Page 1 of 24 December 2023 Ben Lukuyu1, Molly Allen2, Peggy Karimi3, Ronnie Ahumuza1, Martha Businge1 and Chris Jones1 1International Livestock Research Institute 2National Livestock Resources Research Institute (NaLIRRI) 3Alliance of Bioversity and CIAT 2 Affiliations Protocol for multilocation trials Page 2 of 24 ©2023 This publication is copyrighted by the International Livestock Research Institute (ILRI). It is licensed for use under the Creative Commons Attribution 4.0 International Licence. To view this licence, visit https://creativecommons.org/licenses/by/4.0. Unless otherwise noted, you are free to share (copy and redistribute the material in any medium or format), adapt (remix, transform, and build upon the material) for any purpose, even commercially, under the following condition: ATTRIBUTION. The work must be attributed, but not in any way that suggests endorsement by ILRI or the author(s). NOTICE: For any reuse or distribution, the licence terms of this work must be made clear to others. Any of the above conditions can be waived if permission is obtained from the copyright holder. Nothing in this licence impairs or restricts the author’s moral rights. Fair dealing and other rights are in no way affected by the above. The parts used must not misrepresent the meaning of the publication. ILRI would appreciate being sent a copy of any materials in which text, photos etc. have been used. Citation: Ben Lukuyu,B., Allen,M., Karimi,P., Ahumuza,R. Businge, M., and Jones, C. 2023. Evaluation of improved forage varieties in selected countries in East Africa (Kenya and Uganda). Nairobi, Kenya: International Livestock Research Institute (ILRI). Acknowledgments This work was conducted as part of the CGIAR Initiative on Sustainable Animal Productivity for Livelihoods, Nutrition and Gender inclusion (SAPLING). CGIAR research is supported by contributions to the CGIAR Trust Fund. CGIAR is a global research partnership for a food-secure future dedicated to transforming food, land, and water systems in a climate crisis. https://creativecommons.org/licenses/by/4.0 https://www.cgiar.org/funders/ https://www.cgiar.org/funders/ Page 3 of 24 Contents Contents ............................................................................................................................................................................ 3 Project sites ....................................................................................................................................................................... 4 Evaluation of forage species/cultivars ............................................................................................................................... 4 Evaluation of grasses ......................................................................................................................................................... 5 Methodology ................................................................................................................................................................. 5 Sowing and seed rate: ................................................................................................................................................... 7 Plot management .......................................................................................................................................................... 8 Evaluation of lablab purpureus varieties ...................................................................................................................... 9 Data collection ............................................................................................................................................................ 11 Annex 1: meteorological data for experimental site ....................................................................................................... 13 Annex 2: evaluate number of plant population, tillers numbers, height, pests and diseases incidence in different forage cultivars. ............................................................................................................................................................... 14 Annex 3: Evaluate dry matter yield of forage species /cultivars. .................................................................................... 16 Annex 5: Example of farmer participatory evaluation sheet for evaluating forage species / cultivar(s) ........................ 17 Annex 5: Inputs-use data sheet ....................................................................................................................................... 