Alliance Bioversity-CIAT Research Online Accepted Manuscript Enteric methane mitigation and fermentation kinetics of forage species from Southern Mexico: in vitro screening The Alliance of Bioversity International and the International Center for Tropical Agriculture believes that open access contributes to its mission of reducing hunger and poverty, and improving human nutrition in the tropics through research aimed at increasing the eco-efficiency of agriculture. The Alliance is committed to creating and sharing knowledge and information openly and globally. We do this through collaborative research as well as through the open sharing of our data, tools, and publications. Citation: Valencia-Salazar, S.; Jiménez-Ferrer, G.; Arango, J.; Molina-Botero, I.; Chirinda, N.; Piñeiro-Vázquez, A.; Jiménez-Ocampo, R.; Nahed-Toral, J.; Kú-Vera, J. (2021) Enteric methane mitigation and fermentation kinetics of forage species from Southern Mexico: in vitro screening. Agroforestry Systems 95:293–305. ISSN: 0167-4366 Publisher’s DOI: https://doi.org/10.1007/s10457-020-00585-4 Access through CIAT Research Online: https://hdl.handle.net/10568/110990 Terms: © 2021. The Alliance has provided you with this accepted manuscript in line with Alliance’s open access policy and in accordance with the Publisher’s policy on self-archiving. This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. You may re-use or share this manuscript as long as you acknowledge the authors by citing the version of the record listed above. You may not use this manuscript for commercial purposes. For more information, please contact Alliance Bioversity-CIAT - Library Alliancebioversityciat-Library@cgiar.org Agroforestry Systems Enteric methane mitigation potential of forage species from Southern Mexico --Manuscript Draft-- Manuscript Number: AGFO-D-20-00077R1 Full Title: Enteric methane mitigation potential of forage species from Southern Mexico Article Type: Original Research Keywords: Agroforestry; greenhouse gases; Lacandon Rainforests; Mitigation strategies; Silvopastoral systems Corresponding Author: Sara Stephanie Valencia-Salazar Colegio De La Frontera Sur San Cristobal de las Casas, MEXICO Corresponding Author Secondary Information: Corresponding Author's Institution: Colegio De La Frontera Sur Corresponding Author's Secondary Institution: First Author: Sara Stephanie Valencia-Salazar First Author Secondary Information: Order of Authors: Sara Stephanie Valencia-Salazar Guillermo Jiménez-Ferrer Jacobo Arango Isabel Molina-Botero Ngonidzashe Chirinda Angel Piñeiro-Vázquez Rafael Jiménez-Ocampo José Nahed-Toral Juan Ku-Vera Order of Authors Secondary Information: Funding Information: CGIAR Dr Sara Stephanie Valencia-Salazar Abstract: In tropical regions worldwide there is a variety of forage species that have the capacity to improve cattle diet and reduce CH 4  emissions. A screening study was conducted to investigate the nutrient quality, phytochemical composition,  in vitro  gas and CH 4  production of fifteen tropical multipurpose forage species from southern Mexico. The results indicated that the highest crude protein (CP) and  in vitro  digestibility was found in  Gliricidia sepium  with 264 g/kg -1  dry matter (DM) and 709 g/kg -1  DM respectively.  Bursera simaruba  had the lowest CH 4  production with 3.924 mg/g - 1 incubated organic matter (IOM) and 9.077 mg/g -1  degraded organic matter (DOM), condensed tannin (CT) content of 20% and relative low digestibility of 471 g/kg -1  DM. Results found in this study indicate that several species in tropical regions in Mexico are potential candidates in mitigating CH 4  production and can be used as additive or supplementary feed to improve diet quality in cattle raised under grazing conditions in the tropics. Response to Reviewers: Dear Editor, The manuscript has been improved. The species has been cited in the material and methods section. The number of figures and some of the references were modified. Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation Manuscript Click here to download Manuscript Valencia-Salazar et al. 2020. June 13, 2020.docx Click here to view linked References 1 Enteric methane mitigation potential of forage species from Southern Mexico 2 Sara Valencia-Salazar1*, Guillermo Jiménez-Ferrer1, Jacobo Arango2, Isabel Molina-Botero3, Ngonidzashe 3 Chirinda2, Angel Piñeiro-Vázquez4, Rafael Jiménez-Ocampo5, José Nahed-Toral1, Juan Kú-Vera5 4 1 The Southern Border College (ECOSUR), Department of Agriculture, Society and Environment, Chiapas, 5 Mexico. 6 2 International Center for Tropical Agriculture (CIAT), Valle del Cauca, Colombia. 