assumed as 2.65 g/cm3 (Lin et al. 2013). Atmospheric disperse nutrients to the soil through their excreta. Di and temperature, precipitation and evaporation were recorded Cameron (2012) reported that "under a dairy cow urine daily at the meteorological station of the research center. patch, the N-loading rate from a single urination can be as high as 1,000 kg N/ha", which should benefit grass Experimental methodology production. Nitrogen (N) is the highest nutrient requirement for A randomized complete block design was adopted with grasses and is absorbed through the roots in the form of 18 plots of 2 × 2.5 m, in a factorial arrangement with nitrate (NO -3 ) and ammonium (NH +4 ) ions. In general, the repeated measures in time. The factors were 3 regional plants prefer the former over the latter, mainly due to its grass species: signalgrass (Urochloa decumbens) cv. greater solubility in water and because NH +4 ions in high Basilisk (CIAT 606), koroniviagrass (U. humidicola) cv. concentrations can be toxic (Whitehead 2000). Many Humidicola (CIAT 679) and guineagrass (M. maximus) factors can alter the concentration of the ions mentioned cv. Mombasa (BRA-006645) × 2 levels of bovine urine above; precipitation, temperature and humidity are among application (with and without) with 3 replicates. Urine the abiotic factors that influence immobilization and was applied at the beginning of the rainy and dry seasons, mineralization of N, mediated by microbial populations respectively (May 2014 and February 2015). The grasses (Saggar et al. 2004; Dubeux Jr et al. 2007; Cameron et al. were established 2 years prior to the application of the 2013). urine treatments, without any management prior to the The objective of the present investigation was to experiment. evaluate the effects of bovine urine on biomass and N The N applications were separated into dry and rainy production in 3 regionally important grasses, together seasons (DS and RS, respectively). However, the split with the concentrations of available N in the soil. data did not comply with the statistical assumptions for Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) Effect of bovine urine on grass and soil 107 analysis of variance. In consequence, the data were Control plots were established to simulate an area analyzed together to strengthen the model, containing the without animal effects; they received no N application, experimental variation within the grass species to reach i.e. no urine. normality and homoscedasticity. Herbage accumulation and N content Estimation of the amount of bovine urine to apply Every 28 days, herbage mass was determined by In RS and DS separately, urine was collected from 10 harvesting 2 samples per plot, using a 0.5 × 0.5 m quadrat Zebu cows [418 ± 17.4 kg live weight (LW)], grazing a and leaving 10 cm stubble height. Each sample was dried U. decumbens pasture. at 60 °C for 48 h to calculate dry matter (DM) yield. To determine the amount of urine to be applied, a small Samples were then ground in a Wiley mill to pass a 1 mm amount of urine was collected at the beginning of each stainless steel screen for determination of N concentration season and preserved with 5 mL of 5% H2SO4 per 100 mL by the Kjeldahl method. N content was calculated by urine. Thereafter, concentrations of N (Kjeldahl) and multiplying the DM yield by the N concentration in each creatinine (commercial kit) in the urine were determined. sample. Following sampling, additional material within The amount of urine excreted per animal/day was calculated each experimental unit was cut and removed from the using the following formula (Valadares et al. 1999): area, leaving the same stubble height in all plots. VU = W × (Ccreatinine/Mcreatinine) Due to logistical issues in management of the research where: center, sampling periods were restricted to May– VU = Urine volume (L/d); December 2014 (RS) and February–April 2015 (DS). W = Live weight (kg); Ccreatinine = Creatinine coefficient (mg/kg LW/d); and Available nitrogen in soil Mcreatinine = Creatinine concentration in the sample (mg/L). During the first month after urine was applied, soils in each experimental plot were sampled weekly by Creatinine coefficient value was assumed as 17.3 collecting and pooling 8 soil cores to 20 cm depth. mg/kg LW/d (Rennó et al. 2000). Samples were dried at 50 °C and passed through a 2 mm Assuming a theoretical stocking rate of 2 animals/ha sieve. Concentrations of ammonium (NH +4 -N) and nitrate in Urochloa pastures, plus 9 months for RS and 3 months (NO -3 -N) nitrogen were determined by extraction using 1 for DS and a standard area of 1 ha, the total volume of M KCl, with reduction of ammonium for the calculation urine voided in each season was calculated using the of NO -3 -N (AOAC 1998). The analyses were performed following formula: in the Laboratory of Soil and Water of the Universidad U = (V × SR × Period)/A Nacional de Colombia (Bogotá). where: Statistical analyses U = Amount of urine/season (L/m2); V = Volume of urine/animal/d (L); The data did not comply with the statistical assumptions SR = Stocking rate (animals/ha); for linear models due to the presence of outliers, so Period = Season length (days); and outliers were eliminated by confidence intervals with one A = Standard area (m2). standard deviation. Significant effects of the treatments Urine was collected from cows by massaging the vulva were determined by analysis of variance and post-hoc and stored at 2 °C without acid preservation, to obtain the HSD Tukey test. necessary volumes needed for the plots. To determine the level of association between the Urine was sprinkled on the pasture at the start of each climatic variables and N available in soil, a linear season (13 May 2014 and 2 February 2015) in the plots regression model was used, after verification of statistical randomly allocated to the urine treatment. Application assumptions. For the construction of the model, all rates were 1.37 and 0.77 L urine/m2 for RS and DS, variables were evaluated through backward elimination, corresponding to 7.8 and 6.1 g N/m2 (78 and 61 kg N/ha). forward selection and stepwise regression, selecting those The lower amounts of urine/nitrogen applied in the DS factors with lower scores of Akaike Information Criterion were a combination of a higher concentration of N in the (AIC) (Winner 2018).This criterion uses model fit and the urine (0.57 and 0.8% N in RS and DS, respectively) and number of parameters as criteria. In comparing models, the shorter duration of DS. the model with the smallest AIC is considered optimal, Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) 108 J.E. Garzón, O. Pardo and E.A. Cárdenas computing the value with the following regression space (WFPS) values were 51.8 ± 4.3 and 49.5 ± 6.0% in equation (Kaps and Lamberson 2017): the same periods of time (Figure 2). AIC = n log(SSRES/n) + 2 × p where: Herbage accumulation and N content SSRES = Residual sum of squares; n = Number of observations; and The growth of pasture followed a seasonal pattern with p = Number of parameters in the model. highest yields during the rainy season and lowest production in the dry season (Table 1). The response in forage Finally, Pearson’s correlation test was performed accumulation to application of urine was not uniform across among those predictors with the greatest adjustment to species over the total period, with U. decumbens showing a their respective response variable. 33% increase (P<0.05) in growth compared with its Control, All data were analyzed using the RStudio® software, while the remaining species showed no responses (Table 1). with a level of significance of P<0.05. However, during the periods immediately following urine application, i.e. harvests in June 2014 and April 2015, DM Results accumulation averaged over the 3 species increased by 20.8% (June) and 80.8% (April). In the intervening period Environmental variables DM yields each month did not vary significantly between urine and Control treatments. Water balance (precipitation and evaporation) and air Similarly, application of bovine urine significantly temperature are presented in Figure 1. These revealed a increased N content overall in U. decumbens (P<0.05), high rainfall incidence during April‒June 2014 and with no significant responses in U. humidicola and November–December 2014, with a relatively dry period in M. maximus (Table 2). As for DM accumulation, during January–March 2015. Average relative humidity was 86%. the month following application of urine to the grasses, N During both sampling periods, average soil uptake by plants treated with urine was 79% higher than temperatures were 26.5 ± 0.7 and 28.6 ± 0.7 °C for RS by Control in June 2014 and 58% higher than by Control and DS, respectively. Similarly, average water-filled pore in April 2015 (Table 2). 300 35.0 30.0 250 25.0 200 20.0 150 15.0 100 10.0 50 5.0 0 0.0 Precipitation Evaporation Temperature Figure 1. Accumulated precipitation, evaporation and average atmospheric temperature in AGROSAVIA “La Libertad” during the study period. Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) Precipitation and evaporation (mm) Temperature (°C) Effect of bovine urine on grass and soil 109 90 35 30 80 25 70 20 60 15 50 10 40 5 30 0 WFPS Temperature Figure 2. Changes in soil moisture level (WFPS) and temperature during sampling period for ammonium and nitrate concentrations. Data from September to October 2014 were not recorded because of datalogger damage. Table 1. Effects of species and urine application on monthly herbage accumulation (t DM/ha) during 2014–2015. Month Urochloa decumbens Urochloa humidicola Megathyrsus maximus Control Urine Control Urine Control Urine Jun 1.03 1.20 1.43 1.67 1.09 1.42 Jul 0.96 1.75 1.27 1.35 1.90 1.68 Aug 1.45 1.78 1.88 1.68 1.89 1.46 Sep 1.49 1.09 1.67 1.59 0.84 1.05 Oct1 1.65 1.98 2.42 2.45 2.15 1.37 Nov 1.20 1.54 1.43 1.57 1.26 1.02 Dec 1.00 1.36 2.02 1.36 1.10 0.73 Feb 0.30 0.53 1.40 1.29 0.61 0.67 Apr 1.23 2.49 0.99 1.59 0.95 1.65 Mean ± s.e. 1.14 ± 0.1a 1.52 ± 0.1b 1.61 ± 0.1a 1.60 ± 0.1a 1.31± 0.1a 1.23 ± 0.1a Mean ± s.e. 1.3 ± 0.01B 1.6 ± 0.07A 1.2 ± 0.08B Means within species followed by the same lower-case letters are not different (P≥0.05). Overall means for species followed by the same upper-case letters are not different (P≥0.05). 1The harvest in October 2014 was made 36 days after the previous defoliation event, due to logistical issues at the research center. Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) WFPS (%) Soil temperature (°C) 110 J.E. Garzón, O. Pardo and E.A. Cárdenas Table 2. Effects of urine application in May 2014 and February 2015 on N content (kg N/ha) of 3 tropical grasses. Month Urochloa decumbens Urochloa humidicola Megathyrsus maximus Control Urine Control Urine Control Urine Jun 14 14.6 22.3 13.5 25.7 22.3 41.9 Jul 14 17.4 33.8 21.8 20.2 25.6 35.5 Aug 14 23.1 30.8 21.5 23.4 37.1 30.5 Sep 14 30.1 20.8 28.6 26.7 21.0 25.6 Oct 14 26.9 35.9 26.6 25.9 42.9 28.7 Nov 14 21.6 27.0 20.7 21.4 26.9 22.5 Dec 14 16.5 22.2 22.1 19.7 27.8 17.1 Feb 15 5.8 11.3 14.5 24.9 12.4 13.5 Apr 15 17.2 29.9 11.1 20.5 20.6 26.6 Mean ± s.e. 19.2 ± 1.9a 26.0 ± 2.3b 19.8 ± 2.0a 22.9 ± 1.5a 26.3 ± 2.9a 26.3 ± 3.0a Mean ± s.e. 22.6 ± 1.3AB 21.3 ± 1.0B 26.3 ± 1.7A Means within species followed by the same lower-case letter are not different (P≥0.05). Overall means for species followed by the same upper-case letter are not different (P≥0.05). Soil concentrations of NH +4 -N and NO - + 3 -N concentration had the greatest effects on NH4 -N concentration in soil, while rainfall and soil temperature Table 3 shows the concentrations of the two N forms were the factors that best predicted changes in NO -3 -N during the month following treatment application. A peak concentration in the soil. in concentration of NH +4 -N was registered following + - urine application in May 2014 with declining levels Table 4. Best adjusted models for soil NH4 -N and NO3 -N during the following month. The second application concentrations as response variables. 1 2 showed a lesser effect on concentration during DS. Method Best model AIC score In the first week after urine application in RS, the Initial Final NO -3 -N concentrations were minimal, and displayed a model model + - substantial increase later in the season. However, during Backward NH4 = ST + NO3 32.0 25.0 NO - = Rain + AT + 35.1 32.2 the DS, concentrations remained relatively stable during 3 RH + ST all samplings regardless of treatment (Table 3). Forward NH + = ST + NO -4 3 30.7 25.0 Seven variables were evaluated in building the NO -3 = Rain + ST 38.2 30.9 explanatory model: rainfall, air and soil temperatures, relative humidity, WFPS and concentrations of NO -3 -N Stepwise NH +4 = ST + NO -3 30.7 25.0 and NH +4 -N. All initial models presented Akaike NO -3 = Rain + ST 38.2 30.9 Information Criterion (AIC) scores greater than 30, 1AT = Air temperature; ST = Soil temperature; RH = Relative which were reduced once different methodologies were humidity; Rain = Rainfall; applied (Table 4). Soil temperature and NO -3 -N 2AIC = Akaike Information Criterion. Table 3. Effects of urine application on concentrations of NH + -4 -N and NO3 -N (mg/kg) in soil, during the month following application (2014‒2015). Day NH +4 -N NO -3 -N Control Urine Mean ± s.e. Control Urine Mean ± s.e. May 16 17.7 33.3 23.9 ± 3.5a 0.6 1.3 0.9 ± 0.6b May 22 8.0 16.0 11.2 ± 2.2de 20.9 21.2 21.1 ± 6.0a May 29 6.2 9.2 7.7 ± 1.5de 23.4 8.3 17.4 ± 9.5ab Jun 5 6.6 7.5 7.1 ± 0.7e 25.9 10.3 18.1 ± 7.8ab Feb 4 14.7 17.7 16.2 ± 1.0bc 7.6 6.8 7.2 ± 2.1ab Feb 12 16.0 18.6 17.3 ± 1.3b 8.1 8.2 8.1 ± 3.2ab Feb 19 8.9 12.2 10.5 ± 0.9de 6.3 4.0 5.2 ± 2.2ab Feb 25 10.6 14.0 12.3 ± 0.9cd 0.5 7.7 4.1 ± 2.4ab Mean ± s.e. 11.7 ± 1.0A 15.3 ± 1.0B 11.6 ± 3.3A 8.5 ± 1.8A Overall means within columns followed by the same lower-case letter are not different (P≥0.05). Means within N soil ions followed by the same upper-case letter are not different (P≥0.05). Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) Effect of bovine urine on grass and soil 111 Discussion pastures for grazing, as a compromise between biomass production and nutritional value of the forage (Rincón et Herbage accumulation and N content al. 2010). During our experiment, we applied the equivalent of 61 and 78 kg N/ha in DS and RS, The highest herbage accumulation in all pastures was respectively, which would have generated the significant registered in October 2014. During this time, humidity, response in N concentration during these first samplings. precipitation and temperature favored DM production, as While herbage accumulation also increased, the opposed to the following month, when temporary responses were not large enough to detect significant waterlogging in some plots reduced growth rates. differences (Table 1). The absence of differences in the Conversely, the lowest herbage accumulation was following months was possibly due to the transformation registered in all plots during February 2015. This month and use of the N applied within the soil through is part of the DS in the Piedmont plains, when little ammonification and nitrification or by its escape from the rainfall is received (Figure 1), resulting in lower soil system by volatilization or denitrification (Orozco 1999). moisture levels (Figure 2) but elevated temperature. Although the second application of urine occurred in Water is vital in plant metabolic processes and supplies February 2015, none of the pastures showed effects in electrons for the reduction of CO2 in photosynthesis. their productive variables in the following month, and Likewise, its elimination through transpiration is linked to responses were delayed until April 2015. This could the stomatic conductance of the leaves; to avoid water happen due to weather conditions during the DS, with few loss, a lower moisture content within the plant decreases precipitation events (Figure 1) and decreased soil the stomatal opening, reducing the amount of CO2 taken moisture (Figure 2) in January and February 2015. in by the leaf for photosynthesis. All this is reflected in Reduced levels of water in the soil solution suppress reduced plant growth (Berlyn and Cho 1999). activity of the microbial populations responsible for Observing the total N content for the study period, making N available for plants (Cameron et al. 2013), Table 2 shows important differences between plant which could result in loss of N by ammonia volatilization species, with the highest values for M. maximus and (Jantalia et al. 2012) or its inorganic preservation by the U. decumbens. Due to its larger foliar area, type of growth low soil pH and presence of clays. The immobilization of and genetic characteristics, M. maximus normally N by the soil during the DS could preserve this element presents a higher rate of photosynthesis (Silva 2004) and until the return of the rains, when it is mineralized by the efficiency in N use (Pérez 2014) than other tropical microbial populations, making it available for the plants grasses, meaning a high herbage accumulation and crude (Baggs et al. 2010). protein concentration (Fernández et al. 2004). In this study, U. humidicola produced more DM than Concentrations of ammonium and nitrate N in the soil M. maximus (Table 1), which may be explained by its growth habit. U. humidicola grows by stolons, with a According to Table 3, NH +4 -N was always present in the higher DM compared with their leaves but lower soil, since both treatments showed concentrations greater digestibility (Vergara and Araujo 2006). Moreover, than 6 mg/kg. With total N concentration of 0.14%, this Urochloa grasses have morphological adaptations to soil could be considered as low in organic matter. promote better utilization of N available in soils with low However, it is known that soil microorganisms present fertility, such as increased amount and length of roots or adaptive capacities in such situations, fulfilling their lower leaf expansion per unit dry weight (Rao et al. 1995). function as regulators of the N cycle in the soil (Dubeux These factors decrease the dependence of forages on high Jr et al. 2007; Chirinda 2015). Additionally, the applica- N concentrations in the soil, presenting lower tion of urine could increase the concentrations of NH +4 -N concentrations of N in their tissues in consequence (Rao in the soil during the RS, decreasing over time as the N 2001). The effect is more pronounced in U. humidicola, source was transformed into other compounds. probably through its ability to synthesize biological The marked increase in NO -3 -N observed in the RS nitrification inhibitors from its roots (Subbarao et al. after treatment application (Table 3) was possibly due to 2009). the interaction of the biochemical processes of When analyzing responses to treatments over time, ammonification and nitrification (Orozco 1999). significant differences in N content were observed Likewise, Baggs et al. (2010) suggested that lower between the urine treatments and their controls in the first moisture levels in the soil could stimulate nitrate month after urine application (Table 2). The regional ammonification, which would also explain the lower recommendation is to apply 50‒70 kg N/ha to Urochloa nitrate levels during the DS. Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) 112 J.E. Garzón, O. Pardo and E.A. Cárdenas It is noticeable how the concentrations of NH +4 -N and due to the inverse behavior shown by both compounds NO -3 -N differed from the baseline levels. Due to their during the sampling period (Table 3), where ammonium is reductive conditions, oxisol soils could stimulate an initial component for nitrification, resulting in nitrate as ammonification over nitrification, favoring the the final product (Ardakani et al. 1974; Whitehead 1995). production of ammonia and its subsequent loss due to volatilization (Cameron et al. 2013). Table 5. Correlation coefficients between response variables Urochloa grasses showed a higher N content (Table 2) and predictors of the best-fitted models (%). during the RS, which could be explained by a delay of nitrification until the second month after application of Response Factor ρxy + bovine urine. Although tropical grasses are capable of NH4 -N ST -77.4 NO - -57.8 absorbing small amounts of N in NH +4 -N form without 3 NO -3 -N ST 67.0 exhibiting intoxication (Moser et al. 2004), they prefer to Rain 36.2 absorb this element as NO -3 -N, which is more soluble and ST = Soil temperature; Rain = Rainfall. easily transportable in the roots (Azcón-Bieto and Talon 2008). Its higher concentration in the soil would then We conclude that applying urine from cows fed with imply greater absorption, resulting in a greater deposition tropical forages increased herbage accumulation in of N components within the plant. U. decumbens but not in U. humidicola and M. maximus Variation in NH + -4 -N and NO3 -N concentrations was in the Colombian Piedmont plains. However, it did noted in the control plots, despite urine not being applied produce temporary increases in N content in the grasses on these experimental units. This phenomenon requires immediately following application. This supports the further investigation, but Day and Detling (1990) concept that grazing animals play a beneficial role in suggested that forage responses on urine patches relative dispersing nutrients in pastures. However, the effects to unaffected areas may be associated with increased soil seem to be limited in duration possibly because of the loss N availability and root N concentration. The impact of of N through volatilization. Likewise, the edaphoclimatic urine beyond the area of application is aided by the large conditions and regional agricultural management limit the proportion of water in the urine and the mobility and concentrations of N available in the soil, showing the availability of the nutrients it contains (White-Leech et al. validity of using forages adapted to these areas. 2013), reaching areas 3 times or more the area to which it Finally, it is necessary to determine the relationships was deposited (Haynes and Williams 1993). between the concentrations of available N in the soil and Available N values reported in this study differ from in the grasses in the region, to understand better the those reported from other investigations. Conducting an impact of agricultural practices on the N cycle in pastoral experiment to determine ammonifying microbial systems. populations, Verhamme et al. (2011) reported concentra- tions of approximately 190 and 70 g/kg of NH +4 -N and - Acknowledgments NO3 -N, respectively, in a soil in Scotland, while Salazar- Sosa et al. (2009) published values of 50‒100 mg/kg The authors thank the Animal Nutrition Laboratory and nitrates in soils cultivated with a mixture of corn and soy the Research Unit of the Facultad de Medicina Veterinaria in Mexico. Similar results were shown by Trejo-Escareño y de Zootecnia of the Universidad Nacional de Colombia et al. (2013), evaluating the effects of application of (Bogotá) for their support in the forage analyses, and the bovine feces in a corn crop. Those studies suggest that the Fondo Regional de Tecnología Agropecuaria (Fontagro) values reported here can be considered low, although this (FTG/RF-1028-RG) for the funding that made this greatly depends on the management given to the soil and research possible. the external application of nutrients. 