20 Annex 6: Labor record sheet ........................................................................................................................................... 21 Annex 7: Monitoring grass productivity on-farm ............................................................................................................ 22 Annex 8: Feeding trial data record sheet ........................................................................................................................ 24 Page 4 of 24 Project sites Kenya: Nyandarua, Makueni, Kakamega counties. Uganda: Lira, Mbarara, Mukono districts Experimental locations: The experiments will be established at three National Agricultural Research Organization (NARO) zonal research stations namely. Mbarara Zonal Agricultural Research and Development Institute, Mbarara Ngetta Zonal Agricultural Research and Development Institute, Lira National Livestock Resources, Research Institute (NaLIRRI), Nakyesasa, However, since the research stations are far from project sites where up-scaling will be conducted, famers will be transported to the zonal stations to conduct focused group discussions to determine farmers’ preferences of test forages. Evaluation of forage species/cultivars The evaluation of forage cultivars will be carried out in the zonal Research Stations under the supervision of experts from the respective research stations. The performance of forage species/cultivars will be assessed by the farmers and other stakeholders visiting the experimental plots at different stages (e.g., at establishment, full vegetative stage and maturity). Information about soil type and nutrient level of experimental plot will be collected pre- and post- experiment. GPS coordinates and altitude will be recorded for all experimental sites, and monthly rainfall and temperature data will be collected for the experimental period. Land preparation: Ploughing and harrowing will be done twice to a fine tilth. All the perennial grasses should be eliminated. Soil sampling: Before the establishment of the trial site, soil samples from the experimental area should be collected at depths of 0-20 and 20-50. Subsequently, at least one watering should be carried out in the area and if necessary, subsoiling of the soil. After this procedure, the desiccation of the invasive plants that emerge in this area will be performed. Later, grading will be done to level the area, if necessary, one more watering will be made before this procedure. Limestone will be applied according to the need of the soil, considering the goal of increasing the saturation by bases (V%) to 60%, considering the layer 20-40 cm. The capping will be carried out in cover 90 days before sowing. The grasses and legumes in Table 1 are the ones to be tested. Table 1: Grasses and legumes to be tested Legumes Grasses Lablab-Janzi Rhodes grass-Katambora Lablab-Highworth Rhodes grass-Tolgar Page 5 of 24 Lablab-Maridadi (local check Kenya) Panicum Maximum -Mombasa Lablab (Local check Uganda) Brachiaria Cobra Cowpea 1 (ILRI) Rhodes grass (local check) Cowpea 2 (local check) Panicum (local check) Crotalaria juncea (Sunn hemp) Brachiaria Mulato (local check) Evaluation of grasses Methodology Treatments The performance of four perennial varieties will be evaluated in the participating countries to identify suitable cultivars to a given agro ecology or their stability across all the agroecologies. The perennial forages will include two cultivars of Rhodes grass vv. Katambora and cv. Tolgar, Brachiaria Cobra and Panicum Maximum cv. Mombasa from Papalotla seed company in Mexico. One Napier grass variety will be included in the evaluation as local check. Design Randomized Complete Block Design (RCBD) with 3 replications as shown in Table 1 Table 2 and 3). Table 2: Field layouts for grasses Lake Victoria crescent (MUZARDI) Uganda/ Kakamega, Kenya REP 1 Local Rhodes Panicum maximum (Cv Mombasa) Brachiaria Mulato check Rhodes grass Cv Tolgar Local Panicum Cobra Rhodes grass Cv Katambora REP 2 Rhodes grass Cv Tolgar Cobra Local Rhodes Panicum maximum (Cv Mombasa) Rhodes grass Cv Katambora Local panicum Brachiaria Mulato- check REP 3 Cobra Rhodes grass Cv Katambora Rhodes grass Cv Tolgar Brachiaria Mulato- Check Panicum maximum (Cv Mombasa) Local Rhodes Local Panicum Southwestern rangelands (Mbarara) Uganda/Makueni Kenya REP 1 Local Panicum Local Rhodes Cobra Rhodes grass Cv. Katambora Panicum maximum Cv Mombasa Rhodes grass Cv Tolgar Brachiaria Mulato- check Page 6 of 24 REP 2 Cobra Panicum maximum Cv Mombasa Rhodes grass Cv Katambora