7 3 Agrarian National University La Molina, Department of Nutrition, Faculty of Animal Science, Lima, Peru. 8 4 National Technological of Mexico. I.T. Conkal, Conkal, Yucatan, Mexico. 9 5 Faculty of Veterinary Medicine and Animal Science, University of Yucatan, Mexico. 10 11 *Correspondence: 12 Sara S. Valencia-Salazar 13 The Southern Border College (ECOSUR), Department of Agriculture, Society and Environment, San 14 Cristobal de las Casas, Chiapas, Mexico. 15 saraudea@gmail.com 16 17 18 19 20 21 22 23 24 Abstract 25 In tropical regions worldwide there is a variety of forage species that have the capacity to improve cattle diets 26 and reduce enteric CH4 emissions. A screening trial was conducted to investigate the nutrient and phytochemical 27 composition, in vitro gas and CH4 production of fifteen tropical multipurpose forage species from Southern 28 Mexico. The results indicated that the highest crude protein (CP) and in vitro digestibility was found in 29 Gliricidia sepium with 264 g/kg-1 dry matter (DM) and 709 g/kg-1 DM respectively. Bursera simaruba had the 30 lowest CH4 production with 3.924 mg/g-1 incubated organic matter (IOM) and 9.077 mg/g-1 degraded organic 31 matter (DOM), condensed tannin (CT) content of 20% and relative low digestibility of 471 g/kg-1 DM. Results 32 found in this study indicate that several plant species widely available in Southern Mexico are potential 33 candidates for mitigating enteric CH4 production and can be used as additive or supplementary feed to improve 34 diet quality in cattle raised under grazing conditions in the tropics. 35 Keywords: Agroforestry, greenhouse gases, Lacandon Rainforests, Mitigation strategies, Silvopastoral 36 systems. 37 Introduction 38 Livestock production is an increasingly important issue in global research and development agendas (LGA 39 2016, FAO 2019). This activity provides 17% of the protein consumed worldwide, contributes to food security 40 of almost 1,300 million people and more than 800 million poor people in the world subsist on animal husbandry 41 (FAO 2016). However, extensive cattle production systems in tropical regions have generated severe impacts 42 on soil and ecosystems and continue to promote land use change. Those systems are also less efficient from the 43 productive standpoint due to the low quality of the pastures, poor management and its dependence on external 44 inputs. This essentially extractive production system induces high rates of deforestation that has generated loss 45 of biodiversity and a higher contribution to greenhouse gas (GHG) emissions. Pastures used in extensive 46 livestock systems have high content of neutral detergent fiber (NDF), low soluble carbohydrates and crude 47 protein (CP) that induce higher CH4 productions with low production parameters (Kennedy and Charmley 48 2012), therefore, a better management of the grazing system is required to efficiently use solar energy. 49 Nonetheless, tropical regions worldwide display a high forage biomass production from trees, shrubs and 50 herbaceous plants with adequate chemical composition (14-32% CP and <400 g/kg NDF) with potential to be 51 used as suitable feed alternative in sustainable livestock production systems. This diversity of species 52 provides ecosystem services such as natural rehabilitation of degraded soils, N-fixation (in the case of legumes) 53 and decreased CH4 emissions by the improvement of diet quality, addition of phytochemicals in the diet and 54 the possibility to modify the rumen microbiome. Methane is a GHG produced by ruminants during anaerobic 55 fermentation of carbohydrates in the rumen which is quantitatively eructated to the atmosphere. Secondary 56 metabolites contained in many plant species have the capacity to modify the population of microorganisms that 57 synthesize or are related to the formation of CH4 (Melesse et al. 2017, Albores-Moreno et al. 2018) in the rumen. 58 Forestry resources in the tropics with multiple uses and cultural importance can be studied for the mitigation of 59 CH4 and for the implementation of silvospastoral systems. 