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Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) Tropical Grasslands-Forrajes Tropicales (2020) Vol. 8(2):115–124 115 doi: 10.17138/TGFT(8)115-124 Research Paper Effects of swine manure application and row spacing on growth of pearl millet (Cenchrus americanus) during the establishment period and quality of silage produced in Southwest Nigeria Efectos de la aplicación de estiércol de porcinos y de la distancia de siembra en el establecimiento y la calidad del ensilaje de Cenchrus americanus en el suroeste de Nigeria V.O.A. OJO1, F.T. ADESHINA1, G.A. ADETOKUNBO1, S.O. JIMOH1,2,3, T.A. ADEYEMI1, J.L. NJIE4 AND O.S. ONIFADE1 1Department of Pasture and Range Management, College of Animal Science and Livestock Production, Federal University of Agriculture, Abeokuta, Nigeria. unaab.edu.ng 2Institute of Grassland Research, Chinese Academy of Agricultural Sciences, Hohhot, Inner Mongolia, China. caas.cn 3Sustainable Environment Food and Agriculture Initiative (SEFAAI), Lagos, Nigeria. sefaai.org 4School of Agriculture and Environment Sciences, University of The Gambia, Serrekunda, The Gambia. utg.edu.gm Abstract The effects of swine manure application and row spacing on dry matter yields of Cenchrus americanus (pearl millet) at 6 weeks after sowing and chemical composition, fermentative characteristics and in vitro gas production of silage produced from the forage were studied. The design was a 2 × 2 factorial with 2 row spacings (0.5 and 1.0 m) and 2 levels of manure application [no manure (Control) and swine manure at 5 t/ha (22% DM; 0.34% N on DM basis)] replicated 3 times. Swine manure application had no effect (P>0.05) on dry matter yield but a row spacing of 0.5 m produced higher (P<0.05) dry matter yields than 1.0 m spacing (mean 7.05 vs. 5.57 t DM/ha). Fresh forage from manured treatments had significantly higher crude protein concentration (114.9–124.2 g/kg DM) than from unfertilized plots (86.2–95.1 g/kg DM). After being ensiled for 42 days, CP% in the forage had declined by 16–18% but relative differences remained. Quality measurements indicated that silages from the various treatments were all of acceptable standard although CP% of silage from Control plots was barely high enough to provide a maintenance diet. This study suggests that, under the experimental conditions, planting of pearl millet at a spacing of 0.5 m rather than 1.0 m would increase DM yields obtained in the first 6 weeks of growth, while application of swine manure would not affect yields but would increase CP% of forage produced. The laboratory study indicates that the forage produced could be ensiled successfully although there was significant loss of crude protein during the process. Since there were no significant increases in DM yields of forage, other benefits, e.g. increase in N concentration, improved soil organic matter, etc., would need to be considered in justifying the additional cost of drying and applying the manure. Keywords: Fertilization, forage conservation, nutritive value, tropical forages. Resumen En Abeokuta, Oguna State, Nigeria se estudiaron los efectos de la aplicación de estiércol porcino y el espaciado entre hileras sobre los rendimientos de materia seca de Cenchrus americanus a las seis semanas después de la siembra, la composición ___________ Correspondence: S.O. Jimoh, Institute of Grassland Research, Key Laboratory of Grassland Ecology and Restoration, Chinese Academy of Agricultural Sciences, 120 East Wulanchabu Street, Hohhot, Inner Mongolia 010010, China. E-mail: sahjim05@gmail.com Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) 116 V.O.A. Ojo, F.T. Adeshina, G.A. Adetokunbo, S.O. Jimoh, T.A. Adeyemi1, J.L. Njie and O.S. Onifade química, las características fermentativas y la producción de gas in vitro por el forraje ensilado de esta gramínea. Se empleó un diseño factorial 2 × 2 con dos distancias de siembra (0.5 y 1.0 m entre hileras) y dos niveles de aplicación de estiércol [sin y con 5 t/ha (22% MS; 0.34% N con base en MS)] y tres repeticiones. La aplicación de estiércol porcino no mostró ningún efecto (P>0.05) en el rendimiento de materia seca, pero la distancia entre hileras de 0.5 m produjo rendimientos de materia seca más altos (P<0.05) que la distancia de 1.0 m (media 7.05 vs. 5.57 t MS/ha). El forraje fresco en los tratamientos con estiércol presentó mayor (P<0.05) concentración de proteína cruda (114.9–124.2 g/kg de MS) que el de las parcelas no fertilizadas (86.2–95.1 g/kg de MS). Después de estar ensilado durante 42 días, el porcentaje de PC en el forraje se redujo en 16–18%, pero las diferencias relativas se mantuvieron. Las mediciones de calidad indicaron que los ensilajes en los tratamientos eran aceptables; no obstante en el testigo (tratamiento sin estiércol) el porcentaje de PC del ensilaje solo llenaba los requerimientos de mantenimiento para bovinos. Este estudio mostró que en las condiciones experimentales, la siembra de C. americanus a una distancia de 0.5 m aumenta los rendimientos de MS obtenidos en las primeras 6 semanas de crecimiento, mientras que la aplicación de estiércol porcino no afecta los rendimientos, pero aumenta la concentración de CP en el forraje.. El estudio de laboratorio indica que el forraje producido puede ser ensilado con éxito, pero con una pérdida significativa de PC durante el proceso. En vista de que la aplicación de estiércol no mostró aumentos significativos en el rendimiento de forraje, otros beneficios, p.ej. el aumento de la concentración de N y el incremento de la materia orgánica en el suelo, etc., deberían considerarse para justificar el costo adicional del secado y la aplicación del estiércol. Palabras clave: Conservación de forraje, fertilización, forrajes tropicales, valor nutritivo. Introduction production systems. The crop is well adapted to a wide range of agronomic conditions varying from the semi-arid Forages are the primary diets for ruminants in many to the subhumid zone, where annual rainfall varies tropical regions and grasses constitute the bulk of the between 600 and 1,100 mm, with forage yields of 2.7– energy sources for grazing ruminants (Olanite et al. 7.7 t DM/ha (Agishi 1985). Preliminary studies with pearl 2010). High-yielding forages of high quality are the millet confirmed that it could be harvested and conserved most economical feed for ruminants and result in for feeding to ruminant animals during prolonged dry acceptable liveweight gains and animal performance. seasons (Nuru 1989). However, prolonged annual dry seasons negatively To ensure both adequate yields and acceptable quality of affect plant performance, resulting in limited quantity forage, application of fertilizer and optimal row spacing are and poor quality of available forage at this time, leading essential (Miah et al. 1990; Olanite et al. 2010). In the past, to a reduction in voluntary feed intake and nutrient application of inorganic fertilizer has been the normal utilization, with reduced overall performance of practice, but increased costs of these products, combined ruminant animals. with the attendant nutrient depletion and negative residual Ojo et al. (2015a) suggested that the challenge of feed effects of inorganic fertilizers on the environment (Malhi et shortages could be solved through the cultivation of al. 2002), have resulted in increased interest in using organic forage crops with better nutritional values than the manures on soils. Applying organic manures to soils/crops existing native feed resources. Similarly, Bamikole et al. would have multiple benefits by overcoming the problem of (2004) reported that high concentrations of protein, manure management and disposal, improving the nutrient vitamins and minerals in sown forages could markedly status of the soils and possibly improving both yields and improve animal performance. Conservation and nutrient value of the forage produced (Ojo et al. 2013). preservation of these cultivated species, combined with The present study investigated the effects on DM improved management practices, is a possible solution to yields and nutritive value of the resulting forage of the limitations posed by poor quality and quantity of applying swine manure to a crop of pearl millet sown at native forages in the dry season (Ojo et al. 2015b). 2 different row spacings, and quality of silage produced Babayemi (2009) recommended the ensiling of forages, from the forage. at a growth stage when there is a balance between yield and quality of the available crop, for feeding to animals Materials and Methods during times of nutritional stress. Amodu et al. (2005) indicated that pearl millet Location [Cenchrus americanus (L.) Morrone syn. Pennisetum americanum (L.) Leeke] is a promising forage crop with The experiment was conducted at the Pasture Unit of high potential for integration into Nigerian livestock Federal University of Agriculture, Abeokuta (FUNAAB) Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) Manuring, row spacing and ensiling pearl millet 117 Farm, Ogun State, Nigeria (7°58' N, 3°20' E; 75 masl). complete block design was used with a total land area The site is situated in the derived savannah agro- measuring 19 × 17 m divided into 3 equal blocks. Each ecological zone of Southwest Nigeria with average annual block measured 5 × 17 m with a buffer zone of 1 m rainfall of 1,037 mm. Mean monthly temperature ranges between blocks, while each plot measured 4 × 5 m, with from 25.7 ºC in July to 30.2 ºC in February (earth.google. a buffer zone of 1 m between plots. Variability in soil was com/). Data on rainfall, temperature and humidity blocked across the slope and each treatment was covering the period when the study was conducted are replicated 3 times. shown in Table 1. The manure was broadcast onto individual plots according to treatment in a single application and Table 1. Meteorological data for the experimental area during subsequently raked into the soil manually and the plots June–December 2015. were left for 2 weeks before sowing the grass. Seeds of C. americanus were drilled in rows at the predetermined Month Rainfall Mean temp. Mean rel. row spacings at a seeding rate of 20 kg/ha in August 2015. (mm) (°C) humidity (%) No herbicides were applied. Jun 165 26.9 79.4 Jul 66 26.6 80.9 Forage harvest and ensiling process Aug 29 26.3 79.3 Sep 165 26.5 81.3 Oct 159 27.4 81.9 Millet forage was harvested at 6 weeks after sowing by Nov 17 28.6 72.5 cutting the entire forage in each plot at 15 cm above Dec 0 26.1 39.7 ground level. Fresh weight of the forage was determined Source: Agrometeorology Department, FUNAAB, Nigeria. immediately after harvesting, following which 500 g subsamples of the fresh forage from each treatment were Land preparation collected to determine DM% and chemical composition. Thereafter, the harvested forage was bulked within A total land area of 598 m2 was cleared, plowed and treatments and allowed to wilt for 4 hours. Wilting helped allowed to rest for a period of 2 weeks before harrowing, reduce the moisture content of the forage to give a DM following conventional tillage operations to provide a fine concentration of 33%, following which the material was seed bed. Prior to sowing, soil samples were collected at manually chopped to lengths of about 2 cm. The chopped random from the area to a depth of 15 cm using a soil forage was rapidly compressed and sealed into laboratory auger to determine the pre-planting nutrient status of the glass silos of 960 mL capacity with 3 silos per treatment. soil. Analysis of these soil samples indicated that it was a Small glass silos were used as this was an exploratory sandy silt with the following parameters: pH 6.75, organic study and no animal feeding trials were planned. A total carbon 1.27%, available phosphorus (P) 30.4 mg/kg, of 500 g of forage was manually compressed into the silo potassium (K) 0.79 cmol/kg, calcium (Ca) 3.36 cmol/kg, bottles to a density of 0.52 g/mL. The silos were tightly magnesium (Mg) 2.42 cmol/kg, sodium (Na) 1.51 sealed with 6 layers of duct tape to prevent re-entry of air cmol/kg and total nitrogen (N) 0.11%. into the silos, and the ensiled materials were allowed to Swine manure was collected from the Piggery Section stand for a period of 6 weeks, at an ambient room of the Directorate of University Farms, FUNAAB, 14 days temperature of 26 ºC. before application in bi-axially oriented polypropylene bags. Following collection, the manure was spread to dry Quality analyses and stored under a barn to allow for normal decomposition. The pigs had been fed a standard “Pigs finisher diet”. Fresh forage. Analysis of the fresh forage commenced Chemical analysis of the manure revealed that it contained: immediately after sample collection. Subsamples (500 g) 4.76% Ca, 2.40% Mg, 1.92% K, 2.07% Na, 0.34% N and were oven-dried to a constant weight at 65 ºC. The dried 1.2% P on a DM basis. samples were then milled through a 1 mm sieve and crude protein (CP) and ash concentrations were determined Experimental design according to the standard methods of AOAC (2000). Fiber fraction concentrations [neutral detergent fiber The experiment was laid out as a 2 × 2 factorial design (NDF), acid detergent fiber (ADF), and acid detergent comprised of 2 row spacings (drilled at 0.5 and 1.0 m lignin (ADL)] were determined according to Van Soest et inter-row intervals) and 2 levels of swine manure al. (1991). Cellulose concentration was estimated as the application (0 and 5 t/ha at 22% DM). A randomized difference between ADF and ADL concentrations, while Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) 118 V.O.A. Ojo, F.T. Adeshina, G.A. Adetokunbo, S.O. Jimoh, T.A. Adeyemi1, J.L. Njie and O.S. Onifade hemicellulose concentration was estimated as the Results difference between NDF and ADF concentrations. Silage. At the expiration of the ensiling period, the silos Fresh forage were opened and assessed for physical and sensory properties, including color, odor, moisture and presence The narrower row spacing produced significantly (P<0.05) of mold (Appendix 1). Six trained silage experts assessed higher forage yields (6.85 and 7.25 t DM/ha for no manure all treatments using a scoring sheet (Bates 1998). After and manured, respectively) than the wider row spacing opening the silos, about 200 g of silage was weighed into (5.48 and 5.65 t DM/ha for no manure and manured, 500 ml beakers. Color and moldiness scores were respectively) (Figure 1). There was no significant (P>0.05) awarded based on the visual appearance of the silage. response to manure application at either row spacing. Odor score was based on how assessors felt when smelling a sample of silage. For moistness, the assessors 9 – wearing latex gloves – squeezed moisture from about a 30 g of the silage and allocated a score for the level of free 8 a water in the silage. For the scoring schemes, see Appendix 7 1. Ammonia and volatile fatty acid concentrations (acetic, b b propionic, butyric and lactic acid concentrations) in the 6 silages were determined according to the procedures of AOAC (1990) and Mathew et al. (1997), respectively. 5 Immediately after opening of the silos, 10 g samples of silage were taken from each silo and soaked in 100 mL of 4 distilled water for 12 hours. The mixtures were then 3 filtered and the supernatant divided into 4 aliquots each for pH determination using a pH meter (Hanna 2 instruments, pH 211, microprocessor pH meter, K012818, Portugal) (Wilson and Wilkins 1972). 1 Subsamples of 300 g silage were oven-dried to a constant weight at 65 ºC. Concentrations of CP, ash and 0 0.5 m 1.0 m 0.5 m 1.0 m fiber fractions were determined following the same procedures outlined for the fresh samples. In addition, No manure Manure mineral concentrations (Ca, P, K, Mg, iron and copper) of the silage samples were determined according to the Figure 1. Yields of Cenchrus americanus as affected by row standard methods of AOAC (2000). spacing and swine manure application. Error bars indicate To evaluate the degradability of the ensiled forage standard error of the mean. Values for columns with different materials, in vitro gas production was determined according letters are significantly (P<0.05) different. to the procedure of Menke and Steingass (1988). At both row spacings, fertilized pearl millet had Statistical analysis higher (P<0.05) CP percentage than the unfertilized grass (Table 2). There were no consistent patterns between Data collected were subjected to a 2-way analysis of treatments in NDF, ADF, ADL, hemicellulose and variance and treatment means were separated using cellulose levels but unfertilized forage at the wider row Duncan’s Multiple Range Test (SAS 2003) and analyzed spacing tended to have the lower levels of NDF, ADF and using the PROC GLM procedure. cellulose. Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) Dry matter yield (t/ha) Manuring, row spacing and ensiling pearl millet 119 Table 2. Chemical composition (g/kg DM) of fresh Cenchrus americanus forage as affected by row spacing and swine manure application. Row Manure CP Ash NDF ADF ADL Hemi- Cellulose spacing (m) application cellulose 0. 5 Manure 114.9a 1 163 645a 386a 87.7 199b 297b No manure 95.1b 179 668a 390a 82.0 287a 305b 1.0 Manure 124.2a 151 626ab 409a 89.9 203b 321a No ma nure 86.2b 200 586b 365b 80.3 227b 277c s.e.m. 5.76 8.3 22.7 6.2 4.39 30.8 6.3 1Means in the same column followed by different letters are significantly (P<0.05) different. Silage row spacing irrespective of manure application (Table 5), while manure application had no effect on DM% Irrespective of row spacing, silage made from (P>0.05). Manure application at both row spacings unfertilized pearl millet recorded higher (P<0.05) silage increased (P<0.05) CP% of silage. There were no color scores than silage from fertilized forage, with the consistent patterns in the effects of both manure silage having desirable green to yellowish-green color application and row spacing on ADF, ADL, hemi- (Table 3; Appendix 1). All silages except that from the cellulose and cellulose concentrations. fertilized plots at wide row spacing had quite acceptable All mineral concentrations in silage followed the same odors. pattern except for P, in the order Narrow + manure > Fermentative characteristics of the silage were Wider + manure > Wider + no manure > Narrow + no significantly (P<0.05) affected by both row spacing and manure (P<0.05) (Table 6). Concentration of P in silage manure application (Table 4). Fertilized forage from the showed an increase from manure application at narrow narrow row spacing had lower VFA (volatile fatty acid) row spacing but a reduction from manure application at concentrations but higher ammonia N concentrations and the wider spacing. pH values than other silages. Meanwhile, silage from In general, patterns of gas production from silage over unfertilized treatments had the lowest pH regardless of time were inconsistent and not significantly affected by row spacing. either manure application or plant spacing (P>0.05) Silage from the narrow row spacing had significantly (Table 7). Fermentation of substrates was ongoing beyond (P<0.05) higher DM percentage than that from the wider 24 hours of incubation. Table 3. Physical characteristics of Cenchrus americanus silage as affected by row spacing and swine manure application. Row spacing (m) Manure application Color Odor Moistness Moldiness 0. 5 Manure 7.8b 1 23.6b 9.0a 8.0b No manure 9.8a 26.0a 9.0a 9.0a 1.0 Manure 6.7c 14.9d 6.0c 5.0d No m anure 9.6a 22.0c 8.0b 6.0c s.e.m. 0.16 0.13 0.06 0.07 For the scoring schemes, see Appendix 1. 1Means in the same column followed by different letters are significantly (P<0.05) different. Table 4. Fermentative characteristics of Cenchrus americanus silage as affected by row spacing and swine manure application. Row Manure VFA1 Ammonia N Acetic acid Propionic acid Butyric acid Lactic acid pH spacing (m) application (%) 0. 5 Manure 10.8c 2 3.0a 1.30bc 0.87c 0.13b 1.95c 5.17a No manure 12.0a 1.7c 1.44a 0.96a 0.14a 2.16a 4.15c 1.0 Manure 11.7ab 2.2b 1.25c 0.94ab 0.14a 2.11ab 4.70b No ma nure 11.3b 1.4d 1.36ab 0.90c 0.14a 2.03b 4.27c s.e.m. 0.09 0.02 0.05 0.01 0.00 0.02 0.02 P-value 0.0001 <0.0001 0.0001 0.0001 0.0001 0.0001 <0.0001 1Volatile fatty acids. 2Means in the same column followed by different letters are significantly (P<0.05) different. Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) 120 V.O.A. Ojo, F.T. Adeshina, G.A. Adetokunbo, S.O. Jimoh, T.A. Adeyemi1, J.L. Njie and O.S. Onifade Table 5. Chemical composition of Cenchrus americanus silage as affected by row spacing and swine manure application. Row spacing Manure DM CP Ash NDF ADF ADL Hemicellulose Cellulose (m) application (%) (g/kg DM) 0. 5 Manure 68.0a 1 94.6a 137ab 537ab 320 73.3 167 247 No manure 64.1a 79.9b 147ab 557a 320 66.7 237 253 1.0 Manure 59.1b 101.3a 127b 510ab 340 73.3 170 267 No m anure 55.1b 71.2b 167a 490b 300 66.7 190 233 s.e.m. 4.22 2.64 6.37 13.5 11.55 6.67 14.7 11.7 1Means in the same column followed by different letters are significantly (P<0.05) different. Table 6. Mineral concentrations in Cenchrus americanus silage as affected by row spacing and swine manure application. Row spacing Manure Calcium Phosphorus Potassium Magnesium Iron Copper (m) application (g/kg DM) (mg/kg DM) 0. 5 Manure 9.83a 1 5.22a 20.3a 6.25a 74.3a 7.44a No manure 3.83d 3.65c 18.1d 4.58d 36.7d 2.34d 1. 0 Manure 8.09b 3.77c 20.2b 5.45b 62.4b 6.69b No m anure 7.37c 4.33b 19.7c 5.31c 41.9c 4.13c s.e.m. 0.00 0.08 0.00 0.00 0.01 0.02 1Means in the same column followed by different letters are significantly (P<0.05) different. Table 7. In vitro gas production (mL/h) of Cenchrus americanus silage as affected by row spacing and swine manure application. Row spacing (m) Manure application Time (h) 3 6 9 12 24 36 48 0.5 Manure 1.3 3.3 6.7 8.7 20.7 30.0 31.3 No manure 2.0 4.0 8.0 10.7 20.0 24.0 24.0 1.0 Manure 1.3 4.7 8.0 10.7 19.3 24.0 25.3 No ma nure 1.0 4.7 8.7 9.3 22.0 28.7 30.7 s.e.m. 0.77 1.35 2.09 2.48 3.85 4.09 4.10 Discussion In contrast, the higher CP% in fertilized forage was surprising given the low N rates in the manure but This study revealed that planting Cenchrus americanus with corroborates an earlier study by Jimoh et al. (2019) who narrow row spacing under the conditions existing in this reported a 20.1% increase in CP% when swine manure study resulted in about 25–28% increase in DM yield was applied to Panicum maximum cvv. Local and Ntchisi compared with the wider row spacing. This higher DM yield relative to the control in southwestern Nigeria. It is recorded for plants on the narrow-spaced plots may be due possible that P in the manure may have influenced the to higher nutrient use efficiency (Jiang et al. 2013) as a result increased N uptake by the plants since N uptake has been of more effective distribution of plants over the available reported to be influenced by higher P availability surface, as opposed to the wider-spaced plots with a higher (Cleveland et al. 2011; Alkhader and Abu Rayyan 2015). plant density within rows. The quality of grasses remains comparatively stable from Interestingly DM yields for plants on both manured vegetative to early stages of stem elongation (Jones and and control plots were similar. A lack of response to Wilson 1987) and declines as the plant matures (Ojo et al. manure application may be due to the slow nutrient 2016a, 2018; Ali et al. 2019). In this study, forages were release rate associated with swine manure (Chastain et al. harvested at 6 weeks of growth, when forage should have 1999; Silva et al. 2016), implying that perhaps much of ample CP% to meet ruminant demand, allowing multiple the nutrients in the applied manure may not have been harvests within the growing season. CP concentrations in converted to plant usable forms, and the quantities the fresh forage were quite satisfactory for ruminant released to the plant may have been insufficient to induce feeding at 8.6‒12.4%. However, there was a marked higher dry matter yields. Since the quantity of manure reduction in CP% in the corresponding silages with a applied supplied only about 3.7 kg N/ha it is scarcely range of 7.1‒10.1%, which agrees with the findings of surprising that no growth response occurred. Naeini et al. (2014), who reported a reduction in CP% Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) Manuring, row spacing and ensiling pearl millet 121 from 6.2% in fresh sweet sorghum to 5.4% in the silage. composition. In addition, mean concentration values for The critical limit required by rumen microbes to build major limiting nutrients (Ca, P, Mg and Cu) were higher their body protein for effective digestion of forages in than the minimal concentration levels of 2.3–2.9, 2.0–3.5, ruminants is 7.0% (Van Soest 1994). The lower values for 0.4–0.5 g/kg and 4 mg/kg, respectively, suggested for silage in our study are barely high enough to provide dairy cows (NRC 1984). This implies that there would be maintenance levels of protein for ruminants and values no need for supplements if the silage were fed to animals. below this could result in reduction in DM intakes. Usually, deficiencies and imbalances in nutrients are The color of the silages in this study fell within the neutralized by mineral supplements which may be costly range of 5–8 (green to yellowish-green) and 9–12 (yellow for poor resource farmers (Tiemann et al. 2009); however, to brownish) which are regarded as good to acceptable the mineral concentration values recorded for the silages ranges for silage (Bates 1998; Babayemi 2009). Further, in this study, especially those from the manured the odor of the silage also fell within the range of 11–23 treatments, were at favorable levels. (acceptable) and 24–28 (desirable) reported by Bates The observed similarities in the in vitro gas volumes (1998), although better scores were obtained from silage implies that despite the differences in the NDF made from the narrowly spaced plots which could be due concentrations of the ensiled forages, this may not be to the higher dry matter percentage that has been reported sufficient to hinder the degradability of the forages if fed to to influence the stability of ensiled forages (Wilkinson ruminants. Anassori et al. (2012) had asserted that gas and Davies 2013). volume readings could be used to estimate the degradability The pH values recorded for the silages were within the of forages in vitro. This finding further supports the initial range of 4.5–5.5 classified as indicative of good silage proposition that the higher NDF values recorded for the (Meneses et al. 2007). However, concentrations of desirable narrow spaced plants falls within the threshold of what can lactic acid in silage (1.95–2.16%) were below the range of be efficiently degraded by rumen microbes. 