Brachiaria Mulato- Check Local Panicum Local Rhodes Rhodes grass Cv Tolgar REP 3 Brachiaria Mulato- check Rhodes grass Cv Tolgar Cobra Local Panicum Rhodes grass Cv Katambora Panicum Maximum Cv Mombasa Local Rhodes Table 3: Field layout for legumes Lake Victoria crescent MUZARDI) Uganda/ Kakamega, Kenya Rep 1 Local cowpea Sunhemp Janzi Cowpea Highworth Locl lablab Rep 2 Cowpea Highworth Local lablab Janzi Sunhemp Local cowpea Rep 3 local lablab Janzi Local cowpea Sunhemp Cowpea Highworth Southwestern Rangelands (Mbarara) Uganda/ Makueni, Kenya Rep 1 Local Cowpea Local Lablab Sunhemp Highworth Cowpea Janzi Rep 2 Highworth Sunhemp Cowpea local cowpea Janzi Local lablab Northern grasslands (Ngetta) Uganda/ Nyandarua Kenya REP 1 Brachiaria Mulato- check Rhodes grass Cv Katambora Local Rhodes Panicum maximum Cv Mombasa Cobra Local Panicum Rhodes grass Cv Tolgar REP 2 Panicum maximum Cv Mombasa Cobra Rhodes grass Cv Katambora Brachiaria Mulato- check Local Rhodes Rhodes grass Cv Tolgar Local Panicum REP 3 Rhodes grass Cv Tolgar Cobra Rhodes grass Cv Katambora Local Panicum Brachiaria Mulato- check Panicum maximum Cv Mombasa Local Rhodes Page 7 of 24 Rep 3 Sunhemp Local Cowpea Janzi local lablab Cowpea Highworth Northern grass lands (Ngetta) Uganda/ Nyandarua, Kenya Rep 1 Cowpea Local Cowpea Highworth Janzi Local Lablab Sunhemp Rep 2 Highworth local lablab local cowpea Sunhemp Janzi Cowpea Rep 3 Janzi Sunhemp Highworth Cowpea Local Lablab Local Cowpea Sowing and seed rate: How to plant Panicum grass Prepare a good seed bed. Drill (furrows) at 50cm apart or dig holes 15-20 cm deep at a spacing of 50 x 50 cm. Seed rate 2-3kg/ha or 0.8-1.2kg/acre At planting, apply phosphorus at 40 kg N/ha or 36 kg N/acre and Nitrogen at 50kg/ha. Apply one soda bottle-top of DAP/TSP or 2 bottle-tops of TSP fertiliser per hole and mix. Alternatively, add 1-2 handfuls of well-cured farmyard manure (FYM) per hole (4 tonnes FYM/acre) at planting Using seeds - broadcast seed mixed with dry soil/sand for better spread. Using Splits – Plant 2-3 Giant panicum grass root splits pe hole at the onset of rains at a spacing of 50 x 50cm. How to plant Brachiaria grass by direct sowing seeds in the field Brachiaria grass should be planted in a well-prepared seedbed since the seeds are very small. It is advisable to harrow the field to get a desired fine seed bed. If there are notorious weeds e.g. couch grass spray with herbicide such as Round-Up to kill the weeds before ploughing. Make shallow holes (1-2 cm) at 30cm apart between hills/hole and 45 cm between rows. Apply NPK or DAP fertilizer at 200 kg/ha = 80kg/acre Use seed rate of 8 kg/ha which is 3.2 kg/acre. Page 8 of 24 Place 4-5 seeds per hole i.e. pinch with two fingers The seeds should not be buried below 2 cm of depth. How to plant Rhodes grass Sow when the soil is loose (dry). Make furrows 25 cm apart, using a peg to measure. Apply nitrogen fertilizer, preferably during heavy rains, at the rate of 100 kg per hectare. Drill the seeds in the furrows, at a seed rate of 12 kg per hectare (2.5 acres). That means 1.2 kg will sow 0.1 ha or 1/4 acre. Cover the seeds lightly, for example by placing light tree branches over the furrows. For the local checks, use the recommended seed rate. In case of vegetative materials, the spacing between rows should be maintained at 50 cm and 20 cm within the row. For Napier grass production refer fact sheet link Napier/elephant For Rhodes grass refer fact sheet link https://worldagroforestry.org/publication/rhodes-grass For Brachiaria refer fact sheet link https://www.kalro.org/kcsap/docs/bracharia.pdf Sowing time: Preferably at onset of rains for direct seeding. If vegetative materials are used, it is advisable to transplants 1-2 weeks after onset of the rains to make sure the splits are actively growing. Basal fertilizer: Apply fertilizer at the rate of 100:60:30 kg N: P2O5: K2O / ha. In addition, use well decomposed farmyard manure / compost at the rate of 10 ton/ha. Land preparation: Plough and harrow the land twice to a fine tilth. All the perennial grasses should be eliminated preferably using herbicides. Plot management Top dressing fertilizer: Twelve weeks after establishment, apply nitrogenous fertilizer at a rate of 100 kg N/ha per year in two equal split doses. Weed control: Use hand hoe to remove the weeds from the plots. Alternatively, use any recommended herbicides to control broad leaved weeds. Pest and diseases control: Pests and diseases will NOT be controlled in the evaluation plots since important data will be collected to assess their attack on tested forage