60 61 The Lacandon rainforest is located to the East and Northeast of the state of Chiapas in Southern Mexico. One 62 of the main economic activities of the indigenous maya and mestizo peasants of this region is extensive cattle 63 production and has generated the greatest changes in land use in the history of the Lacandon rainforest 64 (Covaleda et al. 2014). Currently, there are only 498,138 ha of forests and preserved forests left (Covaleda et 65 al. 2014). The biodiversity of this region is one of the most important in the country (Jiménez-Ferrer et al. 66 2008), this potential allows a reconversion of livestock towards sustainable production systems through the use 67 of available resources such as fodder trees, shrubs and herbaceous plants that have potential to reduce CH4 68 emission from cattle and regenerate degraded areas. The objective of this study was to evaluate the enteric CH4 69 mitigation potential and nutritional quality of different forage species recognized as suitable for livestock 70 production in the Lacandon rainforest region of Mexico. 71 72 Materials and Methods 73 74 Description of the study area 75 Samples of forage species were taken in the municipality of Ocosingo Valley, Chiapas, Mexico, which is part 76 of the socio-economic region XII Lacandon Rainforest (Flores-González et al. 2018). The climate is warm- 77 humid (23-27 °C) with an altitude that ranges from 10 to 900 MASL (García del Valle et al. 2015). The 78 predominant formation is lower montane rainforest, montane rainforest occurs on the moist, cool upland slopes 79 and riparian zones in the low-lying areas with a unique ecological niche where pines intermingle with lowland, 80 rain- forest species (Cook, 2016). Large areas have been extensively transformed by settlers, and timber and oil 81 companies, creating a patchwork of secondary forests, cultivated fields, and acahuales (fallow fields) in various 82 stages of secondary growth (Cook, 2016). Ocosingo covers the largest region of the Lacandon rainforest and 83 has a predominantly Tzeltal and Chol indigenous population (Flores-González et al. 2018). Indigenous 84 communities in the region have highland and midland rainforest vegetation with patches of cloud forests and 85 pine and oak forests, as well as acahuales (secondary vegetation), maize plots, vegetable cultivars (García del 86 Valle et al. 2015) and grazing cattle. 87 88 Selection of species 89 Species were selected from studies carried out in the region for identification of forage species based on local 90 knowledge and cultural importance for livestock producers (Soulard 2003, Pinto-Ruiz et al. 2005, Velasco- 91 Pérez 2007, Paz-Cortés 2010, Douterlungen, 2010). The species collected were: Gliricidia sepium, Bauhinia 92 variegata, Cecropia obtusifolia, Guazuma ulmifolia, Erythrina goldmanii, Spondias mombin, Acacia 93 pennatula, Parmentiera aculeata, Tithonia diversifolia, Liabum glabrum, Platymiscium dimorphandrum, 94 Ochroma pyramidale, Brosimum alicastrum, Bursera simaruba, and Mucuna pruriens. 95 96 Sampling 97 Leaves were collected from 5 to 9 individuals per species. During the sampling, the location points of each 98 species were taken by a GPS (Garmin® Etrex 30x) to geographically locate the presence of the species. Species 99 were firstly identified with the help of producers and botanical samples were taken for further verification at 100 the herbarium of the Southern Border College (ECOSUR). For chemical analysis, samples were dried in a 101 forced air oven at 55 °C for 48 hours or until constant weight to determine dry matter (DM) content and ground 102 in a Thomas Wiley® laboratory mill at a particle size of 1mm and stored in bags with airtight seal. 103 104 Chemical Analysis 105 Chemical analysis and the in vitro gas production technique were carried out at the Laboratory of Animal 106 Nutrition and Forage Quality at the International Center of Tropical Agriculture (CIAT), Cali, Colombia. 107 Organic matter (OM) content of the samples was determined by combustion in a muffle furnace at 500°C for 4 108 h (AOAC 2005: method 942.05), CP (CP=N×6.25) by Kjeldahl (AN 3001 FOSS; AOAC 1990: method 984.14), 109 NDF and acid detergent fiber (ADF) content were determined using the method proposed by Goering and Van 110 Soest (1970), adapted to an Ankom Fiber Analyzer AN 3805 (Ankom® Technology Corp. USA) and gross 111 energy was determined with an adiabatic bomb calorimeter following the procedure described in ISO 112 9831.1998. Ether extract content was determined by the Soxhlet method. The two-stage in vitro technique 113 (Tilley and Terry 1963) was used for the determination of digestibility. Secondary metabolite quantification 114 was carried out at the Bromatology Laboratory of the Southern Border College (ECOSUR). Tannin content of 115 the species was determined by the vanillin extract assay (Hagerman and Butler 1978). Qualitative quantification 116 of alkaloids, cyanogenic glycosides and saponins were carried out by the methodologies proposed by 117 Domínguez (1979). 