2.37–5.89% reported by Kung and Shaver (2001). High concentrations of lactic acid in silage are a clear indication Conclusion of good preservation, which invariably results in lower loss of DM and energy during storage. Ammonia nitrogen is an While application of swine manure to pearl millet at 5 t/ha important indicator of proteolytic activity during the increased CP% of forage and N recovery rate when fermentation process. Ammonia concentrations in the silage harvested at 42 days, it failed to increase forage yields. were very low and well below 12%, which is considered the However, decreasing row spacing from 1.0 to 0.5 m indicator of an excellent and well preserved forage (Silveira increased DM yield by 25‒28%, indicating significant 1975; Kung and Shaver 2001). Acetic acid concentrations gains to be made by planting pearl millet at a closer row were within the range (0.5–3.0% DM) classified as normal spacing at the sowing rates used in that environment. for grass silage (Kung and Shaver 2001) and also fell in line Plant populations, soil fertility and rainfall levels could with the range of 0.74–1.53% DM for Pennisetum hybrid have significant impacts on these findings. While silage silage reported in another experiment (Ojo et al. 2016b). made from the forage was of a good standard, CP% in the The NDF values of C. americanus silage recorded in forage had declined by 16‒18%. It seems that applying this study were well below the 65% threshold suggested swine manure at 1.1 t DM/ha to pearl millet planted at as the level at which intake and degradability of tropical 0.5 m spacing and cutting at 6 weeks after planting could feeds by ruminants would be limited (Eastridge 2006). produce high yields of forage. Ensiling this material This was not surprising given the growth stage at which would give a product of sufficient quality to be fed as the forage was harvested. Higher NDF concentration in supplements or a complete diet to ruminants especially silage made from the forage planted at 0.5 m intervals during the dry season. Better responses in yield might be could be a function of competition for light owing to the expected from other manure sources with higher N close spacing, resulting in continuous elongation in the content. Cutting at more mature stages of growth would height of the plants, thereby leading to increased fiber be expected to increase DM yields but quality of forage concentration. should decline, especially N concentration, and ability There were general improvements in the Ca, K, Mg, to compress the forage during ensiling would be expected Fe and Cu concentrations of silage made from the to decline as well. Longer-term studies to determine if manured plants. This was expected as the manure may yield advantages from the narrower row spacing could be have made more nutrients available to the plants and maintained as the stand matured as well as the quality corroborates the report by Christophe et al. (2019) that outcomes of repeated cutting at close intervals with those organic amendments tend to improve plant mineral from less frequent cutting seem warranted. Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) 122 V.O.A. Ojo, F.T. Adeshina, G.A. Adetokunbo, S.O. Jimoh, T.A. Adeyemi1, J.L. Njie and O.S. Onifade References Cleveland CC; Townsend AR; Taylor P; Alvarez-Clare S; (Note of the editors: All hyperlinks were verified 4 May 2020.) Bustamante MMC; Chuyong G; Dobrowski SZ; Grierson P; Harms KE; Houlton BZ; Marklein A; Parton W; Porder S; Agishi EG. 1985. Forage resources of Nigerian rangelands. In: Reed SC; Sierra CA; Silver WL; Tanner EVJ; Wieder WR. Adu IE; Taiwo BBA; Osinowo OA; Alhassan WS, eds. 2011. 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Journal of Journal of Animal Science 18(1):230–241. bit.ly/2YBaKdZ Dairy Science 74:3583–3597. doi: 10.3168/jds.S0022-0302 Ojo VOA; Oyebanjo ED; Opayemi OT; Mustapha SO; Adelusi (91)78551-2 OO; Adeoye SA; Anele UY; Ogunsakin AO; Jolaosho AO; Wilkinson JM; Davies DR. 2013. The aerobic stability of Ofinade OS. 2016b. Effects of seasonal changes on the silage: Key findings and recent developments. Grass and nutritive quality of Moringa oleifera and Panicum maximum Forage Science 68:1–19. doi: 10.1111/j.1365-2494.2012. silage. Slovak Journal of Animal Science 49:76–84. 00891.x sjas.ojs.sk/sjas/article/view/164 Wilson RF; Wilkins RJ. 1972. An evaluation of laboratory Ojo VOA; Adelusi OO; Jimoh SO; Yusuf KO; Dele PA; ensiling techniques. Journal of Science of Food and Akinyemi BT. 2018. Effects of proportion and ensiling Agriculture 23:377–385. doi: 10.1002/jsfa.2740230315 Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) 124 V.O.A. Ojo, F.T. Adeshina, G.A. Adetokunbo, S.O. Jimoh, T.A. Adeyemi1, J.L. Njie and O.S. Onifade Appendix 1. Silage physical and sensory evaluation sheet. Factor Description Score range Color Desirable: Green to yellowish-green 9‒12 Acceptable: Yellow to brownish 5‒8 Undesirable: Deep brown or black indicating excessive heating or putrefaction 0‒4 Odor Desirable: Light, pleasant odor with no indication of putrefaction 24‒28 Acceptable: Fruity, yeasty, musty which indicate a slightly improper fermentation; slight burnt 11‒23 odor, sharp vinegar odor Undesirable: strong burnt odor indicating excessive heating; putrid, indicating improper 0‒10 fermentation Moistness No free water when squeezed in hand; well preserved 9‒10 Some moisture can be squeezed from silage or silage dry or musty 5‒8 Silage wet, slimy or soggy, water easily squeezed from sample; silage too dry with a strong burnt 0‒4 odor Moldiness No mold 9‒10 Slightly moldy 5‒8 Highly moldy 0‒4 (Received for publication 18 September 2018; accepted 6 April 2020; published 31 May 2020) © 2020 Tropical Grasslands-Forrajes Tropicales is an open-access journal published by International Center for Tropical Agriculture (CIAT), in association with Chinese Academy of Tropical Agricultural Sciences (CATAS). This work is licensed under the Creative Commons Attribution 4.0 International (CC BY 4.0) license. Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) Tropical Grasslands-Forrajes Tropicales (2020) Vol. 8(2):125–132 125 doi: 10.17138/TGFT(8)125-132 Research Paper Effect of seed storage on seed germination and seedling quality of Festulolium in comparison with related forage grasses Efecto del almacenamiento de la semilla de Festulolium y especies relacionadas en su germinación y la calidad de plántulas RADE STANISAVLJEVIĆ1, DOBRIVOJ POŠTIĆ1, RATIBOR ŠTRBANOVIĆ1, MARIJENKA TABAKOVIĆ2, SNEŽANA JOVANOVIĆ2, JASMINA MILENKOVIĆ3, DRAGOSLAV ĐOKIĆ3 AND DRAGAN TERZIĆ3 1Institute for Plant Protection and Environment (IZBIS), Belgrade, Serbia. izbis.com 2Maize Research Institute, Belgrade, Serbia. mrizp.rs 3Institute of Forage Crops (IKBKS), Krusevac, Serbia. ikbks.com Abstract Tests of seed germination, seed dormancy and seedling growth were performed on 0-, 6-, 20- and 30-months-old seed lots of Festulolium in comparison with Italian ryegrass (Lolium multiflorum) and meadow fescue (Festuca pratensis). Tests were performed on seeds harvested in 2 different years (2014 and 2015) resulting in no major difference between the years. Seed storage affected seed viability and dormancy and seedling growth in all 3 grasses. The maximum germination of Festulolium seeds was achieved 6 months after harvest (95% normal seedlings); germination decreased significantly thereafter. While maximum germination of L. multiflorum and F. pratensis seeds was also achieved following storage for 6 months, these germination rates (93 and 90%, respectively) were retained until at least 20 months in storage. After storage for 30 months, seed germination of Festulolium, L. multiflorum and F. pratensis had declined to 72, 79 and 83%, respectively. High germination in all species was associated with higher rates of seedling growth. In an artificial seed ageing test, a temperature of 41 °C (during 48 and 72 hours) was found to effectively rank seed lots for germination performance in all 3 grasses. This test seems to have application for use in the seed trade to identify seed lots which could deteriorate more rapidly in storage. Further studies are needed to verify this hypothesis. Keywords: Ageing of seed, ageing test, dormancy, embryonic stem and radicle, forage grasses. Resumen Se realizaron pruebas de germinación y de crecimiento de plántulas provenientes de lotes de semillas de Festulolium almacenadas durante 0, 6, 20 y 30 meses, en comparación con raigrás italiano (Lolium multiflorum) y festuca de pradera (Festuca pratensis). En lotes de semillas cosechadas en 2014 y 2015 no se encontraron diferencias entre los años. El almacenamiento afectó la viabilidad y la latencia de las semillas y el crecimiento de las plántulas en las tres especies. La germinación máxima de las semillas de Festulolium se presentó 6 meses después de la cosecha (95% de plántulas normales), a partir de los cuales disminuyó significativamente. También las semillas de L. multiflorum y F. pratensis presentaron máxima germinación después de 6 meses (93 y 90%, respectivamente); estas tasas, sin embargo, se mantuvieron hasta al menos 20 meses de almacenamiento. Después de 30 meses, la germinación de las semillas disminuyó a 72, 79 y 83% para Festulolium, raigrás y festuca, respectivamente. La alta germinación en todas las especies se asoció con mayores tasas de crecimiento de plántulas. En una prueba rápida de envejecimiento artificial de semillas (temperatura de 41 °C durante 48 y 72 horas) fue posible predecir el comportamiento de germinación de las semillas de las tres especies. Esta prueba parece tener aplicación en el comercio para identificar lotes de semillas que podrían deteriorarse más rápidamente durante el almacenamiento. Se necesitan más estudios para verificar esta hipótesis. Palabras clave: Dormancia, envejecimiento de semilla, gramíneas forrajeras, prueba de envejecimiento, radícula y tallo embrionarios. ___________ Correspondence: Rade Stanisavljević, Institute for Plant Protection and Environment, Teodora Drajzera 9, 11000 Belgrade, Serbia. E-mail: stanisavljevicrade@gmail.com Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) 126 R. Stanisavljević, D. Poštić, R. Štrbanović, M. Tabaković, S. Jovanović, J. Milenković, D. Đokić and D. Terzić Introduction establishment of Festulolium in combination with legumes or as a monocrop. Moreover, seed companies Lolium multiflorum, L. perenne, Festuca pratensis and and seed trade companies are concerned about F. arundinacea are widely cultivated forage grasses and have maintenance of seed quality during storage. Literature been studied from the aspects of genetics and breeding, seed searches revealed no studies on changes in Festulolium production, growing practices and utilization. Work on seed quality during the storage period (seed ageing). interspecies hybridization between Lolium and Festuca was In the absence of previous research, studies reported conducted in the 1970s, and a novel hybrid named in this paper were conducted to observe changes in seed Festulolium was recorded in Europe after 2010 (Østrem et al. germination, dormancy and seedling vigor in 2013). According to Humphreys et al. (2013; 2014), the Festulolium, L. multiflorum and F. pratensis over 30 hybrid grass Festulolium was a result of crosses of months of storage. F. pratensis or F. arundinacea with L. perenne or L. multiflorum. As stated by Akgun et al. (2008), Festulolium Materials and Methods is characterized by higher yields of green fodder and dry matter than those of L. multiflorum and F. pratensis, Seeds were sampled from seed crops grown near the city improved resistance to environmental stresses (Abdelhalim of Smederevo, Serbia (44°40'‒44°66' N, 20°56'‒20°93' E; et al. 2016) and improved fodder quality (Skládanka et al. 66‒98 masl). Three seed production lots (I, II and III) 2010). In addition, it displays a different pattern of plant were used for each species tested. All seed lots were growth in the autumn-early winter period; e.g. × Festulolium harvested with a small combine in June 2014 and in June braunii expands its root apical meristem (RAM) and 2015. After harvest, the cleaned seeds were dried to continues to grow, while cellular growth in the RAM of moisture content below 14% and stored under ambient F. pratensis declines and reduces vegetative growth conditions for 30 months (Table 1). Seeds of the (Pašakinskienė and Švėgždienė 2018). Furthermore, seed following species were collected: ‘Perun’ Festulolium yields recorded for Festulolium have been superior to those (Festulolium), ‘Kruševački 21’ meadow fescue (Festuca of L. multiflorum and F. pratensis (Akgun et al. 2008). pratensis) and ‘Kruševački 13’ Italian ryegrass (Lolium Optimum growing systems have been developed for the multiflorum). Storage conditions during the investigation cultivation of Festulolium for seed (Deleuran et al. 2010). period are shown in Table 1. While Festulolium, Festuca arundinacea and L. perenne Kruševački 21 was produced by breeding genotypes are considered temperate grasses, in a subtropical climate originating from indigenous populations of eastern Serbia (central Mexico) analyses of forage quality and milk and Resava. Plant height is about 105 cm and tillering is production in feeding studies with dairy cows showed these strong, while resistance to rust disease (Puccinia sp.) is species compared favorably with kikuyu grass (Cenchrus enhanced. The semi-erect shoots are characterized by a clandestinus syn. Pennisetum clandestinum) (Plata-Reyes et clear pattern of light green leaves and a dark green fine al. 2018). Likewise Mwendia et al. (2019) concluded that stem. The seed is medium-sized and uniform, with 1,000- perennial ryegrass and festulolium have the potential to seed weight of approximately 1.96 g. Under conditions of contribute to improving the forage resource base in the continuous cropping, it produces 9‒10 t DM/ha with highlands of Central Kenya and similar areas. 160 g CP/kg at a sowing rate of 20‒25 kg/ha. It is recom- Fodder manufacturers and seed companies have mended for all types of long-lasting grass and clover- enquired about the relationship between seed quality and grass mowing mixtures. Table 1. Average monthly temperatures (°C) and relative humidity (%) during storage of up to 30 months of Festulolium, Festuca pratensis and Lolium multiflorum seeds harvested in June 2014 (period of seed storage: June 2014‒November 2016) and June 2015 (period of seed storage: June 2015‒November 2017). Year of storage Variable Jan Feb Mar April May June July Aug Sep Oct Nov Dec 2014 T 20.6 22.4 22.2 12.4 11.0 6.0 3.0 RH 64.0 64.3 68.5 74.5 76.4 77.7 79.5 2015 T 2.1 6.2 9.2 11.3 13.8 20.5 23.1 24.1 13.0 11.2 6.2 3.2 RH 81.0 79.2 78.5 66.1 65.6 63.2 64.8 68.6 74.7 76.7 77.4 79.6 2016 T 2.3 6.3 9.3 11.4 13.7 20.5 22.7 23.6 12.7 11.1 6.0 3.3 RH 81.8 79.4 78.6 66.3 65.9 63.6 64.7 68.5 74.6 76.6 77.5 79.6 2017 T 2.3 6.3 9.3 11.4 13.8 20.4 22.5 23.1 12.5 11.2 6.2 RH 81.5 79.3 78.5 66.3 65.8 63.7 64.5 68.3 74.4 76.8 77.3 T - Temperature; RH - Relative humidity. Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) Seed and seedling quality of Festulolium and related species 127 Kruševački 13 was developed by breeding and selection top of a box screen (11 × 11 × 3.5 cm) containing 40 mL of genotypes of introduced populations. The height of of distilled water, which provided a relative humidity of regenerative shoots in the first cutting reaches 1 m, while 98–100%. Grid boxes were used for this test to keep the vegetative shoots are about 80 cm tall. The shoots are seeds moist but not submerged in water. The boxes were covered with many broad, distinctly glossy leaves of clear placed in a water bath at 41 °C for 48 and 72 h. Seed green color. The plants tiller strongly and produce medium moisture contents before the test and after 48 and 72 h were tender, greyish green stems. The spikes are medium in length determined with values of 12.0‒12.7% before placing in the with 6‒8 spikelets, while seeds are easily shed and have a water bath and 36.6‒37.5% after being in the water bath, 1,000-seed weight of 1.99 g. The crop is high-yielding and which is in accordance with ISTA recommendations under favorable conditions can produce in excess of 14‒15 t (Hampton and TeKrony 1995). This test was performed hay/ha with 16% CP. It is an excellent variety for sowing in according to the method applied for the evaluation of seed mixtures with red clover for 3-year leys, is very productive lots of L. multiflorum (Tunes et al. 2011) and Festuca under irrigation and is also suitable for early spring and late pratensis (Stanisavljević et al. 2013). We were unable to autumn utilization. locate any data in the available literature regarding the Seed testing was performed with 2 experiments: application of the ageing test to Festulolium seeds – data are Experiment 1: testing of germination and seedling growth lacking on appropriate temperature and duration of seed (embryonic stem, radicle, seedling weight); and Experiment subjection and seed moisture prior to testing. Following 2: seed ageing test. Both experiments were performed on exposure to this temperature (41 °C) and duration (48 and 72 each seed lot (I, II and III) according to standard h), a germination test was performed as described above. methodology. Given that there were no significant differences in germination and seedling growth between Statistical analysis seed lots, results for individual seed lots are not shown. However, in the ageing test, differences in germination The experimental data were analyzed using 3-way factorial between seed lots were significant. This is significant in analysis of variance (ANOVA). Tukey’s multiple range practice when deciding which seed lot can stay longer in a F test and coefficient of variation were used to test for the warehouse, and which needs to be packaged for market effects of the treatments. Standard error of the mean was earlier. calculated to indicate variation around the mean. Data for germination and dormancy percentages were arcsine Experiment 1: Seed germination and dormancy; seedling transformed [sqr(x/100)] before being subjected to analysis growth of variance. The program Minitab, version 16.1.0 (Minitab Inc., State College, PA, USA) was used to process data (free Germination tests were performed after 0, 6, 18 and 30 version). months of storage. After being chilled at 5 °C for 5 days, seeds were sown in boxes filled with sand (20 × 14 × 4 cm). Results The boxes were then placed into germination cabinets (temperature 20/30 °C; 8 h light at 1,520 lux and 16 h dark; Since there were no significant differences for seed 50 seeds for each of 3 replications). Multiple incubators were germination and seedling growth between the seed lots employed and species were uniformly distributed. Seed tested or for the harvest years 2014 and 2015, data for both germination and dormancy were determined on day 14 after years were combined and means for each species and sowing. Seed was considered to have germinated when a treatment are presented (Tables 2 and 3). Seed age (length of radicle and embryonic stem up to 1 cm had developed. The time in storage) was the only parameter which affected tetrazolium test was applied to distinguish dormant seeds germination percentages of seed of the 3 species and from dead seeds (ISTA 2019) after 14 days. In addition, development of seedlings. Seed age significantly affected primary root length, shoot length and fresh seedling biomass germination and initial growth of seedlings for Festulolium (root + shoot) were measured after the final count. Seedling (P≤0.001), plus L. multiflorum and F. pratensis (P≤0.01 or length was measured using a ruler (Stanisavljević et al. P≤0.05) (Table 4). 2011). Experiment 1 (seed germination and dormancy; seedling Experiment 2: Ageing test growth) A seed ageing test was applied to a subsample of 6-months- Seed germination and dormancy. Immediately after seed old seeds for each species and each seed lot using 4 drying (0 months), germination of Festulolium seeds was replications of 100 seeds that were evenly arranged on the higher by 7 and 4% than that of F. pratensis and Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) 128 R. Stanisavljević, D. Poštić, R. Štrbanović, M. Tabaković, S. Jovanović, J. Milenković, D. Đokić and D. Terzić L. multiflorum seeds, respectively (Table 2). During the first Seedling growth. There were differences between species in 6 months of storage, germination of seeds of all species growth of embryonic stems and radicles, which were increased, reaching peak levels of 95, 93 and 90% for reflected in seedling weights (Table 3). These followed Festulolium, L. multiflorum and F. pratensis, respectively, similar patterns with Festulolium > L. multiflorum > while numbers of dormant seeds declined. Subsequently, F. pratensis for seeds up to 20 months of age and species germination of Festulolium declined, while those of differences generally disappeared for 30-months-old seeds. L. multiflorum and F. pratensis remained constant until 20 For all parameters peak performance was reached after 6 months of storage. By 30 months of storage germination months storage and this level was maintained at 20 months. percentages had declined to 72, 79 and 83% for Festulolium, After 30 months storage, stem and radicle growth and L. multiflorum and F. pratensis, respectively (P<0.05) and seedling weight of Festulolium and L. multiflorum had there were no dormant seeds. declined (P<0.05) but not for F. pratensis (P>0.05) (Table 3). Table 2. Effect of storage time (seed age) on germination and dormancy (± s.e.m.) of seeds of Festulolium, Lolium multiflorum and Festuca pratensis. Species Seed age 0 months Seed age 6 months Seed age 20 months Seed age 30 months Seed germination (%) Festulolium 88 ± 0.69aB 95 ± 0.91aA 89 ± 0.71bB 72 ± 0.66cC Lolium multiflorum 84 ± 0.75bB 93 ± 0.88abA 90 ± 0.70abA 79 ± 0.70bC Festuca pratensis 81 ± 0.79cC 90 ± 0.77bA 92 ± 0.59aA 83 ± 0.58aB Mean 84 93 90 78 CV% 4.16 2.72 1.69 7.14 Dormant seeds (%) Festulolium 10 ± 0.33cA 3 ± 0.21cB 0 ± 0.00cC 0 ± 0.00aC L. multiflorum 13 ± 0.41bA 6 ± 0.35bB 5 ± 0.71bB 0 ± 0.00aC F. pratensis 17 ± 0.42aA 9 ± 0.33aB 7 ± 0.71aB 0 ± 0.00aC Mean 13 6 4 0 CV% 26.3 44.4 90.1 – Within parameters, means within rows with different upper-case letters and means within columns with different lower-case letters are significantly different at P<0.05 by Tukey’s test. Table 3. Effect of storage time (seed age) on growth of embryonic stems (cm), radicles (cm) and seedling weight (mg) of seeds of Festulolium, Lolium multiflorum and Festuca pratensis. Trait Seed age 0 months Seed age 6 months Seed age 20 months Seed age 30 months Embryonic stem Festulolium 6.26 ± 0.43aB 7.79 ± 0.54aA 7.68 ± 0.21aA 5.07 ± 0.36aC L. multiflorum 4.49 ± 0.39bB 5.56 ± 0.39bA 5.51 ± 0.34abA 4.35 ± 0.29aB F. pratensis 3.45 ± 0.33cB 4.45 ± 0.30cA 4.99 ± 0.53bA 4.29 ± 0.39aA Mean 4.73 5.93 6.06 4.57 CV% 30.0 28.7 23.5 9.5 Radicle Festulolium 4.05 ± 0.29aB 4.98 ± 0.53aA 4.88 ± 0.36aA 3.75 ± 0.34aB L. multiflorum 3.42 ± 0.34abB 4.12 ± 0.29bA 4.10 ± 0.71bA 3.46 ± 0.51aB F. pratensis 2.85 ± 0.41bB 3.22 ± 0.61cA 3.39 ± 0.63cA 3.21 ± 0.63aA Mean 3.44 4.11 4.12 3.47 CV% 17.4 21.4 18.1 7.8 Seedling weight Festulolium 14.9 ± 0.53aB 17.5 ± 0.61aA 17.2 ± 0.29aA 13.9 ± 0.53aC L. multiflorum 12.1 ± 0.61bB 14.6 ± 0.39bA 14.0 ± 0.37bA 12.4 ± 0.61bB F. pratensis 10.9 ± 0.48cB 12.3 ± 0.35cA 12.1 ± 0.31cA 11.8 ± 0.35bA Mean 12.6 14.8 14.4 12.7 CV% 16.2 17.6 17.9 8.5 Within parameters, means within rows with different upper-case letters and means within columns with different lower-case letters are significantly different at P<0.05 by Tukey’s test. Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) Seed and seedling quality of Festulolium and related species 129 Experiment 2 (ageing test) under ambient conditions was an important issue. Seed dormancy immediately after harvest or dispersal under Application of the seed ageing test to the 6-months-old natural conditions is a biological trait that prevents seeds showed differences in seed germination (%) germination of some seeds as a survival mechanism in the between seed lots for all 3 species tested. Whereas the event that seedlings cannot cope with environmental classical germination test did not show a significant conditions following germination (Bewley 1997). This difference (F test) between the seed lots of any of the trait is influenced by multiple factors including genetics, tested species, the aging test was able to detect differences physiology, biochemistry and histology (Graeber et al. between seed lots in germination of all tested species 2012; Long et al. 2015; Sah et al. 2016). (Table 4). The test also showed a faster decline in Dormant seeds of plants can reduce germination germination percentage (average for all 3 seed lots) of percentage at any one time (Stanisavljević et al. 2014). Festulolium seed (from 55% after 48 h to 17% after 72 h) For farming systems, where synchronized germination is than of L. multiflorum seed (from 76 to 53%) and desired, farmers sometimes compensate for dormancy by F. pratensis seed (from 83 to 73%) (Table 4). increasing sowing rates (an additional cost) to achieve optimum populations for high yields. An alternative Table 4. Germination percentages (± s.e.m.) of 6-months-old approach is to treat seed prior to sowing, e.g. by seed lots of Festulolium, Lolium multiflorum and Festuca scarification or heat treatment, to break dormancy and pratensis after an ageing test by placing in a water bath at 41 °C ensure more uniform germination. This is particularly for 48 and 72 hours. important for species with high percentages of dormant seeds at harvest, e.g. legume seeds. Species Seed Test duration While seedlings developed from dormant seeds would lot 48 h 72 h normally be healthy, the delay in their emergence and Festulolium I 52 ± 0.45b 18 ± 0.27b development can reduce their capacity to compete with II 68 ± 0.39a 21 ± 0.29a already developed seedlings (seedlings developed from III 45 ± 0.37c 13 ± 0.51c immediately germinated seeds) (Bass et al. 1988). Mean 55 17 According to Adkins et al. (2002), seed dormancy is L. multiflorum I 77 ± 0.29b 53 ± 0.32b II 81 ± 0.63a 59 ± 0.39a common in bred forage grasses, especially soon after III 71 ± 0.43c 48 ± 0.77c harvest, unlike many cereal crops, where breeding has Mean 76 53 been used to reduce seed dormancy. When studying F. pratensis I 86 ± 0.37a 79 ± 0.28a dormancy in F. pratensis, Stanisavljević et al. (2012) II 79 ± 0.44 b 66 ± 0.36c found low variability (CV = 3.6%) among 3 varieties and III 83 ± 0.51ab 74 ± 0.41b 3 populations immediately after harvest but further Mean 83 73 studies showed that, depending on seed moisture content Values within columns and species with different letters are at harvest, seed dormancy varied from 33 to 18% significantly different (P<0.05) by Tukey’s multiple range test. (Stanisavljević et al. 2013). Dormancy percentages in our current study following harvest ranged from 10% Discussion (Festulolium) to 17% (F. pratensis). After the maturation period, the dormant seeds become Experiment 1 germinable, total germination increases and at that stage the utilizable value of seeds is at its peak, i.e. the desired Observed seed lots were from regions geographically number of plants can be obtained from sowing lower close and harvested in a similar way, which may be a amounts of seeds – the most economic establishment of reason for their uniformity in terms of tested traits of seed crops. According to Stanisavljević et al. (2011), a storage and seedling quality. period of 500 days under uncontrolled temperature and While germination rates of seed of all species was humidity conditions is necessary for L. multiflorum seeds quite acceptable following harvest, the declining to mature and achieve maximum germination. The current germination after storage is potentially an issue for the study suggested that storage for 180 days was sufficient seed trade, because the minimum legislated germinations to reach maximum germination percentage. Being able to of F. pratensis and L. multiflorum seeds at sale are 75 and market and plant seed at 180 days following harvest rather 70%, respectively. Following 30 months of storage, than 500 days after harvest is much more cost-efficient. germination rates for Festulolium and L. multiflorum While germination of Festulolium declined from this seeds were 72 and 79%, respectively, so seed ageing time, L. multiflorum (92–90%) and F. pratensis (90–92%) Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) 130 R. Stanisavljević, D. Poštić, R. Štrbanović, M. Tabaković, S. Jovanović, J. Milenković, D. Đokić and D. Terzić remained at peak levels until 20 months of storage (Table rapid test which provided us with information about how 2), showing a small advantage for these species. seed lots might perform after lengthy storage and A significant advantage of sowing non-dormant seeds estimates of the deadlines for use of certain seed lots to is faster and more uniform initial growth of the prevent serious deterioration in germination percentage. established crop. In our studies, an additional outcome Prolonged exposure to adverse conditions during the with high germination was stronger growth of the ageing test inevitably leads to loss of quality and reduced embryonic stem and radicle, as well as increased weight germination (Marcos Filho 2015). In our experiment, by of seedlings (Table 3). These factors are very important applying the 2 seed ageing test durations, germination of because mixtures of forage grasses are commonly grown Festulolium decreased from 42 to 72 h test duration by (Wyszkowska et al. 2019), sometimes with forage 38%, L. multiflorum by 23%, and F. pratensis by 10% legumes as a mixed crop (Neuberg et al. 2011). (Table 4). Tunes et al. (2011) reported that a seed ageing As demonstrated in this study, the stage of maximum test performed on 4 seed lots of L. multiflorum with an germination is followed by ageing of the seed, which increased duration of 72 vs. 48 h reduced germination by involves irreversible changes to seeds, ultimately an average of 38%. The recommended temperatures for resulting in decreased capacity to germinate and grow as the ageing test and the length of seed exposure vary by a seedling. In these studies, the most pronounced ageing forage species: for Brachiaria brizantha, Hernández et al. was observed in Festulolium (germination was 72% after (2017) recommended 40 ºC during 48 or 60 h. For 30 months). Long-term storage of this species is not L. multiflorum, the recommended temperature is 41 ºC recommended. and exposure time 24, 48 or 72 h, depending on whether Seed ageing is specific for each plant species (Nagel a traditional test or an alternative test with NaCl is and Borner 2010). The consequence of seed ageing is not performed (Tunes et al. 2011). In F. pratensis the just a germination reduction but also a reduction in the recommended temperature is 41 °C and exposure time 48 initial growth of the radicle, embryonic stem and seedling or 72 h (Stanisavljević et al. 2013). weight (Rajjou et al. 2008; Stanisavljevic et al. 2011). A seed ageing test measures the ability of a seed lot to resist Conclusions degradation changes and protection mechanisms that are present in the seed (Balešević-Tubić and Tatić 2012). i) According to our results Festulolium seeds stored under According to Hare et al. (2018) germination percentage ambient conditions with seeds of Lolium multiflorum and under laboratory conditions declined to below 50% after Festuca pratensis generally had lower seed dormancy and 3 years of storage of seed for 2 guinea grasses a shorter period was necessary for post-harvest (Megathyrsus maximus syn. Panicum maximum) cvv. maturation, release from dormancy and for achieving Mombasa and Tanzania, 4 years for Ubon paspalum maximum germination. However, Festulolium seeds were (Paspalum atratum) and 4‒5 years for Urochloa syn. less resistant to the ageing process, and germination Brachiaria hybrid cv. Mulato II seed. percentage deteriorated more rapidly with time in storage than for seeds of L. multiflorum and F. pratensis. Whether Experiment 2 this pattern would be changed with control of storage conditions remains unanswered. The difference between seed germination in Experiment ii) High germination performance was accompanied 1 and germination in the seed ageing test is noteworthy as by vigorous growth of seedlings of all species, providing are the differences between the 3 seed lots in the seed an added benefit with good quality seed. ageing test. This should be considered as evidence of the iii) The seed ageing test proved a simple and rapid test sensitivity of the ageing test. According to Marcos Filho to provide estimates of how germination of seed samples (2015), the seed ageing test is one of the most applicable would change over extended periods of storage. More tests for determining how well seeds survive under widespread use of this simple test commercially should storage. Its application in forage seed testing is less identify seed lots which could deteriorate rapidly in significant than in vegetable seeds (Wang et al. 2004). storage. Further testing is needed to verify this hypothesis. The reason for this is probably that expensive hybrid seeds and individual seed sowing are used in the Acknowledgments production of vegetable and some field crops. It is important to ensure that expensive seeds do not decay We thank the Ministry of Education, Science and before sale, so this quick test is a good way to evaluate Technological Development, Republic of Serbia, for longevity. In our case, the ageing test proved useful as a financial support. 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This work is licensed under the Creative Commons Attribution 4.0 International (CC BY 4.0) license. Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) Tropical Grasslands-Forrajes Tropicales (2020) Vol. 8(2):133–140 133 doi: 10.17138/TGFT(8)133-140 Research Paper Selection based on meiotic behavior in Urochloa decumbens hybrids from non-shattered seed Selección con base en el comportamiento meiótico de híbridos procedentes de semillas no dehiscentes de Urochloa decumbens JOANA NERES DA CRUZ BALDISSERA1, ANDRÉA BEATRIZ DIVERIO MENDES2, MARLON MATHIAS DACAL COAN2, CLAUDETE APARECIDA MANGOLIN2, CACILDA BORGES DO VALLE3 AND MARIA SUELY PAGLIARINI2 1Colegiado de Biologia, Instituto Federal do Paraná, Palmas, PR, Brazil. palmas.ifpr.edu.br 2Departamento de Agronomia, Universidade Estadual de Maringá, Maringá, PR, Brazil. dag.uem.br 3Empresa Brasileira de Pesquisa Agropecuária, Embrapa Gado de Corte, Campo Grande, MS, Brazil. cnpgc.embrapa.br Abstract This study aimed to evaluate the end-products of meiosis in sexual and apomictic hybrids of Urochloa decumbens, so as to identify genotypes with good production of viable pollen for use in breeding programs to increase yields of pure viable seed and reduce degree of seed shattering. From 457 intraspecific hybrids of U. decumbens arising from crosses between 3 artificially tetraploidized sexual plants and the apomictic cultivar Basilisk, 27 hybrids from non-shattered seed were selected. Slides were prepared by smearing anthers and staining to determine the presence of abnormalities. The abnormalities found were micronuclei, microcytes and polyads. The data were compared by the Scott-Knott test at P<0.05. Data obtained enabled separation of hybrids into 4 groups depending on the presence of micronuclei and formation of polyads and into 6 groups based on the presence of microcytes in the tetrads. Among the analyzed apomictic hybrids, R179 has the attributes for viable seed production to proceed to cultivar development. Among the sexual hybrids, R161, R181, R193 and S47 are recommended as female parents for use in crossing programs. Keywords: Abnormalities, breeding, cytogenetics, forages, intraspecific crosses. Resumen El estudio tuvo como objetivo evaluar los productos finales de la meiosis en híbridos sexuales y apomícticos de Urochloa decumbens, para identificar genotipos con buena producción de polen viable que puedan ser usados en un programa de mejoramiento genético y aumentar así los rendimientos de semilla pura viable y reducir su grado de dehiscencia. De un total de 457 híbridos intraespecíficos de U. decumbens que resultaron de cruzamientos entre tres plantas sexuales tetraploidizadas artificialmente y el cultivar apomíctico ‘Basilisk’, se seleccionaron 27 híbridos procedentes de semillas no desprendidas. Para el efecto se prepararon portaobjetos con anteras que fueron teñidas para determinar la presencia de anomalías. Las anomalías encontradas fueron micronúcleos, microcitos y políadas. Los datos se compararon mediante la prueba de Scott-Knott (P<0.05). Los resultados permitieron separar los híbridos en cuatro grupos dependiendo de la presencia de micronúcleos y la formación de políadas, y en seis grupos basados en la presencia de microcitos en las tétradas. El híbrido R179, entre los híbridos apomícticos analizados, presentó los atributos necesarios para el desarrollo de cultivares con potencial de producción de semillas viables. Entre los híbridos sexuales, se recomiendan R161, R181, R193 y S47 como progenitores femeninos en programas de cruzamiento. Palabras clave: Anomalías, citogenética, cruces intraespecificos, fitomejoramiento, forrajes tropicales. ___________ Correspondence: Joana Neres da Cruz Baldissera, Colegiado de Biologia, Instituto Federal do Paraná, Campus Palmas, Av. Bento Munhoz da Rocha Neto s/nº - PRT-280, Trevo da Codapar, Palmas, CEP 85555-000, PR, Brazil. Email: jondcb@gmail.com Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) 134 J.N.C. Baldissera, A.B.D. Mendes, M.M.D. Coan, C.A. Mangolin, C.B. do Valle and M.S. Pagliarini Introduction important to ensure the formation of viable seeds. Furthermore, hybridization is successful only if crossings Brazil has 221.8 million head of cattle and produced 9.71 are performed between parents with the same ploidy level, million tonnes of meat, worth R$ 523.25 billion in 2017 using a sexual genotype as a mother-plant and the (ABIEC 2018), making the country one of the main apomictic or another sexual as pollen donor(s) (Barrios et producers of beef in the world. This is a result of the al. 2013; Alves et al. 2014). adoption of new technologies relating to genetics and the Considering the importance of selecting genotypes management and feeding of beef herds (Gomes et al. that combine high levels of seed retention and 2017). quantity/quality of viable seeds, the present study had as Approximately 95% of the animals are raised on its objective evaluation of the final products of meiosis pasture (Araújo et al. 2017), so high quality forages and and pollen viability in hybrids of U. decumbens, in order improvement of existing pastures are a prerequisite to to identify stable genotypes and good pollen producers for efficient livestock production (Ribeiro-Junior et al. 2017). use in the Urochloa breeding program at Embrapa Beef In addition, Brazil occupies a prominent position in the Cattle Research Center (Embrapa Gado de Corte). world as a producer and exporter of tropical forage seed (Pereira et al. 2011). In 2015, 50 thousand tonnes of Materials and Methods certified seed was produced, with 75% destined for the domestic market and 25% for export (Rodrigues 2017). A base population of 457 intraspecific hybrids of Brachiaria (now: Urochloa) breeding in Brazil began U. decumbens was produced from crosses between 3 when CIAT and EMBRAPA achieved compatibility artificially tetraploidized sexual plants and the apomictic between species with different ploidy levels in the late cultivar Basilisk. This population is maintained in an 1980s (Triviño et al. 2017). While a number of cultivars experimental field at Embrapa Beef Cattle Center, in have been released, most have some limitations, so further Campo Grande, Mato Grosso do Sul (20°25'03" S, genetic improvement is warranted. Low seed yields and 54°42'20" W) in an allic Red Latosol type soil. From that seed quality are significant issues for cultivars which have population 27 hybrids from non-shattered seed were been released most recently, especially the hybrids. selected (Table 1) and were cytogenetically analyzed in If a cultivar is to be adopted widely and have a the Laboratory of Cytogenetics from the Universidade significant impact on animal production, adequate Estadual de Maringá, Paraná. supplies of good quality seed are essential (Valle et al. The inflorescences of the hybrids were collected and 2008). One factor affecting seed quality is the occurrence fixed in a mixture of ethanol:chloroform:propionic acid of natural shattering, i.e. seeds detaching from the raceme (6:3:2) for 24 hours and stored in 70% alcohol at 4 °C. on reaching maturity. Seed must be retrieved from the Three anthers from the same hermaphrodite flower, ground, resulting in some being lost plus contamination chosen randomly on the raceme, were used per slide, for by pathogens and impurities. Development of genotypes the analysis of the final products of meiosis and pollen resistant to seed shattering would lead to increases in both viability. Each slide was considered a replication, with seed yields and quality. 5 replications per hybrid. One hundred cells were counted Another factor affecting seed quality and quantity is per slide. polyploidy because, when there is more than a Tetrads of microspores and pollen viability were chromosomal set in a cell, the organization of the same evaluated after squashing, then staining with 1% can be difficult at the time of pairing and segregation propionic carmine and analyzing under light microscopy. causing meiotic abnormalities. In the genus Brachiaria The pollen grains were classified into 2 groups: 1) viable (now: Urochloa), most ecotypes studied are polyploids pollen grains, with the exine intact and the protoplasm (Valle and Savidan 1996; Utsunomiya et al. 2005) and the well stained; and 2) unviable pollen, with weak staining vast majority reproduce by apomixis (Valle and Savidan or shriveled and not stained. 1996; Fuzinatto et al. 2007, 2008; Mendes-Bonato et al. The data on anomalous tetrads of microspores and 2007). Adamowski et al. (2008) revealed many important non-viable pollen grains obtained in percentage (%) were meiotic and post-meiotic abnormalities that compromise, transformed into arcsine function using the square root sometimes seriously, the end-product of meiosis, causing (√x) of the proportion of abnormal tetrads. Data were then pollen sterility. Apomixis in Urochloa is a pseudogamous submitted to analysis of variance using the SAS 9.2 apospory, where, despite the fact that the egg cell program (SAS Institute 2009). The mean percentages circumvents fertilization, the central cell requires it for were compared using the Scott-Knott test at the 5% endosperm formation. Therefore quality of pollen is very probability level using GENES (Cruz 2001). Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) Selection based on meiotic behavior of U. decumbens hybrids 135 Table 1. Hybrids of Urochloa decumbens analyzed. effect was significant by the F-test with 5% probability of error; therefore, there are differences between hybrids in Hybrid Reproduction Female Male the frequencies of chromosomal irregularities in the mode parent parent tetrads of microspores (Table 2). For the variables related R158 Apomictic D24/27 cv. Basilisk to abnormalities in tetrads, the estimated coefficient of R168 Apomictic D24/27 cv. Basilisk variation (CV) was high for the presence of micronucleus R169 Apomictic D24/27 cv. Basilisk in 1 microspore (38.3%), microcytes (41.9%) and polyads R176 Apomictic D24/27 cv. Basilisk (86.7%). These high values for CV can be explained by R177 Apomictic D24/27 cv. Basilisk R179 Apomictic D24/27 cv. Basilisk differences in the numbers of cells found with these R184 Apomictic D24/27 cv. Basilisk abnormalities in each slide (replication). R187 Apomictic D24/27 cv. Basilisk Based on the Scott-Knott test it was possible to R189 Apomictic D24/27 cv. Basilisk separate the hybrids into 4 groups (A, B, C and D) S48 Apomictic D24/27 cv. Basilisk concerning the presence of micronuclei in 1, 2 and 3 T87 Apomictic D24/27 cv. Basilisk microspores and 3 groups concerning the presence in 4 X113 Apomictic D24/45 cv. Basilisk microspores. The groups differ on the basis of minimum Y22 Apomictic D24/45 cv. Basilisk significant difference while the hybrids within the groups Y23 Apomictic D24/45 cv. Basilisk are similar. Z8 Apomictic D24/45 cv. Basilisk R 161 Sexual D24/27 cv. Basilisk Table 2. Analysis of variance of the meiotic abnormalities R163 Sexual D24/27 cv. Basilisk observed in the 27 hybrids of Urochloa decumbens. R165 Sexual D24/27 cv. Basilisk R167 Sexual D24/27 cv. Basilisk Source DF Mean Square of meiotic abnormalities R171 Sexual D24/27 cv. Basilisk 1 2 3 4 5 6 R181 Sexual D24/27 cv. Basilisk Hybrid 26 0.109* 0.123* 0.072* 0.156* 0.208* 0.73* R193 Sexual D24/27 cv. Basilisk Error 108 0.008* 0.007* 0.007* 0.018* 0.007* 0.006* S47 Sexual D24/27 cv. Basilisk Total 134 Y21 Sexual D24/45 cv. Basilisk CV% 38.3 22.4 16.5 21.6 41.9 86.7 Z9 Sexual D24/45 cv. Basilisk 1 = micronuclei in 1 microspore; 2 = micronuclei in 2 micro- X119 - D24/45 cv. Basilisk spores; 3 = micronuclei in 3 microspores; 4 = micronuclei in 4 X122 Sexual- sterile D24/45 cv. Basilisk microspores; 5 = microcyte; 6 = polyad. *Significant by the F-test (P<0.05). The most representative abnormalities in the tetrads and pollen grains were photographed under an Regarding the presence of micronuclei in just 1 OLYMPUS CX 31 capture microscope with attached SC microspore, hybrids of Group D (Table 3) presented the 30 camera, using the AnalySIS getIT software, with 400× lowest frequencies of this abnormality. However, based magnification. on the parameters established by Love (1951), this group should be considered unstable, since more than 10% of Results abnormal tetrads were detected, with the presence of micronuclei in all 4 microspores (Table 3). For the Many abnormalities were observed in the final products of presence of micronuclei in 2 microspores of the tetrad, meiosis of hybrids of U. decumbens analyzed, the main Groups C and D (Table 3) were those with fewer than ones being 1, 2, 3 and 4 micronuclei in the microspores 10% of abnormal tetrads, while also presenting high (Figures 1a‒1d), microcytes (Figures 1e‒1f) and polyads frequency of micronuclei in the tetrad. The same is true (Figures 1g‒1i). for micronuclei in 3 microspores where hybrids R187 Cytogenetic analysis revealed the presence of and R189, despite having fewer micronuclei in 3 micronuclei and microcytes in the same tetrad (Figures microspores, showed 33 and 68% of micronuclei in the 1e‒1f) and polyads with micronuclei (Figures 1g‒1i). tetrads, with high frequency of microcytes and polyads. The analyses of the tetrads of microspores from these The only hybrid that presented fewer than 10% of tetrads hybrids are presented in Table 2. with micronuclei in the 4 microspores was R181, The meiotic abnormalities in tetrads were expressed as although this hybrid did not differ statistically from percentages of abnormal cells and the significant other hybrids of Group C. differences between the irregularities of the hybrids were Hybrids have been classified into 6 groups from A to tested by the Scott-Knott test. In the analysis of variance F on the basis of the presence of microcytes in tetrads for meiotic abnormalities, the mean square for the hybrid (Table 3). Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) 136 J.N.C. Baldissera, A.B.D. Mendes, M.M.D. Coan, C.A. Mangolin, C.B. do Valle and M.S. Pagliarini Figure 1. Meiotic abnormalities observed in tetrads of microspores, due to irregular segregation of chromosomes and genome asynchrony in tetraploid hybrids of Urochloa decumbens: a) micronuclei in 1 microspore; b) micronuclei in 2 microspores; c) micronuclei in 3 microspores; d) micronuclei in 4 microspores; e-f) tetrads with micronuclei in the microspores and microcytes; and g-h-i) polyads with microspores of different sizes and with micronuclei (400× magnification). Hybrids in Groups A and B are expected to have higher lower frequencies, probably not compromising pollen frequency of unbalanced gametes and thus higher pollen fertility. infertility. According to the parameters established by Pollen viability of U. decumbens hybrids was tested Love (1951), hybrids of Groups D, E and F can be using propionic carmine at 1% (Figures 2a‒2c). Pollen considered stable cytogenetically. grains of different sizes and staining patterns were Hybrids were separated into 4 groups, from A to D, on observed in the hybrids analyzed, but in many cases it was the basis of the frequency of polyads (Table 3). Except for not possible to accurately determine whether pollen hybrid R187 with 25% of polyads, these occurred in much grains were viable or non-viable. Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) Selection based on meiotic behavior of U. decumbens hybrids 137 Table 3. Grouping of the 27 Urochloa decumbens hybrids evaluated based on similar behavior regarding mean percentages of incidence of abnormal cells observed at the end of meiosis. Micronuclei in microspores Microcytes Polyads 1 2 3 4 Hybrid % Group Hybrid % Group Hybrid % Group Hybrid % Group Hybrid % Group Hybrid % Group R181 28.0 A R193 33.8 A Y22 38.6 A R189 68.0 A S48 42.0 A R187 25.0 A R179 23.0 A R179 30.1 A X113 36.5 A R163 64.5 A R176 28.0 B X122 7.8 B R 161 19.0 A X119 28.9 A R177 32.9 A R169 56.2 A R187 33.0 B S48 6.6 B R193 19.0 A R181 26.8 A R158 30.4 A R167 53.8 A X122 23.0 B R189 5.5 B Y23 14.0 B R171 26.5 A Z9 30.0 A Y22 50.0 A R 161 15.0 C R167 2.8 C R171 11.0 B Z9 24.8 A X119 29.7 A S47 48.2 A R189 17.0 C R163 2.3 C X119 11.0 B T87 24.5 A R184 29.6 A X122 43.5 B R167 10.0 D Y23 2.0 C R165 10.0 B R 161 24.4 A Z8 28.6 A Z8 43.3 B Y21 8.6 D R169 1.3 C R168 8.3 B R158 23.8 A R171 28.0 A R184 42.0 B R163 3.3 E Y21 1.3 C R176 7.8 B Y23 21.6 A R165 27.7 A R177 39.5 B R165 1.1 E R168 1.2 C R158 7.7 B R165 21.3 A R193 27.1 A S48 37.9 B R168 3.1 E R176 0.5 D Z9 6.2 C R184 19.8 B T87 27.0 A X113 37.1 B R169 6.1 E R 161 0.2 D Z8 6.1 C X113 18.7 B S47 26.2 A Y21 37.0 B R171 1.3 E R165 0.2 D R184 5.4 C S47 18.2 B Y21 25.5 B T87 34.5 B T87 2.0 E R171 0.2 D T87 5.3 C R177 17.9 B Y23 24.6 B R187 33.3 B Y23 2.5 E R181 0.2 D Y21 4.5 C R176 16.9 B R179 24.1 B Z9 33.1 B Z8 2.0 E Z9 0.2 D R177 4.2 C Y21 16.5 B R167 22.9 B R158 30.2 B R158 0.6 F Z8 0.1 D S47 4.0 C R168 14.8 B R169 21.3 B R168 29.5 B R177 0.8 F R158 0.0 D X113 3.7 C Z8 13.8 B R176 20.9 B R165 28.1 B R179 0.1 F R177 0.0 D R167 2.6 C Y22 10.3 B X122 19.4 B R171 25.7 B R181 0.0 F R179 0.0 D R169 1.6 D R169 8.0 C R168 19.1 B X119 24.8 B R184 0.0 F R184 0.0 D R163 0.7 D R163 7.8 C R163 19.0 B Y23 24.3 B R193 0.0 F R193 0.0 D X122 0.4 D R167 6.1 C R 161 15.6 C R176 18.6 C S47 0.0 F S47 0.0 D S48 0.2 D X122 3.6 C R181 13.2 C R193 12.1 C X113 0.2 F T87 0.0 D Y22 0.2 D S48 1.6 D S48 10.2 C R179 11.7 C X119 0.5 F X113 0.0 D R187 0.0 D R187 0.3 D R189 5.1 D R 161 10.9 C Y22 0.0 F X119 0.0 D R189 0.0 D R189 0.3 D R187 1.8 D R181 7.1 C Z9 0.7 F Y22 0.0 D 1 = micronuclei in 1 microspore; 2 = micronuclei in 2 microspores; 3 = micronuclei in 3 microspores; 4 = micronuclei in 4 microspores. Grouping based on significance by the Scott-Knott test (P<0.05). Figure 2. Pollen viability of the 27 Urochloa decumbens tetraploid hybrids determined by staining with 1% propionic carmine: a) viable pollen grain strongly stained; b) non-viable pollen grains unstained; c) viable and non-viable pollen grains (400× magnifications). Discussion reported by Risso-Pascotto et al. (2004), micronuclei in 1 or more microspores are the most common cytological Micronuclei are a consequence of segregation abnormality resulting from irregular chromosome irregularities occurring in different phases of meiosis. As segregation in higher plants. When formed, the Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) 138 J.N.C. Baldissera, A.B.D. Mendes, M.M.D. Coan, C.A. Mangolin, C.B. do Valle and M.S. Pagliarini micronuclei can remain in tetrads of microspores even more promising, since the other 3 microspores of the tetrad after the dissolution of the callose wall and the release of may contain balanced genetic material. microspores impairing normal gamete formation (Valle Using this basis for selection, the best hybrids would be and Pagliarini 2009). Micronuclei can also be eliminated those with no micronuclei or a high frequency of tetrads from the tetrads as microcytes by cytokinesis. In the with micronuclei in only 1 microspore. That was not the hybrids analyzed, the elimination of micronuclei by case in the hybrids studied, where the important criteria additional cytokinesis gave rise to microcytes in tetrads were to select hybrids with fewer micronuclei throughout and polyads. and also absence of microcytes and polyads. Micronuclei in microspores of tetrads, tetrads with The formation of microcytes in tetrads and polyads is microcytes and polyads have often been reported in meiotic much more serious than the presence of micronuclei in studies of interspecific hybrids of Urochloa (Risso- microspores of the tetrad. When additional cytokinesis Pascotto et al. 2004; Mendes-Bonato et al. 2007), which, forms microcytes and polyads, all microspores are depending on the frequency of occurrence, results in the abnormal due to uneven division of the genomes. Tetrads formation of unbalanced gametes. We expected that with microcytes and polyads generate unbalanced pollen intraspecific hybridizations would produce fewer grains of different sizes. anomalies in meiosis than with interspecific hybrids, since Pollen viability is an accepted measure of male fertility chromosome sets were supposedly homologous. The and can be estimated by staining methods using mature occurrence of abnormalities in these intraspecific hybrids pollen grains. Although several authors, e.g. Ricci et al. of U. decumbens could be due to the recent artificial (2010); Simioni and Valle (2011); Souza et al. (2015), have replication of the chromosomes of their female parent. already tested pollen viability in Urochloa using this Artificial chromosome duplication using colchicine can staining method and were able to discriminate between cause loss of chromosomes or chromosomal viable and non-viable pollen grains, the method is often rearrangements such as deletions or inversions, as well as unreliable, because in addition to meiotic irregularities, sterility and abnormal growth (Luckett 1989). pollen viability can be affected by failures in the The analysis of meiotic behavior of artificially microgametogenesis process (Twel 1995), natural water tetraploidized accessions of U. decumbens, U. brizantha loss that occurs during the collection and storage of and U. ruziziensis has shown a rate of meiotic inflorescences (Tecchio et al. 2006) and the storage time of abnormalities varying from 5 to 60%, and a high rate of the inflorescences (Stanley and Linskens 1974). According abnormalities in interspecific hybrids using tetraploidized to Souza et al. (2002), pollen grain is fully viable at the parents (Fuzinatto et al. 2007; Souza et al. 2015). These opening of the flower, and as time progresses, the viability culminated in abnormal tetrads and in the formation of a decreases, reducing its efficiency. high rate of unviable pollen grains. Hybridizations performed in the Urochloa breeding Love (1951) indicated that the analysis of tetrads easily program of Embrapa Beef Cattle use sexual genotypes as proved the degree of stability of the meiotic process, since mother plants and apomictic ones as pollen donors it demonstrated the pattern of chromosome behavior during (Mendes-Bonato et al. 2004). According to Souza et al. the phases of meiosis. According to this author, a plant with (2015), sexual hybrids that have a low frequency of 90‒100% of normal tetrads is considered stable, whereas abnormalities in tetrads and good viable pollen production plants with fewer than 90% of normal meiotic products may be included in polycross blocks with other sexual limit breeding, because this hampers production of viable hybrids for the recombination of alleles or used in crosses seeds. with other elite apomictic genotypes to generate new Although Love´s meiotic index is widely used to populations from which to select future apomictic determine the meiotic stability and consequently the cultivars. Superior apomictic hybrids can be evaluated fertility of a plant, a more detailed analysis of the final agronomically to select new cultivars or can be used as products of meiosis may result in much more accurate pollen donors in new crosses. information, especially for polyploid plants, which have a Among the apomictic hybrids analyzed, R179 could be high rate of abnormalities in tetrads of microspores. This regarded as a good pollen donor, since it had a high can be explained by the fact that a tetrad with micronuclei percentage of tetrads with micronuclei in only 1 or 2 in 1 microspore can theoretically have 3 other normal microspores (Group A), and a low percentage of tetrads microspores. These hybrids would thus produce viable with micronuclei in the 4 microspores (Group C), tetrads pollen in the ratio of 3:1 (viable:unviable pollen). with microcytes (Group F) and polyads (Group D). According to Souza et al. (2015), genotypes with a high Apomictic hybrids R187, R189 and S48, however, with frequency of micronuclei in only 1 microspore would be high rates of tetrads with micronuclei in the 4 microspores, Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) Selection based on meiotic behavior of U. decumbens hybrids 139 microcytes and polyads must be discarded as parents for Técnica. Embrapa Gado de Corte, Campo Grande, MS, crossing. Among the sexual hybrids R161, R181, R193 and Brazil. bit.ly/2yKXitt S47 may be considered for crossing blocks and the next Barrios SCL; Valle CB do; Alves GF; Simeão RM; Jank L. generation evaluated to confirm potential fertility. 2013. Reciprocal recurrent selection in the breeding of Brachiaria decumbens. Tropical Grasslands-Forrajes Absence of seed shattering was a key factor in the Tropicales 1:52–54. doi: 10.17138/tgft(1)52-54 domestication of major grasses because humans could Cruz CD. 2001. Programa Genes: Aplicativo computacional em collect seed throughout the long summer season, making genética e estatística. Editora UFV, Viçosa, MG, Brazil. them preadapted candidates for domestication (Kislev et al. Fonseca DM da; Martuscello JÁ. 2011. Plantas forrageiras. 2004). The inheritance of non-shattering behavior, which Editora UFV, Viçosa, MG, Brazil. in some grasses seems to be controlled by few genes or Fuzinatto VA; Pagliarini MS; Valle CB do. 2007. Evidence of transcription factors (Konishi et al. 2006; Li et al. 2006), is programmed cell death during microsporogenesis in an an important trait to be a focus in evaluation of the interspecific Brachiaria (Poaceae: Panicoideae: Paniceae) intraspecific hybrids of U. decumbens analyzed. Given the hybrid. Genetics and Molecular Research 6:308–315. importance of this character for the improvement of this geneticsmr.com/articles/356 Fuzinatto VA; Pagliarini MS; Valle CB do. 2008. Evaluation of forage, the detailed analysis of the tetrads of microspores microsporogenesis in an interspecific Brachiaria hybrid and pollen viability is essential in selecting hybrids that (Poaceae) collected in distinct years. Genetics and Molecular could produce larger quantities of fertile seeds that could Research 7:424–432. geneticsmr.com/articles/490 be harvested conventionally. Hybrids resistant to shattering Gomes RC; Feijó GLD; Chiari L. 2017. Evolução e qualidade would improve significantly the harvesting of viable seed da pecuária brasileira. Nota Técnica. Embrapa Gado de for either the breeding program or commercial purposes. Corte, Campo Grande, MS, Brazil. bit.ly/3bKWa7z Furthermore, selection of future cultivars with better Kislev ME; Weiss E; Hartmann A. 2004. Impetus for sowing potential production of directly harvested seed should and the beginning of agriculture: Ground collecting of wild reduce cost of seed, resulting in greater adoption rates and cereals. 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CRC Press, New York, USA. p. 103–151 (Received for publication 20 June 2019; accepted 4 April 2020; published 31 May 2020) © 2020 Tropical Grasslands-Forrajes Tropicales is an open-access journal published by International Center for Tropical Agriculture (CIAT), in association with Chinese Academy of Tropical Agricultural Sciences (CATAS). This work is licensed under the Creative Commons Attribution 4.0 International (CC BY 4.0) license. Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) Tropical Grasslands-Forrajes Tropicales (2020) Vol. 8(2):141–146 141 doi: 10.17138/TGFT(8)141-146 Short Communication The effect of stage of regrowth on the physical composition and nutritive value of the various vertical strata of kikuyu (Cenchrus clandestinus) pastures Efecto de la edad de rebrote en la composición física y el valor nutritivo de los diferentes estratos verticales de pasturas de kikuyo (Cenchrus clandestinus) MARCELO A. BENVENUTTI1, CRAIG FINDSEN1, JEAN V. SAVIAN2,3, DAVID G. MAYER1 AND DAVID G. BARBER1 1Queensland Department of Agriculture and Fisheries, Gatton Campus, Lawes, QLD, Australia. daf.qld.gov.au 2Instituto Nacional de Investigación Agropecuaria (INIA), Treinta y Tres, Uruguay. inia.uy 3Grupo de Pesquisa Ecologia do Pastejo, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil. ufrgs.br/gpep Abstract A plot study was conducted at the Gatton Research Dairy, Queensland, Australia, to quantify the effects of 5 regrowth periods (9, 11, 14, 16 and 18 days) and 4 vertical strata on the composition and nutritive value of kikuyu (Cenchrus clandestinus) pastures using a block factorial design with 4 replicates. Pasture samples were analyzed for crude protein (CP), ethanol-soluble carbohydrates (ESC), acid detergent fiber (ADF), neutral detergent fiber (aNDFom), in vitro indigestible neutral detergent fibre (iNDF240) and minerals. Metabolizable energy (ME) was then calculated from the concentrations of other nutrients. Regardless of the stage of regrowth, stems were located mainly in the bottom 1 or 2 strata, while leaves were present mainly in the top 2 or 3 strata. CP, ESC and ME declined, but aNDFom, ADF and iNDF240 increased with stage of regrowth and from top to bottom of the swards (P<0.05). While herbage quality variables were affected by both factors, vertical stratum had a much larger impact on quality than stage of regrowth. These results indicate that grazing management of kikuyu pastures should be based not only on stage of regrowth but also on level of defoliation, as both have strong impacts on the nutritive value of the consumed forage. Keywords: Chemical composition, grazing management, leaf stage, sward structure, tropical pastures. Resumen En Gatton Research Dairy, Queensland, Australia, en un diseño factorial en bloques con cuatro repeticiones, se evaluó el efecto de cinco periodos de rebrote (9, 11, 14, 16 y 18 días) y cuatro estratos verticales en el valor nutritivo de pasturas de kikuyo (Cenchrus clandestinus). En muestras de pasturas se determinaron la concentración de proteína cruda (PC), carbohidratos solubles en etanol (CSE), fibra detergente acido (FDA) y neutro (FDN), FDN indigestible (FDNi), energía metabolizable (EM) y minerales. Los resultados mostraron que los tallos estuvieron localizados principalmente en los dos estratos inferiores y las hojas en los dos o tres estratos superiores de las pasturas. Consecuentemente, la PC, CSE y EM se redujeron, y FDA, FDN y FDNi se incrementaron con el estado de rebrote y desde la parte superior a la inferior de la pastura. A pesar de que ambos factores experimentales afectaron la calidad del forraje, los estratos verticales afectaron más a la calidad que el estado de rebrote. Estos resultados indican que los dos factores deben ser considerados para el manejo del kikuyo ya que ambos afectan significativamente a la calidad del forraje ingerido. Palabras clave: Composición química, estructura de pasto, manejo de pastoreo, pasturas tropicales. ___________ Correspondence: M.A. Benvenutti, Queensland Department of Agriculture and Fisheries, Gatton Campus, Lawes, QLD 4343, Australia. Email: Marcelo.Benvenutti@daf.qld.gov.au Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) 142 M.A. Benvenutti, C. Findsen, J.V. Savian, D.G. Mayer and D.G. Barber Introduction irrigation was applied. A single 1 m2 sampling area was used on each occasion to assess physical attributes and Kikuyu (Cenchrus clandestinus syn. Pennisetum perform harvests to determine yield and quality attributes clandestinum) is one of the main improved pasture of pasture in each of the 4 blocks for the 5 regrowth species used by the grazing industries in the northeast of periods. Australia and other tropical and subtropical areas of the Prior to harvesting the 1 m2 sampling area, 30 random world. Previous studies examined the effects of stage of tillers were measured for total non-extended height, stem regrowth on herbage quality of leaves, stems and whole height and number of fully-expanded leaves (Figure 1). plants of kikuyu pastures (Reeves et al. 1996). These early Stem height was defined as the height from ground level studies provided essential data for the understanding of to the base of the lamina (ligula) of the top fully-expanded factors driving herbage quality in kikuyu, such as leaf. The averages of total sward height, stem height and differences in quality between plant parts at different number of leaves were then calculated. stages of regrowth; however, these herbage quality results may not necessarily represent the quality of herbage actually consumed by animals grazing these pastures. It is well known from previous studies that beef and dairy cattle graze pastures in strata, and herbage quality declines from the top to the bottom of the swards (Ungar and Ravid 1999; Benvenutti et al. 2016, 2017; Ison et al. 2018). These studies found that, when grazing pressure increased, cattle were forced to graze forage in the lower strata which was of lower quality and as a result diet quality and animal performance declined. It is important to quantify the herbage quality of different strata of kikuyu pastures to assist members of the grazing industry Figure 1. Heights of tillers and plant parts. in making informed decisions on grazing management, based on a sound understanding of the effects of sward Pasture inside the sampling quadrat was then cut in 4 structure on diet quality. The aim of this study was to vertical strata. The strata were numbered from top to quantify the effects of stage of regrowth and vertical bottom of the sward so that stratum 1 was the top stratum. distribution in the sward on physical composition and The bottom stratum consisted of plant material collected nutritive value of kikuyu pastures. from ground level to 5 cm high. The top 3 strata were equal vertical proportions of pasture above 5 cm. Average Materials and Methods total sward height minus 5 cm was divided by 3 to form the 3 strata, indicating that the depth of each stratum Experimental design and pasture assessment varied with the total height of the sward. Within each stratum, harvested material was dried at 60 °C to The study was conducted during 18 days on established determine dry matter (DM) yield. Samples from each kikuyu pastures at the Gatton Research Dairy, Queens- stratum were combined in pairs of replicates (blocks 1 and land, Australia (27°32'45'' S, 152°19'44'' E; 104 masl) 2 and blocks 3 and 4). Combined subsamples from each in January–February 2016. The kikuyu pasture was stratum were then sent for analysis to the Dairy One planted in 2010 into a black alluvial soil. The effects of 5 Forage Lab (Ithaca, NY, USA) to determine crude protein regrowth periods (9, 11, 14, 16 and 18 days) and 4 vertical (CP), ethanol-soluble carbohydrates (ESC), acid strata on physical structure and nutritive value of the detergent fiber (ADF), amylase, sodium sulphite-treated pasture were assessed using a blocked factorial design neutral detergent fiber expressed on an ash-free basis with 4 replicates. On 18 January 2016 the experimental (aNDFom) and minerals. Subsamples of each stratum area (0.2 ha) of kikuyu pasture was divided into 4 blocks were also analyzed for in vitro indigestible NDF from of 0.05 ha each and mown to 5 cm in height before 100 240-hour incubations with rumen fluid (iNDF240). kg urea/ha was applied. Plots of 9 m2 within blocks were Metabolizable energy (ME) values were calculated using randomly allocated to differing regrowth periods. The equations and relationships with other nutrients. The pasture was irrigated regularly to replace evapotran- Dairy One Forage Lab uses a multiple component spiration losses. During the 3 weeks of the experimental summative approach for its ruminant energy prediction period 44 mm of rainfall was received and 65 mm system: Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) Vertical strata forage quality in kikuyu 143 ME (MJ/kg DM) = (1.01 × 0.04409 × TDN – 0.45) × Statistical analysis 4.184, where: TDN is total digestible nutrients (%). Nutritive values for herbage components were analyzed according to a blocked 5 × 4 factorial design using Chemical analysis and in vitro rumen fermentation analysis of variance. Here the fixed effects were stage of regrowth at the main-plot level, and vertical stratum as a The ADF and aNDFom concentrations were determined split-plot effect. GenStat (2016) was used for the using the ANKOM Model 200 and the fiber bag technique analyses. developed by ANKOM (ANKOM Technology, Macedon, NY, USA). The acid and neutral solutions for Results and Discussion these analyses were prepared as per AOAC 973.18 (AOAC 1984) and Van Soest et al. (1991), respectively. Pasture structure ESC was determined by the method of Hall et al. (1999). Samples were analyzed for CP using the AOAC Leaf numbers in the pastures ranged from 2.2 to 4.7 leaves per tiller (Table 1). This range included the currently procedure 990.03 (AOAC 1984). To determine mineral recommended stage of regrowth of 4.5 leaves per tiller for concentrations, samples were digested using CEM grazing kikuyu pastures (Reeves et al. 1996). Regardless of Microwave Accelerated Reaction System (MARS6) with stage of regrowth, all pastures consisted of the same MarsXpress Temperature Control using 50 ml calibrated general structure, with stems predominantly located at the Xpress Teflon PFA vessels with Kevlar/fiberglass bottom of the sward and leaves at the top of the sward insulating sleeves then analyzed by ICP using a Thermo (Table 1). The average height of the stems, as a proportion iCAP 6300 Inductively Coupled Plasma Radial of total sward height, increased from 0.32 to 0.44 as stage Spectrometer. of growth advanced from 1 to 5 (Table 1). This indicated The iNDF240 concentration was determined using that the proportion of stems increased from top to bottom 240-hour in vitro fermentation in Daisy Incubators of the sward as well as with the stage of regrowth. A large (ANKOM Technology, Macedon, NY, USA) set at 39 °C. proportion of the bottom 1 or 2 vertical strata consisted of Each Daisy incubation cupboard can incubate 4 bottles stems and the top 2 or 3 strata consisted mainly of leaves (24 bags of samples per bottle) at a time with 1,520 mL (Table 2). Similarly, Benvenutti et al. (2016) found that buffer solution added to each bottle and then combined pastures of Axonopus catarinensis had a similar sward with 400 mL rumen fluid taken from a fistulated steer. structure, where the stems were located in the bottom one- After 120 h fresh rumen fluid was collected and solutions third of the sward for a range of regrowth stages. The were replenished. After 240 h, bags were removed and results are also consistent with the study by Reeves et al. rinsed until clear. NDF concentration in the residue was (1996), who found that the proportion of stems increased analyzed using the Ankom Fiber Analyzer. with the stage of regrowth of kikuyu pastures. Table 1. Physical structure of kikuyu swards at different stages of regrowth. See also Figure 1. Stage of regrowth Pasture height Stem height Stem height (proportion Leaf height Fully expanded (no. of days) (cm) (cm) of pasture height) (cm) leaves (no.) 1 (9) 13.9 4.4 0.32 9.5 2.2 2 (11) 16.3 5.3 0.32 11.0 2.5 3 (14) 23.7 9.1 0.38 14.6 3.3 4 (16) 29.1 12.5 0.43 16.5 4.1 5 (18) 35.1 15.6 0.44 19.5 4.7 Probability <0.001 <0.001 <0.001 <0.001 <0.001 s.e.m. 0.798 0.563 0.011 0.423 0.068 LSD (P<0.05) 2.46 1.74 0.033 1.30 0.21 Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) 144 M.A. Benvenutti, C. Findsen, J.V. Savian, D.G. Mayer and D.G. Barber Table 2. The effects of stage of regrowth (SoR) on stem proportion and herbage quality (DM basis) of vertical strata (S1‒S4) of kikuyu pastures. S1 to S4: top to bottom stratum. Regrowth Stem height Crude ADF aNDFom iNDF240 ESC ME Calcium Phosphorus Potassium Sodium stage and (proportion protein (g/kg) (g/kg) (g/kg) (g/kg) (MJ/kg) (g/kg) (g/kg) (g/kg) (g/kg) stratum of stratum (g/kg) height) SoR 1 (9 days) S1 0.0 340 212 371 0.05 68.5 11.8 4.60 4.60 29.9 1.35 S2 0.0 338 252 375 0.05 54.5 10.8 4.35 4.90 35.6 1.58 S3 0.0 310 277 445 0.08 46.0 9.8 4.85 5.10 38.3 1.86 S4 0.8 25 5 33 3 56 1 0.2 1 37 .5 8. 7 6.9 0 4.5 5 36 .7 2.4 6 SoR 2 (11 days) S1 0.0 357 228 417 0.07 32.5 11.9 4.35 5.20 39.7 1.25 S2 0.0 335 300 462 0.08 30.0 9.5 4.10 5.70 46.6 1.61 S3 0.0 302 352 512 0.10 26.0 8.6 4.90 5.90 49.5 1.95 S4 0.9 24 7 36 6 59 5 0.2 4 34 .0 8. 0 7.5 0 4.6 5 37 .2 2.2 9 SoR 3 (14 days) S1 0.0 350 294 422 0.06 39.0 10.7 3.90 5.25 40.7 0.94 S2 0.0 314 319 462 0.09 22.0 9.4 3.35 6.00 51.4 1.16 S3 0.5 295 309 495 0.13 18.0 9.5 3.85 6.05 54.9 1.52 S4 1.0 24 2 35 2 60 0 0.2 8 28 .0 8. 4 6.8 0 5.0 0 41 .5 2.2 0 SoR 4 (16 days) S1 0.0 329 287 446 0.07 39.5 9.6 3.90 4.60 45.1 0.75 S2 0.0 303 280 491 0.08 31.5 9.8 3.05 5.15 55.3 0.98 S3 0.9 279 322 510 0.13 26.0 8.9 3.90 5.40 59.3 1.47 S4 1.0 22 8 36 2 60 2 0.2 8 19 .0 8. 0 6.7 5 3.9 5 40 .7 2.1 4 SoR 5 (18 days) S1 0.0 314 285 451 0.07 42.5 10.3 3.95 4.60 43.2 1.13 S2 0.0 293 284 497 0.08 37.5 10.0 3.35 5.25 54.5 1.23 S3 0.9 268 329 522 0.13 27.5 8.8 4.15 5.05 59.2 1.60 S4 1. 0 22 6 36 9 60 0 0.2 6 28 .0 7. 5 6.7 0 4.2 0 45 .8 2.2 9 P SoR 0.002 0.049 0.072 0.014 <0.001 0.15 0.009 0.008 0.029 0.01 <0.001 P Stratum <0.001 <0.001 <0.001 <0.001 <0.001 0.01 <0.001 <0.001 <0.001 <0.001 <0.001 P SoR × <0.001 0.046 0.068 0.032 0.104 0.735 0.004 0.389 <0.001 <0.001 0.053 Stratum s.e.m. 0.04 7.18 15.14 11.49 0.007 8.29 0.25 0.20 0.14 1.70 0.05 LSD 0.12 24.1 45.3 36 0.022 25.2 0.74 0.59 0.50 6.09 0.16 (P<0.05) ESC – ethanol-soluble carbohydrates; ADF – acid detergent fiber; aNDFom – amylase and sodium sulphite-treated neutral detergent fiber expressed on an ash-free basis; iNDF240 – in vitro indigestible neutral detergent fiber from 240-hour incubations with rumen fluid; ME – metabolizable energy; stratum numbering from top (S1) to bottom (S4). Nutritive value in the proportions of leaves and stems in the different strata (Table 2). Consistently, Fulkerson et al. (2010) Since there was a significant interaction (P<0.05) between reported a significant difference in herbage quality experimental factors (stages of regrowth and vertical strata) between leaves and stems in kikuyu pastures. Since for most herbage quality variables, results are shown for all herbage quality differed between stages of regrowth for combinations of factors in Table 2. While herbage quality all strata, the observed differences in quality between was significantly affected by both factors, vertical stratum stages of regrowth could be attributed to changes in had a much larger impact on quality than stage of regrowth quality of individual plant parts as the pasture matured. for most quality variables. This is explained in detail below Reeves et al. (1996) found that the CP and mineral for each herbage quality variable. concentrations of leaves changed significantly with the It is likely that the observed differences in herbage stage of regrowth of kikuyu pastures. quality between vertical strata and between stages of CP and ESC concentrations declined significantly as regrowth were due to the differences in the proportion of stage of regrowth increased and from top to bottom of the different plant parts or to their changes in quality as the swards (P<0.05). These quality variables were more pasture matured (Table 2). The differences in herbage affected by the vertical stratum than the stage of regrowth. quality between strata were largely due to the differences While the average decreases in CP and ESC concen- Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) Vertical strata forage quality in kikuyu 145 trations from stage 1 to stage 5 of regrowth were 11 and grazed. In addition, herbage quality declines significantly 34%, respectively, the average declines from the top to with the stage of regrowth for all vertical strata, probably the bottom stratum were 25 and 44%, respectively. as a result of changes in nutritive value within plant parts Previous studies also found that CP% declined with the as the pasture matures. While herbage quality was stage of regrowth in kikuyu pastures (Reeves et al. 1996) affected by both factors, the vertical stratum had a much and from top to bottom in pastures of Axonopus larger impact on nutritive value than stage of regrowth. catarinensis (Benvenutti et al. 2016). We conclude that grazing management of kikuyu In contrast, concentrations of aNDFom, ADF and pastures should be based not only on the stage of regrowth iNDF240 increased significantly with stage of regrowth but also on the level of defoliation, as both have strong and from top to bottom of the sward (P<0.05). These impacts on the herbage quality of forage consumed by the variables were also more affected by the vertical stratum animals. By considering these issues, members of the than the stage of regrowth. While the average increases in grazing industry can now exert greater control over the concentrations of aNDFom, ADF and iNDF240 from nutritive value of the forage consumed by the animals by stage 1 of regrowth to stage 5 were 20, 19 and 48%, the controlling the level of defoliation of the pasture according average increases from the top to the bottom stratum were to its stage of regrowth. Similar studies on other pasture 40, 36 and 300%, respectively. These results indicate that species, e.g. erect species, would verify if these concepts the digestibility of the plant material would decrease at are relevant to species with different growth habits. later stages of regrowth and from top to bottom of the sward. This confirms the work of Reeves et al. (1996), Acknowledgments who found that organic matter digestibility decreased as stage of regrowth increased in kikuyu pastures. We are grateful to Dairy Australia who provided the As might be expected, ME decreased consistently operating funds for this project. when ESC decreased and aNDFom, ADF and iNDF240 increased. Therefore, ME significantly declined with References stage of regrowth and from top to bottom of the sward (Note of the editors: All hyperlinks were verified 15 March 2020.) (P<0.05). This quality variable was also more affected by the vertical stratum than stage of regrowth. While the AOAC (Association of Official Analytical Chemists). 