species/cultivars. https://cgspace.cgiar.org/bitstream/handle/10568/109013/Napier_flyer.pdf?sequence=6&isAllowed=y https://worldagroforestry.org/publication/rhodes-grass https://www.kalro.org/kcsap/docs/bracharia.pdf Page 9 of 24 Research data collection (for all varieties) Rainfall and weather data: Collect rainfall, temperature (mean maximum and minimum) and relative humidity data monthly from the nearest meteorological station for the entire experimental period (Annex 2). Sowing date: Take note on date of sowing/transplanting. Standardization cut: To maintain uniformity on the initial growth among forage species/ cultivars tested perform standardization harvest at six weeks of germination by cutting 5 cm above the ground level. Then take following observations on regrowth. Plant population: Count plants within a 1m x 1m frame, randomly placed over the 2 central rows at 4 weeks interval during establishment phase and at 8 weeks interval during the production phase (Annex3). Number of tillers: Count the number of tillers per plant from 4 plants within the 1 x 1 m frame after every 4 weeks during the establishment phase and after every 8 weeks (at harvest) during the production phase (Annex 2). Plant height: Similarly measure the height of 4 plants within the 1 x 1 m frame at the same interval as tiller numbers above (Annex 3). Pests and diseases: Assess pests and diseases incidences and record data every month throughout the experimental period (Annex 2). It is advisable to take the digital photographs of pest and diseases in each forage species /cultivars. Dry matter yield: The first dry matter yield will be taken at maturity from 2 x 2 m2 net harvest area per plot, weigh the harvested fresh material and collected a sub-sample (400-500g) and dry at 105oC for 48 hours for dry weight determination. Conduct subsequent harvests after every 8 weeks (Annex 4). Analysis for nutritive values: Each forage species/cultivar evaluated will be subjected to nutritive value analysis including Crude Protein (CP), Neutral Detergent Fiber (NDF), Acid Detergent Fiber (ADF), Acid Detergent Lignin (ADL), Metabolizable Energy (ME), Ash, Calcium (Ca), Phosphorus (P) and others. For this purpose, herbage collected from 8 weeks harvest in wet and dry season of a year will be used. Where applicable, forage samples will be subjected to Near Infrared Spectroscopy (NIRS) analysis to determine the nutritive values. Evaluation of Lablab purpureus varieties Treatments: The Lablab purpureus in the evaluation will include two accessions (14423 and 14439) identified in the multi-location trials in Ethiopia. Two local checks, one each in Kenya and Uganda will be included in the evaluation. The lablab genotypes in the evaluation are best bet genotypes identified for lowland rainfed conditions in various locations of Ethiopia for two years. To verify the adaptability for different Page 10 of 24 environments the identified promising lablab genotypes will be evaluated under different climatic conditions and environments in Kenya and Uganda. Design Two advanced lablab genotypes with different maturity time and two local check lablab cultivars (1 from Kenya and from Uganda) will be selected for multilocation variety trials across 3 different agroecology/ environments in both Kenya and Uganda shown in Table 4. Table 4: Planting materials No Accessions Remark 1 Highworth (ILRI No. 147) From ILRI Addis 2 Jhansi (ILRI No. 652) From ILRI Addis 3 Local check From Eldoret Kenya 4 Local check From NaLIRRI, Uganda Randomized Complete Block Design (RCBD) with three replications will be used for the study. The plot size for each genotype will be 3.75m width and 4.5m length to allow six rows and 10 plants within a row. Spacing between plants and rows is 0.5m and 0.75m respectively. The distance between plots 0.75m to 1m and distance between replications (Blocks) 1.5m to 2m as shown in Table 5. Page 11 of 24 Table 5: Field layout Data collection Three middle rows will be used for measurement leaving the edge rows as border. The following are the parameters that will be measured at final harvest: plant height, biomass yield (fresh and dry) and feed quality traits (NDF, CP, IVOMD and Metabolizable Energy). Lablab production fact sheet Sample collection for nutritional analysis The determination of the nutritional quality of the forages will be carried out at least two times in each year of evaluation; an analysis in the wet period and the second analysis in the dry season. The transition periods between seasons should be avoided. Collect samples in the most characteristic period of the rainy and dry seasons. The collection of samples for nutritional analysis will always take place concurrently with an evaluation of forage biomass production. As a result, the same sampled fodder will be used. Chop the forage harvested for biomass estimation with the aid of a panga or machete, into approximate particle sizes of 2 - 5 cm. Sample two to three points of the chopped forage. Mix the two samples thoroughly. From this sample take a subsample of approximately 200g for nutritional analysis. This procedure should be performed for all replicates, so four subsamples of each evaluated forage and consequently four samples will be collected per variety. Transfer samples into a paper bag, with known weight and weigh green matter weight of the sub sample. After this process the sub sample should be taken to the lab for drying at 60°C for 72 hours or until the weights stabilize. At the end of the drying process, the subsample will be weighed, disregarding the weight of the paper bag, to determine the dry weight and calculated the percentage of dry matter. After drying, samples should be sent immediately to the nutritional analysis laboratory, for determination of crude protein (CP), neutral detergent fiber (NDF), acid detergent fiber (ADF), in vitro dry matter digestibility (DIVMS). 17m Rep 1 Rep 2 Rep 3 27.5m https://cgspace.cgiar.org/bitstream/handle/10568/110316/Lablab_flyer.pdf?sequence=1&isAllowed=y Page 12 of 24 Participatory evaluation of forage cultivars in the trials Farmers will be invited to the MLT sites at the first harvest to assess farmer preferences of the forages under test. The procedure will be as follows. Invite 20-25 farmers (with at least 30% representation of women farmers) to the experimental site. Conduct an ice breaking session, introduction and explain to them the objective of the exercise. Develop forage selection criteria with farmers through focus group discussions. First, let the farmers list all the criteria they consider when selecting forages for their livestock and then rank the criteria in order of importance using a pairwise ranking matrix. Provide each farmer with an evaluation score data sheet and train them on how to use the Likert scale (1- 4) where 1=poor 2=fair 3=good and 4=very good; to rate each cultivar. Divide the farmers into group of 10 people. Guide them through a specific route within the experimental field, to score each cultivar using the apparent visible criteria (identified in iii above) in the data sheet. The participatory evaluation will be carried out three times based on the growth and vigour of the grasses; (i) during the wet season when the grass is lush , (ii) at the beginning of the dry season and (iii) during the peak of dry season when growth is poor (Annex 4). Identify the preferred varieties by analysing data using appropriate statistical software. Select farmers/farmer groups for bulking the planting material. Prepare an upscaling plan for better performing forages (for 2024). Page 13 of 24 Annex 1: Meteorological data for experimental site Site: _____________________ Month Rainfall (mm) Temperature (°C) Relative humidity (%) Remarks Minimum Maximum Jul 2022 Aug 2022 Sept 2022 Oct 2022 Nov 2022 Dec 2022 Jan 2023 Feb 2023 Mar 2023 Apr 2023 May 2023 Jun 2023 Jul 2023 Aug 2023 Sept 2023 Oct 2023 Nov 2023 Dec 2023 Page 14 of 24 Annex 2: Evaluate number of plant population, tillers numbers, height, pests and diseases incidence in different forage cultivars Site: _____________________ Date of evaluation ______________ Weeks _____ Treatment (Grass species and cultivar) _______________________________________ Parameter Definition Units REP 1 REP 2 REP 3 REP 4 Plant numbers Plant numbers Plant numbers Plant numbers 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1. Plant numbers Within 1m x 1m quadrat No 2. Tiller numbers Tillers per plant No 3. Plant height Base of plant to flag tip excluding inflorescence cm 4. Insect Incidence 0-5 where 0=no insect pest, 1=few plants have insect and 5=75% of plants have insects Rating (0-5) 5. Extent of insect damage 1-5 where, 1=slight damage on plant(s) that has/have insect and 5=damage is severe on plant(s) that has/have insects. Rating (1-5) 6. Disease Incidence 0-5 where 0=no disease present, 1=few plants have disease and 5=75% of plants are diseased Rating (0-5) 7. Extent of disease damage 1-5 where 1=slight damage on plant(s) that has/have disease Rating (1-5) Page 15 of 24 and 5=damage is severe on plant(s) that has/have disease. Page 16 of 24 Annex 3: Evaluate dry matter yield of forage species /cultivars Site: _____________________ Date