1984. average decrease of ME from stage 1 of regrowth to stage Official methods of analysis. AOAC Inc., Arlington, VA, 5 was 11%, the average decline from the top to the bottom USA. stratum was 25%. Benvenutti MA; Pavetti DR; Poppi DP; Gordon IJ; Cangiano CA. 2016. Defoliation patterns and their implications for the The effects of stage of regrowth and vertical stratum management of vegetative tropical pastures to control intake on mineral concentration in the plant material differed and diet quality by cattle. Grass and Forage Science 71:424– between minerals (Table 2). Concentrations of calcium 436. doi: 10.1111/gfs.12186 and sodium increased from top to bottom of the sward, Benvenutti MA; Pavetti DR; Poppi DP; Mayer DG; Gordon IJ. while concentrations of potassium and phosphorus 2017. Ingestive behaviour and forage intake responses of increased from stratum 1 to stratum 3 and then decreased young and mature steers to the vertical differentiation of for the bottom stratum. Unlike potassium, concentrations sugarcane in pen and grazing studies. The Journal of of calcium and sodium decreased with stage of regrowth. Agricultural Science 155:1677–1688. doi: 10.1017/S00218 In turn, the concentration of phosphorus increased from 59617000673 stage 1 of regrowth to stage 3 and then decreased. On the Fulkerson WJ; Griffiths N; Sinclair K; Beale PN. 2010. Milk contrary, Reeves et al. (1996) found that, as leaves of production from kikuyu grass based pastures. Primefact No. 1068. NSW Department of Primary Industries, Orange, kikuyu pastures matured, calcium concentration increased NSW, Australia. bit.ly/39VFjOF while those of potassium and phosphorus decreased, but GenStat. 2016. GenStat Statistical Package for Windows. 2016 sodium concentration did not change with maturity. Edn. Lawes Agricultural Trust, Rothamsted Experimental Station, Harpenden, UK. Conclusion Hall MB; Hoover WH; Jennings JP; Webster TKM. 1999. A method for partitioning neutral detergent-soluble This study has shown that, regardless of the stage of carbohydrates. Journal of the Science of Food and regrowth, stems were located mainly in the bottom one or Agriculture 79:2079–2086. DOI: 10.1002/(sici)1097-0010 two strata, while leaves were present mainly in the top two (199912)79:15<2079::aid-jsfa502>3.3.co;2-q or three strata. This indicates that herbage quality declines Ison K; Barber DG; Benvenutti MA; Mayer DG; Findsen C. significantly from top to bottom of the sward as it is 2018. The effect of grazing intensity of lucerne (Medicago Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) 146 M.A. Benvenutti, C. Findsen, J.V. Savian, D.G. Mayer and D.G. Barber sativa) pasture on defoliation dynamics and pasture intake Ungar ED; Ravid N. 1999. Bite horizons and dimensions for in dairy cows. Proceedings of the 32nd Biennial Conference cattle grazing herbage to high levels of depletion. Grass and of the Australian Society of Animal Production, Wagga Forage Science 54:357–364. doi: 10.1046/j.1365-2494. Wagga, New South Wales, Australia, 2–6 July 2018. p. 87. 1999.00188.x doi: 10.1071/ANv58n8abs Van Soest PJ; Robertson JB; Lewis BA. 1991. Methods for Reeves M; Fulkerson WJ; Kellaway RC. 1996. Forage quality dietary fiber, neutral detergent fiber, and nonstarch of kikuyu (Pennisetum clandestinum): The effect of time of polysaccharides in relation to animal nutrition. Journal of defoliation and nitrogen fertiliser application and in Dairy Science 74:3583–3597. doi: 10.3168/jds.S0022-0302 comparison with perennial ryegrass (Lolium perenne). (91)78551-2 Australian Journal of Agricultural Research 47:1349–1359. doi: 10.1071/AR9961349 (Received for publication 2 July 2019; accepted 15 February 2020; published 31 May 2020) © 2020 Tropical Grasslands-Forrajes Tropicales is an open-access journal published by International Center for Tropical Agriculture (CIAT), in association with Chinese Academy of Tropical Agricultural Sciences (CATAS). This work is licensed under the Creative Commons Attribution 4.0 International (CC BY 4.0) license. Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) Tropical Grasslands-Forrajes Tropicales (2020) Vol. 8(2):147–152 147 doi: 10.17138/TGFT(8)147-152 Short Communication In vitro digestion characteristics of various combinations of elephant grass hay, gliricidia hay or silage, soybean meal and corn meal in rations for sheep Características de la digestión in vitro de varias combinaciones de heno del pasto elefante, heno o ensilaje de gliricidia, harina de soya y harina de maíz en raciones para ovinos JULIANA CAROLINE SANTOS SANTANA1, JUCILEIA APARECIDA DA SILVA MORAIS2, GELSON DOS SANTOS DIFANTE1, LUÍS CARLOS VINHAS ÍTAVO1, ANTONIO LEANDRO CHAVES GURGEL1, VINICIUS DA SILVA OLIVEIRA2 AND MARIA JUCIARA SILVA TELES RODRIGUES2 1Faculdade de Medicina Veterinária e Zootecnia, Universidade Federal de Mato Grosso do Sul, Campo Grande, MS, Brazil. famez.ufms.br 2Departamento de Zootecnia, Universidade Federal de Sergipe, Aracaju, SE, Brazil. ufs.br Abstract This study examined fermentation rates and kinetics of sheep rations based on combinations of elephant grass hay, gliricidia (Gliricidia sepium) hay or silage, soybean meal and corn meal using in vitro techniques. Three rations were prepared, namely: Control (elephant grass hay + soybean meal + corn meal); gliricidia hay (elephant grass hay + soybean meal + corn meal + gliricidia hay); and gliricidia silage (elephant grass hay + soybean meal + corn meal + gliricidia silage). A fixed ratio of roughage:concentrate of 55:45 was maintained for all rations, which were isocaloric and designed to support sheep gains of 200 g/day. The gliricidia replaced 57.6% of the soybean meal in the rations containing gliricidia and 81.8% of the elephant grass hay. Fermentation rates and kinetics, in vitro dry matter digestibility (IVDMD) and degradability of the rations were evaluated. Rations containing gliricidia as both hay and silage had higher (P<0.05) IVDMD than the Control ration (67.8 and 66.2 vs. 59.8%). The degradability of the ration containing gliricidia hay was higher (P<0.05) than that of the gliricidia silage ration (57.8 vs. 50.5%), whereas the Control showed an intermediate value (54.4%). The ration containing gliricidia hay and the Control produced more gas in the first 24 h than the ration containing gliricidia silage, and the gliricidia hay ration showed the shortest colonization time. Peak gas production occurred for the ration with gliricidia silage later than for the other rations. The study showed that substituting soybean meal with preserved gliricidia can result in higher digestibility of sheep rations. Feeding studies with animals are now warranted to verify these laboratory findings. Keywords: Degradability, digestibility, gas production, Gliricidia sepium, sheep farming, tropical legumes. Resumen En el estudio se determinaron las tasas y la cinética de fermentación ruminal de raciones para ovinos basadas en combinaciones de heno de pasto elefante, heno o ensilaje de gliricidia (Gliricidia sepium), harina de soya y harina de maíz, utilizando técnicas in vitro. Se prepararon tres raciones: Control (heno de pasto elefante + harina de soya + harina de maíz); heno de gliricidia (heno de pasto elefante + harina de soya + harina de maíz + heno de gliricidia); y ensilaje ___________ Correspondence: Juliana Caroline Santos Santana, Faculdade de Medicina Veterinária e Zootecnia, Universidade Federal de Mato Grosso do Sul, Avenida Senador Filinto Müller 2.443, Campo Grande, CEP 79074-460, MS, Brazil. Email: jukrol_@hotmail.com Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) 148 J.C.S. Santana, J.A.S. Morais, G.S. Difante, L.C.V. Ítavo, A.L.C. Gurgel, V.S. Oliveira and M.J.S.T. Rodrigues de gliricidia (heno de pasto elefante + harina de soya + harina de maíz + ensilaje de gliricidia). Se mantuvo una proporción fija de forraje:concentrado de 55:45 para todas las raciones las cuales fueron isocalóricas y diseñadas para producir ganancias diarias de 200 g peso vivo de ovinos. En ambos tratamientos con gliricidia el 57.6% de la harina de soya fue reemplazado y el 81.8% del heno de pasto elefante. Se evaluaron las tasas y cinética de la fermentación ruminal, la digestibilidad in vitro de la materia seca (IVDMD) y la degradabilidad de las dietas. Las raciones que contenían gliricidia como heno y ensilaje tuvieron una IVDMD más alta (P<0.05) que el Control (67.8 y 66.2 vs. 59.8%). La degradabilidad de la ración con heno de gliricidia fue mayor (P<0.05) que la de la ración con ensilaje de gliricidia (57.8 vs. 50.5%), mientras que el Control presentó un valor intermedio (54.4%). La ración con heno de gliricidia y el Control produjeron más gas en las primeras 24 horas que la ración con ensilaje de gliricidia, y la ración con heno de gliricidia mostró el menor tiempo de colonización. La producción máxima de gas ocurrió en la ración con ensilaje de gliricidia más tarde que en las otras raciones. El estudio mostró que sustituyendo la harina de soya por forraje preservado de gliricidia puede resultar en una digestibilidad más alta de raciones para ovinos. Para corroborar estos resultados obtenidos a nivel de laboratorio se requieren estudios de alimentación con animales. Palabras clave: Degradabilidad, digestibilidad, ganadería ovina, Gliricidia sepium, producción de gas. Introduction gas released from feedstuffs inoculated with rumen fluid reflects microbial activity, as gas is a product of Pasture-based production systems are limited primarily fermentation. by variations in climatic conditions, which directly We conducted this study to obtain preliminary data on interfere with plant growth (Euclides et al. 2019), the fermentation rates and kinetics, dry matter resulting in negative impacts on animal performance, digestibility and degradability of 3 sheep rations made up especially during the dry season (Emerenciano Neto of mixtures of hay of elephant grass (Cenchrus purpureus 2018; Braga et al. 2019). One possible solution is to syn. Pennisetum purpureum), gliricidia as hay or silage, conserve forage in periods of high availability to be used soybean meal and corn meal using in vitro techniques. during times of scarcity (Bueno et al. 2018), along with feeding of protein supplements. Materials and Methods The use of high-protein feedstuffs in sheep rations is a common practice worldwide and soybean meal ranks The experiment was conducted at the Laboratories of highly as a protein source. Since soybeans are also used Animal Nutrition and Rumen Fermentation at the for human consumption, costs of this product for feeding Department of Animal Science (DZO), Federal livestock are high. Identifying a less expensive plant- University of Sergipe (UFS), Aracaju, Sergipe. It was set derived protein would be of great benefit to ruminant up as a completely randomized design with 3 treatments, production worldwide. i.e. 3 rations formulated for sheep, namely: Control – Shrub legumes are possible options as alternative basal ration of elephant grass hay-soybean meal-corn sources of fodder which are high in protein, e.g. gliricidia meal; gliricidia hay – elephant grass hay-gliricidia hay- (Gliricidia sepium), which grows well in tropical climates soybean meal-corn meal with only 42.6% of the soybean and is relatively drought-tolerant. Since its chemical and meal supplement contained in Control; and gliricidia productive characteristics are similar to those of other silage – elephant grass hay-gliricidia silage-soybean leguminous species, it is a viable option for animal feed- meal-corn meal with 42.6% of the soybean meal ing, especially in regions where water deficit is a constant supplement contained in Control. These rations were problem (Fernandes et al. 2017; Santana et al. 2019; formulated for sheep to achieve an estimated dry matter Fernandes et al. 2020). Incorporating it in rations should intake of 3.5% of body weight and 200 g/day liveweight allow a reduction in the level of soybean meal required to gain (NRC 2007) and are described in Table 1. Since the supply the protein needs of the sheep. roughage:concentrate ratio was fixed at 55:45, including As a prelude to conducting feeding trials with animals, gliricidia hay or silage reduced percentage of elephant which also are expensive, in vitro studies in the laboratory grass hay and soybean meal in the ration, while can provide preliminary data on likely outcomes from percentage of corn meal was increased. The feeding various rations. The in vitro gas production concentrations of crude protein in elephant grass hay, method described by Theodorou et al. (1994) consists of gliricidia hay and gliricidia silage, soybean meal and corn incubating samples of feedstuffs in bottles attached to meal were, respectively: 12.9; 15.7; 17.5; 42.5 and 8.4% a gas meter. According to Tagliapietra et al. (2010), the on a dry matter basis. Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) In vitro digestion of sheep diets with preserved gliricidia 149 Table 1. Proportions of ingredients and nutrient concentrations For each ration 5 samples were incubated in penicillin- in the experimental rations. type glass bottles with a capacity of 100 mL. Each bottle contained 670 mg of sample in 67 mL of incubation Ration solution. The incubation solution was prepared as Control GH GS described by Theodorou et al. (1994) using cysteine-HLC Ingredient (% DM basis) as a reducing agent (Mould et al. 2005). Elephant grass hay 55.0 10.0 10.0 Rumen fluid formed 20% of the incubation solution. A Gliricidia hay - 45.0 - constant flow of CO was maintained. Each bottle was Gliricidia silage - - 45.0 2 Soybean meal 23.5 10.0 10.0 inoculated manually by using a graduated syringe, and Corn meal 21.5 35.0 35.0 bottles were closed with rubber corks (14 mm), sealed Nutrient concentration (% DM) with an aluminum seal and kept in a water bath at 39 ºC. Organic matter 93.9 94.7 93.9 In addition to the bottles with the samples, an additional Crude protein 21.7 18.3 18.3 4 bottles containing incubation medium without samples Hemicellulose 28.2 30.3 27.0 (blanks) were evaluated. Cellulose 28.6 22.0 21.2 Gas production was measured for a period of 48 h, and Lignin 2.6 6.6 8.2 the pressure within the bottle was recorded with a digital GH = Gliricida hay; GS = Gliricidia silage. manometer coupled to a 3-way valve. Immediately after the pressure readings, gas volume was measured using a The gliricidia used to produce the hay and silage was graduated syringe attached to the valve. The syringe obtained from trees at approximately 12 months after plunger was extracted until the transducer pressure planting, by cutting and selecting tender branches (≤8 mm returned to zero. thick) with leaves, from the EVA (Interdisciplinary Space Gas volume and pressure data were tabulated to obtain of Agroecological Experience) area adjacent to the the linear, quadratic and cubic statistical models and then Department of Animal Science (DZO) at UFS. The forage determine the correlation between gas volume and was then chopped to produce an average particle size of pressure reading using Excel software. The model 2 cm, spread on plastic sheeting in the sun and turned considered satisfactory was that which showed the every 30 min. After 2 days, the hay was bagged. The highest R2 value (R2 = 0.95). elephant grass was cut approximately 5 cm above ground The following equation was obtained: level at 45 days growth and the hay-making process was y = -0.382x2 + 6.087x - 0.772, the same as that for gliricidia. where: Three experimental PVC silos 10 cm in diameter and 30 y is the final gas volume in mL; and cm long with PVC caps at each end were used, and were x is the gas pressure in kilopascal at the respective times. sealed with metal clips. The fresh chopped gliricidia was Gas production on each occasion was corrected for the compacted in these silos to a specific mass of 600 kg/m3. To average gas production from bottles containing the ensure anaerobic conditions in the silos, adhesive tape was incubation medium without diet samples. Mean gas used to promote better sealing than with the metal clips production volumes for each treatment at the respective alone. The silos were weighed before and just after sealing. incubation times were adjusted to the 2-compartment There was a layer of sand at the bottom of the silo to retain logistic model proposed by Pell and Schofield (1993), as effluents and prevent contamination of the silage; a follows: permeable mesh between the silage and the sand layer TV = Vf1/(1 + exp (2 - 4*c1*(T - L))) + Vf2/(1 + exp(2 prevented contact between the sand and the silage. - 4*c2*(T - L))), In vitro gas production from the various rations was where: determined in accordance with the methodology TV = total gas volume (mL/100 mg DM) accumulated described by Theodorou et al. (1994). To this end, after by time T; being dried in a forced-air oven, ration samples were Vf1 = gas volume (mL) of the rapidly digested fraction; ground through a Wiley mill with a 5-mm sieve. c1 = degradation rate of the rapidly digested fraction Rumen fluid was collected from 3 sheep maintained on (L/h); a diet of corn, soybean meal and fresh gliricidia and L = lag time, or fiber colonization time (h); samples were mixed/bulked to form the rumen inoculum, Vf2 = gas volume (mL) of the slowly digested fraction; which was filtered through gauze and stored in a thermos and previously heated in water at 38 ºC. A constant flow of c2 = degradation rate of the slowly digested fraction CO2 was maintained during the preparation process. (L/h). Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) 150 J.C.S. Santana, J.A.S. Morais, G.S. Difante, L.C.V. Ítavo, A.L.C. Gurgel, V.S. Oliveira and M.J.S.T. Rodrigues After 48 hours of incubation, the contents of the bottles gas production occurred later for the ration containing (which had been tared previously) were filtered through gliricidia silage (Figure 1). non-woven fabric (‘TNT’) of known dimensions. Dry The ration containing gliricidia silage produced the matter degradability was determined based on the smallest volume of gas from the highly soluble fraction difference between the constant weight obtained by (12.4 mL/100 mg of incubated DM) and showed the drying at 105 ºC and the weight of the incubated material. longest fiber colonization (lag) time (3.8 h). This ration To determine the in vitro dry matter digestibility also produced the largest volume of gas from the lowly (IVDMD), the residue was washed with neutral detergent soluble fraction (10.7 mL/100 mg of incubated DM). and weighed after drying at 105 ºC in a forced-air oven for 24 h and expressed as a percentage of the initial weight 25 of the sample. The ash concentration in the residue was determined by method 942.05 of AOAC (1990). 20 The parameters estimated by the mathematical model were obtained using iterative non-linear methods. Results 15 were adjusted by least-squares estimates, adopting the Marquardt method, by the PROC NLIN procedure of SAS statistical package (SAS Institute Inc., Cary, NC, USA). 10 Data pertaining to degradability, digestibility and residual ash were subjected to analysis of variance and means were 5 compared by Tukey’s test at the 5% significance level. 0 Results 1 2 3 4 6 8 10 12 16 18 24 36 48 Incubation time (h) Rations containing gliricidia had higher IVDMD levels Control Gliricidia hay Gliricidia silage than the Control (P<0.001; Table 2). While degradability of the ration containing gliricidia hay was higher than that Control: GP = 15.6185/{1 + exp[2+4*0.1925*(2.8873−Time)]} + 7.4599/{1 + exp[2+4*0.05*(2.8873−Time)] 2 of the ration containing gliricidia silage (P = 0.015), }, R = 0.99; Hay: GP = 15.5116/{1 + exp[2+4*0.1608*(2.7619−Time)]} + 7.2305/{1 + degradability of the Control was intermediate. exp[2+4*0.0677*(2.7619−Time)]}, R2 = 0.99; Silage: GP = 12.4415/{1 + exp[2+4*0.2037*(3.8231−Time)]} + 10.6961/{1 + Table 2. In vitro dry matter digestibility (IVDMD) and exp[2+4*0.0438*(3.8231−Time)]}, R2 = 0.99. degradability of rations comprised of varying combinations of elephant grass hay, gliricidia hay or silage, soybean meal and Figure 1. In vitro gas production for Control (elephant grass corn meal designed to produce 200 g/d gain in sheep. hay + soybean meal + corn meal), gliricidia hay treatment (elephant grass hay + soybean meal + corn meal + gliricidia Ration P hay) and gliricidia silage treatment (elephant grass hay + s.e.m. Control GH GS value soybean meal + corn meal + gliricidia silage) following incubation in buffered rumen fluid. IVDMD (%) 59.8b 67.8a 66.2a <0.001 0.70 Degradability 54.4ab 57.8a 50.5b 0.015 1.59 (%) Discussion Means within rows followed by different letters differ (P<0.05) according to Tukey’s test. Control (elephant grass hay + soybean This study suggests that using gliricidia hay or silage to meal + corn meal); GH: gliricidia hay treatment (elephant grass replace some of the soybean meal and elephant grass hay hay + soybean meal + corn meal + gliricidia hay); GS: gliricidia components of an elephant grass-soybean meal-corn meal silage treatment (elephant grass hay + soybean meal + corn meal ration (Control) for growing sheep could result in an + gliricidia silage). increase in IVDMD of the ration, while degradability of the ration might be improved only in the case of gliricidia Significant (P<0.05) differences in accumulation of hay. gas over time were observed for the different rations. The The lower IVDMD of the Control ration could be a Control treatment and the ration containing gliricidia hay function of a higher proportion of cellulose provided by produced more gas in the first 24 h than the ration the elephant grass hay in this ration (Table 1), since this containing gliricidia silage, while the ration containing component reduces both the level and rate of fiber gliricidia hay showed the shortest colonization time. Peak degradation (Díaz et al. 2018). In general, tropical forages Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) In vitro gas production (mL/100 mg DM) In vitro digestion of sheep diets with preserved gliricidia 151 are poorly digestible (Gerdes et al. 2000; Oliveira et al. The time taken by dietary microorganisms to colonize 2017), which is due mainly to the high concentration of the material influences gas production, since this para- cell wall components such as cellulose and low meter determines how long the rumen microorganisms concentration of potentially digestible compounds such as will act on the substrate. Thus, peak production may be non-fibrous carbohydrates, proteins, ether extract, achieved sooner or later (Díaz et al. 2018). The shorter vitamins and minerals (Oliveira et al. 2017). In the case colonization time observed in the ration containing of elephant grass hay the material used was whole plant gliricidia hay allowed the microorganisms to act for a material cut at 45 days of age, while the gliricidia was longer period, with peak production occurring sooner in comprised of only fine stems and leaves. Inclusion of this treatment. gliricidia in the ration allowed the proportion of soybean The ration containing gliricidia silage showed the meal to be reduced, while the proportion of corn lowest values for fermentation parameters, which may be increased, increasing the supply of soluble carbohydrates explained by its higher lignin and lower hemicellulose to the population of microorganisms, which may have concentrations (Table 1). Lignin present in the cell wall increased microbial activity. complexes itself to carbohydrates (mainly hemicellulose) The higher degradability of the ration including through covalent bonds, forming a mechanical barrier to gliricidia hay could be a consequence of longer de- rumen microorganisms, thus reducing the fermentation of gradation time for the gliricidia hay, given the shorter carbohydrates (Oliveira et al. 2017; Díaz et al. 2018). This colonization time on the particles by the incubated micro- fact also explains why the Control and gliricidia-hay organisms. On the other hand, the lower degradability of rations presented shorter lag phases, higher gas volumes the ration containing gliricidia silage may be a reflection and higher rates of gas production from rapidly digested of fermentation of carbohydrates during the ensiling fractions. process, since soluble carbohydrates serve as substrates for the growth of anaerobic bacteria. This, in turn, Conclusions prompts a decline in the pH of the medium, resulting in the preservation of the material (Gomes et al. 2018; Incorporating gliricidia as hay or silage in traditional Santana et al. 2019). As a consequence, the levels of non- sheep rations based on elephant grass hay, soybean meal fibrous carbohydrates (NFC) (a dietary component with and corn meal should allow a reduction in amounts of higher rumen degradation rates; Oliveira et al. 2017) elephant grass and more importantly soybean meal in the decrease during the ensiling process (Ribeiro et al. 2014). ration. These rations should also be more digestible than Conversely, the higher gas production during the initial the traditional ones, which should result in better animal 24 h of fermentation from both the Control ration and that performance. Added to this, reducing the proportion of containing gliricidia hay was likely a consequence of their expensive soybean meal should lower the cost, while higher NFC concentration in comparison with the increasing the corn meal component would cancel out gliricidia silage diet, resulting in increased IVDMD. some cost advantages. Oliveira et al. (2017) measured in vitro gas production Further studies with sheep to determine feed intakes from a range of forage plants and observed that higher and animal performance on these or similar rations are fermentation rates within the first hours of incubation needed to confirm if these laboratory findings can be were detected in plants with higher soluble carbohydrate reflected in improved production. Other shrub legumes concentrations. might also be used depending on availability. If feeding The different results for digestibility found in this studies are successful, the likely financial benefits to study between the Control ration and rations containing farmers would depend on the relative costs of the ration gliricidia (hay or silage) demonstrate the beneficial components, i.e. elephant grass hay, gliricidia hay or effects of this plant, since incorporating gliricidia in the silage and soybean meal and corn meal. ration increased the amount of digested material without affecting gas production. Acknowledgments Since NFCs are considered the main substrate for lactic fermentation within the silo, their concentration The authors thank the Federal University of Sergipe for decreases throughout the ensiling process (Zardin et al. the support in executing the research; The National 2017). The ration containing gliricidia silage showed Council for Scientific and Technological Development longer fermentation times and higher gas production from (CNPq) for the fellowship grant; and the Coordination for the fibrous material, which is considered a slowly the Improvement of Higher Education Personnel - Brazil degraded component. (CAPES) for the financial support (funding code 001). Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) 152 J.C.S. Santana, J.A.S. Morais, G.S. Difante, L.C.V. Ítavo, A.L.C. Gurgel, V.S. Oliveira and M.J.S.T. Rodrigues References inoculant on the formation of volatile organic compounds, (Note of the editors: All hyperlinks were verified 7 April 2020.) fermentative losses and aerobic stability of oat silage. Animal Feed Science and Technology 247:194–198. doi: AOAC (Association of Official Analytical Chemists). 1990. 10.1016/j.anifeedsci.2018.11.016 Official methods of analysis. 15th Edn. 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Effects of light wilting and heterolactic 38:503–512. doi: 10.5433/1679-0359.2017v38n1p503 (Received for publication 23 July 2019; accepted 28 February 2020; published 31 May 2020) © 2020 Tropical Grasslands-Forrajes Tropicales is an open-access journal published by International Center for Tropical Agriculture (CIAT), in association with Chinese Academy of Tropical Agricultural Sciences (CATAS). This work is licensed under the Creative Commons Attribution 4.0 International (CC BY 4.0) license. Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) Tropical Grasslands-Forrajes Tropicales (2020) Vol. 8(2):153–157 153 doi: 10.17138/TGFT(8)153-157 Short Communication Evaluation of Asystasia gangetica as a potential forage in terms of growth, yield and nutrient concentration at different harvest ages Evaluación del potencial forrajero de Asystasia gangetica a diferentes edades de cosecha N.R. KUMALASARI, L. ABDULLAH, L. KHOTIJAH, L. WAHYUNI, INDRIYANI, N. ILMAN AND F. JANATO Department of Nutrition and Feed Technology, Faculty of Animal Science, Bogor Agricultural University, Bogor, Indonesia. www.intp.fapet.ipb.ac.id Abstract The objective of this experiment was to analyze growth dynamics, yield and nutrient concentration of Asystasia gangetica (L.) T. Anderson at different harvest ages. A pot experiment was conducted at Green House Laboratory of Agrostology, Faculty of Animal Science, Bogor Agricultural University, Indonesia, during the growing season of 2018. Seedlings were transplanted into 115 polybags arranged in a completely randomized design with 23 replications. Plant height, number of leaves, number of branches, dry matter (DM) yields and nutrient concentrations at 30, 40, 50, 70 and 90 days after transplanting (DAT) were determined. Whereas plant height, number of leaves, number of branches and DM yields increased with age, nutrient concentrations followed different patterns. Crude protein % in leaf peaked at 24.2% at 40 DAT then decreased progressively to 8.4% at 90 DAT, while corresponding figures for stem were 10.6 and 2.8%, respectively. Crude fiber concentrations in leaf increased from 10.6% at 30 days to 17.3% at 90 days; corresponding figures for stem were 23.2 and 39.2%. From this pot study, cutting between 40 and 50 days after planting seemed to represent a suitable compromise between DM yield and protein percentage. Studies are needed to determine the repeatability of these results under field conditions and the regrowth potential of plants following harvesting. Keywords: Growth dynamics, nutritive value, forage production. Resumen En condiciones de invernadero, en el Green House Laboratory of Agrostology, Faculty of Animal Science, Bogor Agricultural University, Indonesia, se analizaron la dinámica de crecimiento, el rendimiento y la concentración de nutrientes de Asystasia gangetica (L.) T. Anderson a diferentes edades de las plantas. Las plantas crecieron en 115 bolsas de plástico que fueron dispuestas en un diseño completamente al azar con 23 repeticiones. Se evaluaron la altura de la planta, el número de hojas y de ramas, el rendimiento de materia seca (MS) y las concentraciones de nutrientes a los 30, 40, 50, 70 y 90 días después de la siembra. Mientras que la altura de planta, el número de hojas y de ramas, y el rendimiento de MS aumentaron con la edad de las plantas, las concentraciones de nutrientes mostraron tendencias diferentes. El porcentaje de proteína cruda en la hoja alcanzó un valor máximo (24.3%) a los 40 días y luego disminuyó progresivamente a 8.4% (90 días), mientras que los valores correspondientes para el tallo fueron 10.6 y 2.8%, respectivamente. La concentración de fibra cruda en la hoja aumentó de 10.6% (30 días) a 17.3% (90 días), mientras que la del tallo fue de 23.2 y 39.2%, respectivamente. De este estudio a nivel de invernadero se puede concluir que una cosecha entre 40 y 50 días después de la siembra representa un compromiso aceptable entre el rendimiento de MS y el valor nutritivo. Se sugieren estudios complementarios para determinar la repetibilidad de estos resultados en condiciones de campo y el potential de rebrote de las plantas después de un corte o pastoreo. Palabras clave: Dinámica de crecimiento, valor nutritivo, producción. ___________ Correspondence: N.R. Kumalasari, Department of Nutrition and Feed Technology, Faculty of Animal Science, Bogor Agricultural University, Jl. Agatis, Kampus IPB Darmaga, Bogor 16680, Indonesia. Email: nurrkumala@gmail.com Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) 154 N.R. Kumalasari, L. Abdullah, L. Khotijah, L. Wahyuni, Indriyani, N. Ilman and F. Janato Introduction MgO and 6% CaO) at the rate of 2.5 g/polybag, which was mixed with soil 2 weeks before transplanting. The Asystasia gangetica (L.) T. Anderson (Acanthaceae) is an polybags were arranged in a completely randomized attractive herbaceous ground cover that grows from 30 to design with 23 replications. Treatments consisted of 5 60 cm in height. It is widely distributed from tropical Asia different harvesting ages, i.e. 30, 40, 50, 70 and 90 days to Africa including Nigeria (Lithudzha 2004; GRIN 2007) after transplanting (DAT). and in Indonesia is commonly known as ‘ara sungsang’ in Growth attributes were measured on all plants during Sumatra Island or ‘bayaman’ in Java. It is a fast-growing, the growth period as follows: plant height (cm) from the spreading, perennial herb, with usually erect, branched, base of the plant to the tip of the central spike tassel; and square stems up to 2 m long, often rooting at the lower numbers of leaves and branches. Branches were nodes (Shu et al. 2011). It is a soft weed species that is categorized into 3 types, i.e. primary, secondary and widely grown as ground cover in Indonesian palm tertiary branches. At the predetermined ages, plants were plantations (Ramdani et al. 2017). cut approximately 5 cm from the ground and weighed to This plant can dominate over huge areas because it is determine fresh yields. Plants were then separated into highly tolerant of low soil fertility and shade (Samedani branches (stems) and leaves, and weighed to obtain the et al. 2013). It has potential for use as a commercial forage relevant contributions to total yield. Samples were plant due to its ability to reliably grow from seed selected, air dried and weighed to calculate DM yields. (Kumalasari et al. 2018). The plant can grow rapidly as Fresh herbage samples from each treatment were cover crop (Asbur et al. 2018a) and minimize erosion selected, air dried under sunlight for 2 × 12 h, before (Asbur et al. 2018b). drying in an air-forced oven at 60 °C for 48 h, and ground It has many medicinal, nutritional and local values to pass through a 1 mm sieve for chemical analyses. Dry including its use as forage (Adetula 2004). Norlindawati et matter, crude protein, crude fat, crude fiber and ash al. (2019) reported it has high production and crude protein concentrations were determined according to AOAC concentration, which can reach 23.5% and can be higher in International (2005) procedures. the dry season than in the rainy season (Adjorlolo et al. Data were analyzed statistically as a completely 2014). High mineral concentrations (Khalil et al. 2018) and randomized design with R i386 3.6.1 using Analysis of high palatability for animals (Sobayo et al. 2012) are other Variance Test (ANOVA); if there was a significant desirable attributes. Considerable research has been difference, the analyses were continued with the Tukey conducted on its benefit as feed for animals, e.g. broilers Honest Significant Difference Test (HSD). (Sobayo et al. 2012) and ruminants (Wigati et al. 2016). However, there is a lack of information on its Results morphological characteristics and quality as forage at different growth stages. Plant height Research was carried out with A. gangetica to assess plant growth dynamics, dry matter (DM) yields and Plant height of A. gangetica increased progressively with nutrient concentrations at different ages as a guide to age according to a quadratic relationship (P<0.001; Table identifying optimal times for harvesting. 1), reaching a mean of 131 cm at 90 DAT. Materials and Methods Number of leaves per plant The research was conducted at Green House Laboratory Number of leaves per plant of A. gangetica increased of Agrostology, Faculty of Animal Science, Bogor progressively with age (P<0.001; Table 1) reaching a mean Agricultural University, during the growing season of of 635 leaves at 90 DAT. 2018. Forage quality was analyzed at Laboratory of University Center (PAU). Number of branches per plant Seedlings were prepared in trays for 21 days in a nursery until they reached the 4-leaf stage, before being Total number of branches per plant increased progressively transplanted into 115 polybags with capacity of 5 kg filled with time after transplanting (Table 1; P<0.001). Number with latosol. The basal fertilizer was fresh cattle manure of primary branches increased until 50 days after (with 11.2% organic C, 0.46% total N, 0.24% P2O5 and transplanting then decreased with time, while numbers of 0.29% K2O), at the rate of 250 g/polybag, and inorganic both secondary and tertiary branches increased fertilizer (Mutiara - 16% N, 16% P2O5, 16% K2O, 0.5% progressively over time. Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) Evaluation of Asystasia gangetica at different ages 155 Table 1. Effects of plant age on Asystasia gangetica growth indicators. Parameter Age (days after transplanting) s.e.m. P 30 40 50 70 90 Plant height (cm) 27.9d 72.1c 95.6b 97.9b 131a 0.55 <0.001 Number of leaves 66.4d 306c 372bc 423b 635a 3.19 <0.001 Number of branches Primary 2.3b 2.6a 2.8a 2.3b 2.1c 0.06 <0.001 Secondary 3.8c 7.2bc 13.8b 23.7a 24.1a 0.18 <0.001 Tertiary 2.7e 6.0d 11.2c 31.1b 36.1a 0.27 <0.001 Dry weight (g/plant) Leaf 1.3c 5.5c 8.0b 8.6b 11.6a 0.07 <0.001 Stem 0.6d 5.9c 9.5b 10.4b 19.0a 0.11 <0.001 Total 1.8d 11.4c 17.6bc 19.0b 30.6a 0.09 <0.001 Leaf:stem ratio (dry weight basis) 2.3:1 0.95:1 0.84:1 0.82:1 0.61:1 Means in the same row without common letters are different at P<0.001. Forage yield Forage quality Dry matter (DM) yields of both leaf and stem increased Leaf crude protein (CP) concentration peaked at 24.2% at (P<0.001) progressively with age and reached 11.6 g leaf 40 DAT, then declined progressively to 8.4% at 90 DAT DM and 19.0 g stem DM/plant at 90 DAT (Table 1). (P<0.01; Table 2). The pattern for stem CP was similar but Leaf:stem ratio (DM basis) declined progressively with concentrations were much lower (peak of 10.6% at 40 age from 2.3:1 at 30 DAT to 0.6:1 at 90 DAT. Figure 1 DAT, declining to 2.8% at 90 DAT) (P<0.01). demonstrates changes in appearance of plants as they Crude fiber (CF) concentrations increased with age aged, with changes in leaf:stem ratio and senescence of (P<0.01; Table 2) for both leaf and stem; stem CF leaves by 90 DAT being quite noticeable. concentrations, however, were much higher than in leaf. Figure 1. Asystasia gangetica plants of different ages (DAT, days after transplanting). Table 2. Effects of plant age on nutrient concentrations (% DM) in leaf and stem of Asystasia gangetica forage. Plant part/Nutrient Age (days after transplanting) s.e.m. P 30 40 50 70 90 Leaf Crude protein 16.6b 24.2a 20.7b 10.4c 8.4c 0.59 <0.01 Crude fat 2.1 2.7 1.8 3.4 4.7 0.11 <0.01 Crude fiber 10.6c 9.7c 12.0b 11.9b 17.3a 0.21 <0.01 Ash 15.5 14.7 12.3 13.3 15.4 0.14 <0.01 Stem Crude protein 7.6b 10.6a 7.7b 3.6c 2.8c 0.28 <0.01 Crude fat 1.2 0.8 4.1 1.8 0.9 0.11 <0.01 Crude fiber 23.2b 31.3ab 39.0a 39.4a 39.2a 0.46 <0.01 Ash 17.3a 11.0ab 7.7b 9.8b 14.0a 0.27 <0.01 Means in the same row without a common letter are different at P<0.01. Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) 156 N.R. Kumalasari, L. Abdullah, L. Khotijah, L. Wahyuni, Indriyani, N. Ilman and F. Janato Discussion stem, occurred after 70 DAT, CP% had dropped to low levels by this time with CP% of stems (3.6%) being well This study has provided useful information on changes in below maintenance levels. nutrient concentrations with age in A. gangetica forage. As can be seen in Figure 2, decisions on when to utilize As was expected, increasing age had positive effects on the forage would be a compromise between DM yields of plant height, number of leaves and numbers of secondary both leaf and stem and CP% of these 2 components. While and tertiary branches, resulting in marked increases in dry this is a pot study, it would seem that harvesting between matter yields. Plant height and number of branches in this 40 and 50 DAT would give a reasonable compromise study followed a different pattern from that reported by between DM yield and CP% in the available forage, as Asbur et al. (2018b). Plants in our study were taller and leaf:stem ratio still exceeded 0.8 at 50 DAT. The leaf displayed much greater branching due to being grown in material produced would be of high CP% and should full sunlight (Samedani et al. 2013) as opposed to former provide an excellent supplement to low quality roughage research where plants were grown under palm plantation for ruminants. However, feeding studies with animals are shading (Asbur et al. 2018b). While biomass yields of needed to determine responses of animals when this forage both leaf and stem increased progressively with age, is fed as supplements with other forage sources or as a leaf:stem ratio declined progressively. As can be observed complete ration. in Table 1, the number of primary branches declined from It must be stressed that these data are for single plants 50 to 70 DAT as a result of senescence. grown in polybags and competition between plants would As expected, nutrient concentration in A. gangetica not have been expressed as would occur in swards in the leaves was better than in stems (Table 2). While crude field. Further studies under field situations are needed to protein concentrations (CP%) in both plant parts declined verify that the results obtained in our study represent those progressively from 40 DAT, at 50 DAT CP% still exceeded obtained in commercial situations and how the situation the critical level of 7% for satisfactory functioning of might change when grown under shade in palm rumen microflora (Ansah et al. 2018). Peak protein plantations. concentrations (24.2%) in leaf at 40 DAT was higher than 17.5% reported by Herilimiansyah (2019) for 50 DAT but Acknowledgments DM yields were still relatively low at this stage. Growth for the next 10 days was not rapid and protein concentration of This research was funded by The Ministry of Research, forage declined to a greater degree. While marked Technology and Higher Education of Indonesia through the increases in DM yields of both components, especially Prime Basic Research of University (PDUPT) Grant 2018. 30 20 leaf-CP stem-CP leaf-DM stem-DM 18 25 16 14 20 12 15 10 8 10 6 4 5 2 0 0 30 40 50 70 90 Harvest time (days after transplanting) Figure 2. Effects of harvest time (days after transplanting) on DM yields and crude protein concentrations (% DM) in leaf and stem of Asystasia gangetica. Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) Dry weight (g/plant) Crude protein (%) Evaluation of Asystasia gangetica at different ages 157 References Seminar on Animal Industry, Bogor, Indonesia, 28–30 (Note of the editors: All hyperlinks were verified 28 April 2020.) August 2018. p. 189–192. repo.unand.ac.id/29178 Kumalasari NR; Wahyuni L; Abdullah L. 2018. Germination Adetula OA. 2004. Asystasia gangetica (L.) Anderson. In: of Asystasia gangetica seeds exposed to different source, Grubben GJH; Denton OA, eds. 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Proceeding of the 4th International Proc.Intsem.LPVT-2016-p.284-290 (Received for publication 8 February 2019; accepted 1 April 2020; published 31 May 2020) © 2020 Tropical Grasslands-Forrajes Tropicales is an open-access journal published by International Center for Tropical Agriculture (CIAT), in association with Chinese Academy of Tropical Agricultural Sciences (CATAS). This work is licensed under the Creative Commons Attribution 4.0 International (CC BY 4.0) license. Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) Tropical Grasslands-Forrajes Tropicales (2020) Vol. 8(2):158–161 158 doi: 10.17138/TGFT(8)158-161 Short Communication Pest insects in natural and sown pastures of Paraguay Insectos plagas en pasturas naturales y cultivadas de Paraguay HUMBERTO J. SARUBBI AND MARÍA B. RAMÍREZ Facultad de Ciencias Agrarias, Universidad Nacional de Asunción, San Lorenzo, Paraguay. agr.una.py Abstract Paraguayan livestock production is based mainly on the use of natural and sown pastures as basic cattle feed. Several genera of harmful insects reported in forage grasses can cause damage to both yield and quality of forage. A review of the insect collection of the Plant Protection Area of the Faculty of Agrarian Sciences, National University of Asunción was carried out, in order to prepare a list of insects with incidence in grasses. Then random sampling of different species of Poaceae showing insect damage in open areas of paddocks grazed by cattle was carried out during 2014‒2017 in all Regions of Paraguay. Thirteen different genera and species of pastures were collected and 20 species of insects were identified in the following orders: Hymenoptera (Formicidae family: 5 species); Isoptera (Termitidae: 3 species); Hemiptera (Cercopidae: 6 species; Lygaeidae: 1 species); Lepidoptera (Noctuidae: 2 species); and Orthoptera (Acrididae: 3 species). The most common forms of damage observed in pastures were: leaf consumption (25%), leaf cutting (25%) and leaf yellowing-drying (35%). Keywords: Families, identification, predation, sampling. Resumen En Paraguay la producción pecuaria se basa principalmente en el uso de pasturas naturales y cultivadas como alimento base para ganado vacuno. En el país se han identificado varios géneros de insectos dañinos que pueden ocasionar daños tanto en la cantidad como la calidad de las gramíneas forrajeras. Inicialmente se realizó una revisión de la colección de insectos del Área de Protección Vegetal de la Facultad de Ciencias Agrarias, Universidad Nacional de Asunción, con el objeto de elaborar un listado de insectos con incidencia en pasturas. Posteriormente, durante los años 2014‒2017, se realizaron muestreos al azar en diferentes especies de gramíneas que presentaban daños por insectos en áreas abiertas destinadas al pastoreo de vacunos en los diferentes departamentos de Paraguay. En total fueron colectados 13 diferentes géneros y especies de pastos y se identificaron 20 especies de insectos, de los órdenes Hymenoptera (familia Formicidae, 5 especies); Isoptera (Termitidae, 3 especies); Hemiptera (Lygaeidae y Cercopidae, 1 y 6 especies, respectivamente); Lepidoptera (Noctuidae, 2 especies); y Orthoptera (Acrididae, 3 especies). Los daños más comunes observados en las pasturas fueron daños por consumo de follaje (25%), corte de láminas foliares (25%) y amarillamiento y secado de hojas (35%). Palabras clave: Familias, identificación, muestreo. Introduction ha) and natural pastures (10 million ha) are the primary feed source for cattle, since it is the most economic and Paraguayan cattle ranching has experienced a significant practical approach to meat production (Glatzle and improvement in number and quality in the last 20 years, Stosiek 2001; ARP 2017). This has created an ideal and Paraguay is the seventh largest beef exporter in the environment for the proliferation of different genera of world (ARP 2017). The country currently has 15 million insects, which can be harmful to forage crops (Fowler hectares being used for livestock, and sown (5.6 million 1979; Glatzle 1999; Benítez 2002; Sarubbi 2016). Many ___________ Correspondence: H.J. Sarubbi, Facultad de Ciencias Agrarias, Universidad Nacional de Asunción, Campus Universitario, San Lorenzo, Paraguay. Email: humberto.sarubbi@agr.una.py Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) Insect pests in Paraguayan pastures 159 of these insects are widely distributed over the American Results and Discussion continent and cause a range of symptoms from defoliation to death of plants (Gallo et al. 2002; Brandão et al. 2011). Twenty (20) species of insects in the orders Hymenoptera, Knowing the distribution and potential hosts of insects Isoptera, Hemiptera, Lepidoptera and Orthoptera were and damage caused is an important step in the found on 12 pasture hosts (Table 1). In Hymenoptera, 6 development of adequate management strategies (Picanço were in Formicidae family; in Isoptera, 3 were in et al. 1999; Nakano 2011). Termitidae; in Hemiptera, 1 was in Lygaeidae and 6 in The objective of this work was to identify harmful Cercopidae; in Lepidoptera, 2 were in Noctuidae; and in insects and determine their distribution throughout Orthoptera, 3 were in Acrididae. Paraguay, plant hosts infested and description of damage The Urochloa and Megathyrsus genera represent the caused. most important pasture grasses in the country, and consequently the greatest variety of insects was found on Materials and Methods them. A unique case was observed in Urochloa arrecta × U. mutica (tangola grass), which was the exclusive host First, a review of the insect collection of the Plant Protection of Blissus antillus (grass bug). In relation to the Area of the Faculty of Agricultural Sciences, National occurrence of insects across Regions, some species, such University of Asunción was carried out, in order to produce as defoliating ants and cicadas, cover the whole country, a list of registered pests causing damage to pastures. while other species are confined to certain regions or Subsequently, insect collections were carried out at random, places, such as the locust (Staurorhectus longicornis) and during the years 2014‒2017, in the 4 seasons of the year (1 the cutter ant (Atta vollenweideri) in Chaco. The types of collection per season, 16 in total), on different species of damage to pastures most commonly observed were: Poaceae showing insect damage. The work was carried out leaf consumption 25% [caterpillars (Mocis latipes, in open areas of paddocks destined for cattle grazing in the Spodoptera frugiperda) and locusts], cutting 25% (cutter following Regions: Western Region or Chaco: Alto ants) and leaf yellowing and drying 35% [grass bug and Paraguay (APY), Boquerón (BOQ), Villa Hayes (VHA); spittlebug (Notozulia entreriana and Mahanarva and Eastern Region: Amambay (AMA), Concepción fimbriolata)] (Table 1). (CON), San Pedro (SPE), Canindeyú (CAN), Caaguazú Insects with the highest number of species (12) and (CAG) ), Alto Paraná (APA), Central (CEN), Cordillera distribution were cutter ants and spittlebug as mentioned by (COR), Paraguarí (PAR), Guairá (GUA), Caazapá (CAZ), Fowler (1979), Kidono (1982), Glatzle (1999), Benítez Itapúa (ITA), Misiones (MIS) and Ñeembucú (ÑEE). (2002) and Sarubbi (2016). Valério (2006) and Tolloti et Pasture samples were collected from the following species: al. (2018) consider that spittlebugs are among the most Cenchrus ciliaris (CC), Cenchrus purpureus (CP), Chloris important harmful insects of tropical pastures, attacking gayana (CG), Cynodon nlemfuensis (CN), Digitaria several genera, species and varieties, as observed in this eriantha (DE), Megathyrsus maximus (MM, a range of research, as they were present in the whole territory of cultivars), Paspalum notatum (PN), Urochloa brizantha Paraguay and with a wide host range. (UB, a range of cultivars), Urochloa decumbens (UD), Incidence of Blissus antillus in tangola pasture agrees Urochloa mosambicensis (UM), Urochloa ruziziensis (UR) with reports of Valério et al. (1999) and Fazolin et al. and Urochloa arrecta × Urochloa mutica (UA × UM; (2012), who found that tangola grass (a natural Urochloa tangola grass). hybrid) and Urochloa arrecta were the only hosts of Insect pests were collected, recording date, location, Blissus antillus in Brazil. host and type and extent of damage, were photographed The damage caused by termites is considered indirect and immediately deposited in plastic containers for since this species develops mounds that are obstacles for identification. The collection was manual using an agricultural machinery, causing loss of useful area in the entomological sweep net (50 cm ring diameter and 1 m paddocks. bag length). The samples were transported to the Occurrence of most of these insects is seasonal and Entomology Laboratory and examined with a stereoscope some appear in large numbers at specific times. Some for identification. Insects were identified using the which can cause serious damage are: caterpillars (Mocis following reference sources: Fowler (1979), Kidono latipes), bug (Blissus antillus) and cicadas (Notozulia (1982), Glatzle (1999), Valério et al. (1999), Gallo et al. entreriana and Mahanarva fimbriolata), as was (2002), Sarubbi (2016) and Tolotti et al. (2018). mentioned by Gallo et al. (2002) and Tolotti et al. (2018). Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) 160 H.J. Sarubbi and M.B. Ramírez Table 1. Insect pests (common name in parenthesis) identified in different pasture species of Paraguay. Insect order, family and species Host1 Occurrence (Region and collection site)2 Hemiptera: Cercopidae Deois flavopicta (Salivazo) CN, MM, UB AMA 22°08'03.7" S 56°28'41.4" W SPE 23°44'11.4" S 56°29'38.4" W CAG 24°57'06.9" S 56°21'46.8" W APA 25°27'36.2" S 55°02'34.4" W ITA 27°04'07.2" S 56°36'21.7" W Deois mourei (Salivazo) CN, MM, UB, UD CEN 25°19'41.1" S 57°31'11.0" W SPE 24°05'05.6" S 57°06'17.7" W CAG 25°28'10.8" S 56°32'18.3" W APA 25°26'06.7" S 54°46'37.3" W MIS 27°07'19.8" S 56°46'40.2" W Deois rubropicta (Salivazo) CN AMA 22°40'15.7" S 56°02'41.8" W Deois schach (Salivazo) PN, MM, UB CAG 25°24'37.4" S 55°34'16.6" W Mahanarva fimbriolata (Salivazo) CP, UB AMA 22°07'39.0" S 56°27'47.4" W CON 23°27'44.1" S 57°24'28.7" W SPE 24°02'53.0" S 56°27'33.4" W CEN 25°19'40.8" S 57°31'09.8" W COR 25°14'51.4" S 57°08'32.7" W PAR 25°30'18.1" S 57°10'07.0" W CAG 24°57'55.3" S 56°20'58.7" W CAZ 26°09'30.2" S 56°21'49.5" W APA 25°25'09.7" S 55°23'28.5" W GUA 25°43'09.9" S 56°11'01.5" W MIS 26°59'38.1" S 56°47'25.5" W ITA 26°23'41.0" S 55°30'54.4" W Notozulia entreriana (Salivazo) CC, CN, CG, DE, BOQ 23°26'20.4" S 60°75'12.5" W APY 20°11'03.6" S 59°32'23.8" W MM, UB, UD, UR VHA 24°55'58.2" S 57°33'50.1" W SPE 23°45'02.0" S 56°29'34.2" W PAR 26°08'35.0" S 56°42'49.9" W CEN 25°19'28.8" S 57°31'16.4" W CAG 24°57'08.2" S 56°21'40.9" W APA 25°25'48.6" S 55°22'52.6" W ITA 27°12'03.1" S 56°06'51.8" W Hemiptera: Lygaeidae Blissus antillus (Chinche de las gramíneas) UA×UM (tangola) VHA 23°31'54.0" S 58°36'44.2" W Hymenoptera: Formicidae Acromyrmex heyeri (Akekẽ) PN MIS 27°07'35.6" S 56°41'57.3" W Acromyrmex landolti fracticornis (Akekẽ CC, CN, CG, DE, APY 20°10'59.8" S 59°32'18.0" W VHA 23°31'53.7" S 58°36'06.7" W kapi´i) MM, PN, UB, UD, BOQ 21°58'13.9" S 59°59'59.7" W CON 23°24'23.5" S 57°19'29.0" W UM, UR SPE 23°43'54.8" S 56°29'31.4" W CEN 25°12'24.8" S 57°24'31.7" W COR 25°14'56.0" S 57°08'51.6" W PAR 26°08'38.7" S 56°42'52.1" W GUA 25°43'08.3" S 56°11'01.9" W MIS 27°07'15.0" S 56°46'44.7" W Atta capiguara (Ysaú kapi´i) CN, MM, PN, UB, SPE 23°44'20.7" S 56°29'30.3" W CAG 25°24'14.1" S 55°31'56.1" W UD, UR APA 25°10'24.3" S 54°42'01.2" W Atta laevigata (Ysaú akã vidrio) CN, MM, PN, UB, CON 22°24'58.6" S 56°41'37.6" W AMA 22°40'21.7" S 55°55'25.5" W UD, UR SPE 24°43'13.8" S 56°30'11.3" W Atta vollenweideri (Ysaú chaco) CC, CN, CG, DE, APY 20°15'55.1" S 59°32'42.7"W BOQ 22°00'31.8" S 60°00'15.3" W MM, UM VHA 23°31'60.0" S 58°36'58.2" W Isoptera: Termitidae Cornitermes bequaerti (Kupi´i takuru CN, MM, PN, UB, CON 23°01'33.2" S 56°35'46.4" W CAN 24°21'23.3" S 55°04'11.6" W chimenea) UR SPE 24°20'37.5" S 56°25'13.2" W CAG 25°24'06.7" S 55°48'49.8" W Cornitermes cumulans (Kupi´i takuru) CN, MM, PN, UB, CON 23°24'26.9" S 57°19'36.7" W CAN 24°21'34.3" S 55°04'28.6" W UD, UR SPE 24°22'43.4" S 56°25'07.8" W CEN 25°19'46.5" S 57°31'23.8" W COR 25°04'56.6" S 57°23'22.4" W PAR 25°23'26.2" S 57°03'34.1" W CAZ 26°09'42.5" S 56°21'44.6" W CAG 24°58'48.2" S 56°20'58.9" W APA 25°29'15.5" S 54°49'21.4" W MIS 26°40'39.7" S 57°06'04.4" W Procornitermes striatus (Yvy kupi´i) CN, MM, PN, UB CEN 25°19'43.5" S 57°31'10.0" W Lepitoptera: Noctuidae Mocis latipes (Falsa medidora) CC, CN, DE, MM, APY 22°02'00.1" S 59°53'14.3" W BOQ 21°58'49.4" S 60°00'24.1" W UB MIS 26°34'33.7" S 56°54'46.0" W CAG 24°58'03.7" S 56°21'02.3" W Spodoptera frugiperda (Cogollero del maíz) CC, CN, DE, MM, BOQ 23°15'53.6" S 60°43'53.8" W CAG 24°58'12.1" S 56°21'00.3" W UB, UD, UM, UR PAR 25°27'44.9" S 57°15'57.8" W ÑEE 25°57'07.5" S 57°46'37.8" W APA 25°27'32.4" S 55°02'57.5" W ITA 27°01'10.2" S 55°55'12.6" W Orthoptera: Acrididae Rammatocerus pictus (Langosta) CC, CN, MM APY 20°11'31.4" S 59°31'48.1" W BOQ 21°06'46.5" S 60°31'23.6" W Schistocerca cancellata (Langosta migratoria) CC, CN, CG, MM APY 20°16'36.3" S 59°07'22.0" W BOQ 21°19'15.7" S 60°27'10.6" W Staurorhectus longicornis (Langosta de CC, CN, CG, MM APY 20°11'55.7" S 59°32'03.2" W BOQ 23°14'35.4" S 60°45'49.6" W pastura) 1Hosts: CC (Cenchrus ciliaris), CP (Cenchrus purpureus), CG (Chloris gayana), CN (Cynodon nlemfuensis), DE (Digitaria eriantha), MM (Megathyrsus maximus), PN (Paspalum notatum), UB (Urochloa brizantha), UD (Urochloa decumbens), UA×UM (Urochloa arrecta × Urochloa mutica), UM (Urochloa mosambicensis) and UR (Urochloa ruziziensis). 2Occurrence according to the records of the entomological collection of the Plant Protection Area of the Facultad de Ciencias Agrarias, Universidad Nacional de Asunción and collections made by the authors. Regions: AMA = Amambay; APA = Alto Paraná; APY = Alto Paraguay; BOQ = Boquerón; CAG = Caaguazú; CAN = Canindeyú; CAZ = Caazapá; CEN = Central; CON = Concepción; COR = Cordillera; GUA = Guairá; ITA = Itapuá; MIS = Misiones; ÑEE = Ñeembucú, PAR = Paraguarí, SPE = San Pedro; and VHA = Villa Hayes. Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) Insect pests in Paraguayan pastures 161 Conclusions Berti Filho E; Parra JRP; Zucchi RA; Alves SB; Vendramim JD; Marchini LC; Lopes JRS; Omoto C. 2002. Entomologia This study has provided an overview of the range of agrícola. p. 484‒493. FEALQ, Piracicaba, SP, Brazil. insects which occur in pastures in Paraguay. Whether or Glatzle A. 1999. Compendio para el manejo de pasturas en el not active measures to control them should be undertaken Chaco. Editorial El Lector, Asunción, Paraguay. Glatzle A; Stosiek L. 2001. Perfiles por país del recurso would depend on the extent of damage they cause and the pastura/forraje Paraguay. Food and Agriculture impact on both pasture production and resultant animal Organization of the United Nations (FAO), Rome, Italy. performance. Observations on degree of damage to bit.ly/2xG6zTo pastures under a range of conditions should supply some Kidono H. 1982. Algunas observaciones sobre las cigarritas de information on which to base decisions. las pasturas, salivazo en el Paraguay. Universidad Nacional de Asunción, San Lorenzo, Paraguay. References Nakano O. 2011. Entomologia econômica. p. 280‒293. (Note of the editors: All hyperlinks were verified 5 May 2020.) ESALQ, Piracicaba, SP, Brazil. Picanço M; Leite GLD; Mendes MC; Borges VE. 1999. Attack ARP (Asociación Rural del Paraguay). 2017. Introducción a of Atarsocoris brachiariae Becker, a new pest of pasture- Paraguay y su sector cárnico. ARP, Asunción, Paraguay. lands in Mato Grosso, Brazil. Pesquisa Agropecuária bit.ly/35wlZ9h Brasileira 34:885‒890. (In Portuguese). doi: 10.1590/S0100 Benítez E. 2002. Listado de nombres científicos y vulgares de -204X1999000500022 plagas agrícolas y forestales del Paraguay. Universidad Sarubbi H. 2016. Plagas de pasturas en Paraguay. Universidad Nacional de Asunción, San Lorenzo, Paraguay. Nacional de Asunción, San Lorenzo, Paraguay. Brandão CRF; Maye-Nunes A; Sanhudo CED. 2011. Tolotti A; Azevedo WS; Valiati VH; Carvalho GS. 2018. Taxonomia e filogenia das formigas-cortadeiras. In: Della Cigarrinhas das pastagens en gramineas forrageiras no Lucia TMC, ed. Formigas cortadeiras: Da biología ao Brasil. Editora Evangraff, Porto Alegre, RS, Brazil. manejo. Editora UFV, Viçosa, MG, Brazil. p. 27‒48. Valério JR. 2006. Considerações sobre a morte de pastagens de Fazolin M; Tomazini MJ; Estrela JLV. 2012. Pragas das Brachiaria brizantha cv. Marandu em alguns estados do culturas de importância econômica para o Estado do Acre. Centro e Norte do Brasil: Enfoque entomológico. Documentos 127. Embrapa Acre, Rio Branco, AC, Brazil. Comunicado Técnico 98. Embrapa Gado de Corte, Campo bit.ly/3fp26Fp Grande, MS, Brazil. bit.ly/3fmm2sx Fowler H. 1979. Las hormigas cortadoras del Paraguay de los Valério JR; Vieira JM; Valle LCS. 1999. The occurrence of géneros Atta Fabricius y Acromyrmex Mayr: Bionómico, Blissus antillus Leonard (Hemiptera: Lygaeidae: Blissinae) distribución y sistemática. Informes científicos ICB-UNA in Tangola pasture in Mato Grosso do Sul State, Brazil. 2:30‒57. Anais da Sociedade Entomológica do Brasil 28:527‒529. Gallo D; Nakano O; Silveira Neto S; Carvalho RPL; Baptista GC; (In Portuguese). doi: 10.1590/S0301-80591999000300020 (Received for publication 8 February 2019; accepted 30 April 2020; published 31 May 2020) © 2020 Tropical Grasslands-Forrajes Tropicales is an open-access journal published by International Center for Tropical Agriculture (CIAT), in association with Chinese Academy of Tropical Agricultural Sciences (CATAS). This work is licensed under the Creative Commons Attribution 4.0 International (CC BY 4.0) license. Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) Tropical Grasslands-Forrajes Tropicales (2020) Vol. 8(2):162–166 162 doi: 10.17138/TGFT(8)162-166 Short Communication Pasto Certo® version 2.0 - An application about Brazilian tropical forage cultivars for mobile and desktop devices Pasto Certo® version 2.0 – Una aplicación sobre cultivares brasileños de especies forrajeras tropicales para dispositivos móviles y de escritorio SANZIO CARVALHO LIMA BARRIOS1, CAMILO CARROMEU1, MÁRCIO APARECIDO INÁCIO DA SILVA2, EDSON TAKASHI MATSUBARA2, CACILDA BORGES DO VALLE1, LIANA JANK1, MATEUS FIGUEIREDO SANTOS1, GISELLE MARIANO LESSA DE ASSIS3, LEONARDO LAZARINO CRIVELLARO2, THALLYSON DANCHEN TEIXEIRA GONÇALVES4, JOSÉ MARCOS QUEIROZ JÚNIOR5, ANDERSON RAMIRES CANDIDO6, WYVERSON KIM ROCHA MACHADO6, BEATRIZ TOMÉ GOUVEIA7, ALANA APARECIDA AMARILHA NOBRE2 AND AYHAN LIELL ZANELLA5 1Embrapa Gado de Corte, Campo Grande, MS, Brazil. embrapa.br/gado-de-corte 2Universidade Federal de Mato Grosso do Sul, Campo Grande, MS, Brazil. ufms.br 3Embrapa Acre, Rio Branco, AC, Brazil. embrapa.br/acre 4Universidade para o Desenvolvimento do Estado e da Região do Pantanal – Anhanguera/Uniderp, Campo Grande, MS, Brazil. uniderp.com.br 5Universidade Católica Dom Bosco, Campo Grande, MS, Brazil. site.ucdb.br 6Universidade Estadual de Mato Grosso do Sul (UEMS), Aquidauana, MS, Brazil. uems.br 7Universidade Federal de Lavras, Lavras, MG, Brazil. ufla.br Abstract A brief outline of the second version of Pasto Certo®, released by Embrapa and partners in February 2019, is presented. It is an improved and updated version of Pasto Certo® 1.0, an application that describes Brazilian commercial tropical forage cultivars. The application helps the user to identify and differentiate cultivars, provides recommendations and information on use restrictions of each cultivar, and compares different cultivars in terms of a number of characteristics. In comparison with the first version (published in 2017), new features of Pasto Certo® 2.0 are: (1) 7 cultivars of forage legumes (genera Arachis, Cajanus and Stylosanthes) were added to the original 16 grass cultivars (Urochloa spp. and Megathyrsus maximus); (2) the user can choose between Portuguese, Spanish and English languages; (3) information on commercial seed sources in Brazil is included; (4) a guide to selecting the most suitable cultivar for specific conditions is provided; and (5) the application is available for different platforms (Android, iOS and WEB - www.pastocerto.com). Keywords: Grasses, legumes, Megathyrsus maximus, pastures, software, Urochloa. Resumen Como una ayuda para la selección y manejo de cultivares brasileños de forrajeras, dirigida a productores ganaderos, técnicos, agrónomos, zootecnistas y el comercio de semilla en zonas tropicales, en Febrero 2019 la Empresa Brasileira de Pesquisa Agropecuária (Embrapa) con la colaboración de entidades asociadas, puso a disposición la aplicación Pasto Certo® 2.0, una versión mejorada y actualizada de Pasto Certo® 1.0. La aplicación asiste en la selección de cultivares comerciales, proporciona recomendaciones para cada uno de ellos y suministra información sobre posibles restricciones de uso, teniendo en cuenta las características de los diferentes cultivares. En comparación con la primera versión, ___________ Correspondence: Sanzio Carvalho Lima Barrios, Embrapa Gado de Corte, Av. Rádio Maia, 830, Zona Rural, Campo Grande, CEP 79.106-550, MS, Brazil. Email: sanzio.barrios@embrapa.br Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) Pasto Certo® version 2.0 163 publicada en 2017, nuevas funcionalidades de Pasto Certo® 2.0 son: (1) la inclusión de siete cultivares de leguminosas forrajeras de los géneros Arachis, Cajanus y Stylosanthes, al total de 16 cultivares de gramíneas (Urochloa spp. y Megathyrsus maximus); (2) el usuario puede seleccionar para consulta entre los idiomas portugués, español e inglés; (3) se presenta información sobre fuentes comerciales de semilla en Brasil; (4) se incorporó una herramienta de selección de cultivares para diferentes condiciones específicas; y (5) la aplicación está disponible para varias plataformas (Android, iOS y WEB - www.pastocerto.com). Palabras clave: Gramíneas, leguminosas, Megathyrsus maximus, pasturas, software, Urochloa. Background and Development received, which motivated the development team to release version 2.0. Both the brand and the software Pasto Certo® - Brazil is the world’s leader in tropical forage seed versions 1 and 2 are the property of Embrapa at the ‘Instituto production and exports, with a local market of about US$ Nacional da Propriedade Industrial’ (INPI) in Brazil. 600 million/year. Cultivars of Urochloa and Megathyrsus Representatives of the co-operating institutions agreed that maximus represent more than 90% of this market and Pasto Certo® would be an open-source software with BSD consequently the cultivated pasture area in Brazil, which (Berkeley Software Distribution) license. The application covers roughly 100 million hectares (José 2012). Despite can be downloaded at Google Play and Apple Store or this unquestionable significance there is no easily accessed directly at www.pastocerto.com. accessible platform for farmers, technicians, agronomists, veterinarians and seed dealers, plus other users, which Description describes the main characteristics of Urochloa and Megathyrsus maximus cultivars, either released by Version 2.0 of Pasto Certo® is comprised of 23 forage Embrapa or in the public domain. While this information cultivars (10 of Urochloa, 6 of Megathyrsus maximus, 3 of exists, it is scattered in various types of publications, such Arachis, 2 of Cajanus and 2 of Stylosanthes) and more than as Embrapa series (Valle et al. 2004, 2017; Jank et al. 160 variables, which describe these cultivars, grouped 2017), folders (various Embrapa cultivar information into 6 categories: identity, morphology, agronomy, folders) and several scientific articles, but generally performance under grazing, use in integrated systems and relates to a single cultivar. There are other excellent images of the plant from germination to adult stages. The forage platforms, such as Tropical Forages cultivars are arranged in rectangular cards, represented by (tropicalforages.info/) and Feedipedia (feedipedia.org/), a photograph and the respective common and scientific which describe various species and provide considerable names. To access data on a cultivar, either tap on the related information. However Pasto Certo® offers an photograph or type the cultivar´s common name (or part electronic software platform composed of a web tool and thereof) (Figure 1A). a mobile application for users, which allows quick and Three useful interactive features are available on Pasto integrated access to the characteristics of the main tropical Certo® 2.0: Firstly, a comparison between cultivars for forage cultivars released by Embrapa and others in the different variables, where the user can select up to 4 cultivars public domain. to compare simultaneously for as many variables as are of The software platform was constructed by students of interest (Figure 1B); Secondly, the feature ‘choice of forage the Computer Science College (FACOM) of the Federal cultivars for pasture establishment’, where the user answers University of Mato Grosso do Sul under the auspices of 8 technical questions and is presented with cultivar the Association for the Promotion of Research in Forage suggestions for his/her specific needs (available only for Breeding (UNIPASTO). It followed the steps of Urochloa and Megathyrsus maximus cultivars) (Figure 2); computational requirements, inclusion of forage technical and Thirdly, a contact list of more than 30 partner companies information, software architecture design, construction of in Brazil, indicating where to obtain seeds of these tropical the software itself and finally validation by Embrapa forage cultivars. Several of these companies have employees. At the completion of these steps, the platform subsidiaries in Latin American countries. was created, validated and the mobile application (version The application is user-friendly, has Portuguese, 1.0, initially available only for Android operating Spanish and English versions and is automatically systems) was released to users in March 2017 (Barrios et updated where an internet connection is available once al. 2017). the application is opened and usage starts. However, Since its release, the application gained great acceptance Pasto Certo® 2.0 is designed to work both online and by users and several suggestions for improvements were offline, i.e. does not require internet connection. Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) 164 S.C.L. Barrios et al. A B Figure 1. A) Main screen of Pasto Certo® with figure cards indicating the different cultivars in the app. At the left top is the MENU and at the right top are the interactive functions: compare cultivars; choice of forage cultivars for pasture establishment; where to buy tropical forage seeds in Brazil; and video gallery. B) Screen showing the comparison between Megathyrsus maximus cvv. Mombaça, BRS Zuri, BRS Tamani and BRS Quênia for several variables. The application can be accessed free of charge and is Moreover, the application provides the capability for the available on 3 different platforms (Android, iOS and user to send questions and suggestions to the WEB - www.pastocerto.com). Pasto Certo® is thus an administrative team responsible for Pasto Certo®, and efficient tool to assist users in the comparison, choice, thus to contribute to the continuous improvement of the establishment and management of tropical pastures. package. Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) Pasto Certo® version 2.0 165 A B Figure 2. A) Main screen of “choice of forage cultivars for pasture establishment” function. Six of the 8 questions (tolerance of waterlogging, fertility requirement, technological level, leaf diseases, forage production in the dry season, resistance to pasture spittlebugs, frost tolerance and rainfall) are shown on the screen. This function is currently available only for Urochloa and Megathyrsus maximus cultivars. B) Screen showing an example of output from the “choice of forage cultivars for pasture establishment” function, based on the input (responses) of a user. Perspectives inclusion of other forage genera (Andropogon, Paspalum and Cenchrus); and 2 new interactive functions. Pasto Certo® 2.0 has had a broad public acceptance, confirmed by the positive evaluation of the application Acknowledgments and number of downloads surpassing 30 thousand. Moreover, improvements and adjustments have been The authors are grateful for the financial support given by requested by users, which will be incorporated into the Association for the Promotion of Research in Forage version 3.0. This new version is already under Breeding – UNIPASTO (Associação para o Fomento à construction and the following items will be incorporated: Pesquisa de Melhoramento de Forrageiras Tropicais). Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) 166 S.C.L. Barrios et al. References agronegócio. Anuário 2012. Associação Brasileira de (Note of the editors: All hyperlinks were verified 19 May 2020.) Sementes e Mudas (Abrasem), Brasília, DF, Brazil. p. 22–23. Valle CB do; Euclides VPB; Pereira JM; Valério JR; Pagliarini Barrios SCL; Carromeu C; Silva MAI da; Santos MF; Valle CB MS; Macedo MCM; Leite GG; Lourenço AJ; Fernandes do; Jank L. 2017. Pasto Certo – versão 1.0® aplicativo para CD; Dias Filho MB; Lempp B; Pott A; Souza MA de. 2004. dispositivos móveis sobre forrageiras tropicais. O capim-xaraés (Brachiaria brizantha cv. Xaraés) na Comunicado técnico No. 142. Embrapa Gado de Corte, diversificação das pastagens de braquiária. Série Campo Grande, MS, Brazil. bit.ly/2ZnFShB Documentos No. 149. Embrapa Gado de Corte, Campo Jank L; Andrade CMS de; Barbosa RA; Macedo MCM; Grande, MS, Brazil. goo.gl/dQXGwU Valério JR; Verzignassi JR; Zimmer AH; Fernandes CD; Valle CB do; Euclides VBP; Montagner DB; Valério JR; Santos MF; Resende RMS. 2017. O capim-BRS Quênia Mendes-Bonato AB; Verzignassi JR; Torres FZV; Macedo (Panicum maximum Jacq.) na diversificação e MCM; Fernandes CD; Barrios SCL; Dias Filho MB; intensificação das pastagens. Comunicado técnico No. Machado LAZ; Zimmer AH. 2017. BRS Ipyporã (“belo 138. Embrapa Gado de Corte, Campo Grande, MS, começo” em guarani): Híbrido de Brachiaria da Embrapa. Brazil. goo.gl/RHsH1v Comunicado técnico No. 137. Embrapa Gado de Corte, José MR. 2012. Forrageiras: Uma grande parceira para o Campo Grande, MS, Brazil. bit.ly/2ZkfhlJ (Received for publication 1 April 2020; accepted 18 May 2020; published 31 May 2020) © 2020 Tropical Grasslands-Forrajes Tropicales is an open-access journal published by International Center for Tropical Agriculture (CIAT), in association with Chinese Academy of Tropical Agricultural Sciences (CATAS). This work is licensed under the Creative Commons Attribution 4.0 International (CC BY 4.0) license. Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775) TGFT Editorial Team A.A. 6713, Km 17 Recta Cali-Palmira, Cali, Valle del Cauca, Colombia. Phone: +57 2 4450100 Ext. 3084 Email: CIAT-TGFT-Journal@cgiar.org