of evaluation ______________ Weeks _____ Forage s Fresh weight (Main sample) Fresh weight (sub- sample) Dry weight (sub-sample) REP 1 REP 2 REP 3 REP 4 REP 1 REP 2 REP 3 REP 4 REP 1 REP 2 REP 3 REP 4 Page 17 of 24 Annex 5: Example of farmer participatory evaluation sheet for evaluating forage species / cultivar(s) Name of Station: ________________________________ Site: __________________ Date: _____________ Gender: ________________ (Male/Female) Age: _____________ (years) Plot Species/cultivar Selection criteria (√) tick one for each criterion 1=Poor 2=Fair 3=Good 4=Very good 1 Drought tolerant Herbage yield High milk yield Soil erosion control Height at harvest Disease tolerant Pest tolerant Palatability Growth habit 2 Drought tolerant Herbage yield High milk yield Soil erosion control Height at harvest Disease tolerant Pest tolerant Palatability Growth habit 3 Drought tolerant Herbage yield High milk yield Soil erosion control Height at harvest Disease tolerant Pest tolerant Palatability Growth habit 4 Drought tolerant Page 18 of 24 Plot Species/cultivar Selection criteria (√) tick one for each criterion 1=Poor 2=Fair 3=Good 4=Very good Herbage yield High milk yield Soil erosion control Height at harvest Disease tolerant Pest tolerant Palatability Growth habit 5 Drought tolerant Herbage yield High milk yield Soil erosion control Height at harvest Disease tolerant Pest tolerant Palatability Growth habit 6 Drought tolerant Herbage yield High milk yield Soil erosion control Height at harvest Disease tolerant Pest tolerant Palatability Growth habit 7 Drought tolerant Herbage yield High milk yield Soil erosion control Height at harvest Disease tolerant Pest tolerant Palatability Growth habit 8 Drought tolerant Page 19 of 24 Plot Species/cultivar Selection criteria (√) tick one for each criterion 1=Poor 2=Fair 3=Good 4=Very good Herbage yield High milk yield Soil erosion control Height at harvest Disease tolerant Pest tolerant Palatability Growth habit 9 Drought tolerant Herbage yield High milk yield Soil erosion control Height at harvest Disease tolerant Pest tolerant Palatability Growth habit Page 20 of 24 Annex 5: Inputs-use data sheet Site: ____________________________________ Name of location _____________________ Name of farmer: ___________________________________Date: __________ Plot/Species / Cultivar Input used (1=Fertilizer, 2=Manure, 3=Herbicide) Source (1=Own, 2=Bought) Quantity used (Specify unit) Price per unit Total cost of input e.g., Urochloa brizantha cv. Piata (t1) N.B: Please indicate inputs used in each plot or species/variety e.g., fertilizer, manure, herbicide. Page 21 of 24 Annex 6: Labor record sheet Site: ____________________________________ Name of location _____________________ Name of farmer: __________________________ Size of experimental plot: __________(m2) Date Operation Number of persons Labor type Hired=H Family=F Gender M/F Hours/min worked. If hired labor, cost per day Land preparation Planting First weeding Second weeding Third weeding Harvesting Page 22 of 24 Annex 7: Monitoring grass productivity on-farm Site: ....................................................... Date: ................................................... The grass will be sampled using the nestling sampling procedures. Three points for each forage cultivar will be harvested in each farm; where the growth is best, medium growth and poor growth using 1 m x 1 m quadrant and the fresh weight taken separately. A sub-sample will be taken for drying to determine the dry matter yield. Name of Farmer: _______________________________________ Plot Fresh weight of main sample (kg) Fresh weight of sub-sample (kg) Dry weight of sub- sample (kg) Grass height Grass colour What farmer like about the grass What farmer do not like about the grass Suggestion on what farmers would like about the grass Forage species/cultivar: Best growth Medium growth Poor growth Forage species/cultivar: Best growth Medium growth Poor growth Forage species/cultivar: Best growth Medium growth Poor growth Medium growth Page 23 of 24 Poor growth Page 24 of 24 Annex 8: Feeding trial data record sheet Name of farmer: _________________________________ Site: ________________________________ Name of location: ___________________ Name of buffalo/cow: __________________________ Last calving date:___________ Day Type of local forage used Amount (kg) Type of concentrate used Amount (kg) Amount of milk (Litres) Morning Evening 1 2 3 4 5 6 7 Improved forage species/cultivar Day Cultivar Amount Type of concentrate used Amount Amount of milk (Litres) Morning Evening 8 9 10 11 12 13 14