118 119 In vitro gas production technique 120 Cattle were treated in accordance to the Colombian normative num. 84 of 1989 and following the protocol 121 approved by the Ethics Committee of the International Center of Tropical Agriculture (CIAT). Gas production 122 was determined using the in vitro technique proposed by Menke and Steingass (1988) as modified by Theodorou 123 et al. (1994). For the screening, leaves and petioles of 15 species were used. Rumen liquor was obtained from 124 three rumen cannulated Brahman bulls of 550 kg live weight, fed Cynodon plectostachyus. Rumen liquor was 125 filtered through 10 layers of gauze and mixed in a 1:9 ratio with a reduced mineral solution (Menke and 126 Steingass 1988). Ruminal solid and liquid content was liquefied and filtered to ensure the presence of 127 microorganisms of both the liquid and solid phase in the inoculum. For each treatment, 1000 mg of samples 128 were incubated in bottles of 160 ml capacity by triplicate including blanks. Bottles were kept under constant 129 flow of CO2, sealed with a rubber stopper and aluminum ring and placed in a water bath at 39°C for 72 hours. 130 Gas pressure and volume in the headspace of the bottles were measured with a pressure transducer connected 131 to a digital reader (Sper Scientific®, USA) and a three-way valve connected to a hypodermic needle that was 132 inserted into the bottles and a 60 ml syringe to measure gas volume. Gas pressure and volume were measured 133 at 0, 4, 8, 12, 24, 36, 48, 56 and 72 h. Gas volume was stored in amber bottles with a capacity of 125 ml from 134 samples collected from the accumulated gas at 12, 24 and 48 h fermentation. CH4 concentration was quantified 135 using a gas chromatograph (GC-2014 Shimadzu, Japan). For the degradation of DM and OM, the content of 136 the bottles was withdrawn from fermentation at different times (12, 24, 48 and 72 h) and filtered in crucibles 137 with fiberglass filter. The crucibles with the fiberglass filter were then dried in a forced air oven at 65°C for 48 138 h and weighed. 139 Data obtained from the pressure and volume of the experiments was used to generate the following polynomial 140 equation for the correction of the volume of gas produced: 141 𝑦 = 0.0209𝑥2+5.9023x – 2.984 142 𝑅2 = 0.9729 143 144 Gas production data was adjusted to the modified Gompertz model with the following equation: 𝑏−𝑐𝑥 145 𝑦 = 𝑎𝑒−𝑒 146 147 Where, y is equal to the cumulative gas production at a time x, a> 0 is the maximum gas production, parameter 148 b> 0 is the difference between the initial gas and the final gas at a time x and the parameter c> 0 describes the 149 specific rate of gas accumulation. The practical application of this model requires the conversion of parameters 150 a, b, c into parameters with biological significance. The parameters were: time at the inflection point (TIP, 151 hours), gas at the inflection point (GIP, ml), maximum gas production rate (MGPR ml/h) and Lag phase (LP or 152 the microbial establishment, h). For its estimation the following formulas were used: TIP = b / c; GIP = a / e; 153 MGPR = (a * c) / e; LP = ((b / c) - (1 / c)); where "e" is Euler's number, equivalent to ≈ 2,718281828459. 154 155 Statistical analysis 156 For the statistical analysis, a randomized block design was used with 15 treatments, three replicates per hour 157 and three different inoculums as a blocking factor. To assess the behavior of the variables, the PROC GLM 158 procedure of SAS® software, version 9.4 (SAS, 2012) was used. Treatments means were compared with the 159 Tukey test with an Alpha of 0.05. 160 Results 161 Sampling and forage species 162 Plant species were identified and collected in Ocosingo Valley, Chiapas, Mexico in paddocks, acahuales, 163 orchards and live fences. Species were mostly collected in induced grasslands, cultivated areas and mesophyll 164 mountain forest as shown in Figure 1. Forage species were sampled between 683 and 1059 MASL. A total of 165 15 species were identified and collected (Table 1). From the species collected, 40% belonged to the Fabaceae 166 family. 167 Chemical composition 168 Organic matter of the species ranged from 821.28 to 934.51 g kg-1 DM. Species with the highest content of OM 169 were M. pruriens, P. dimorphandrum and A. pennatula (Table 2). Crude protein content was higher for T. 170 diversifolia, G. sepium, L. glabrum with 289.54, 261.41 and 240.16 g kg-1 DM respectively. B. simaruba had 171 the lowest CP content (99.07 g kg-1 DM). The lowest NDF contents were 275.19, 298.17, 305.88 g kg-1 DM for 172 B. variegata, B. alicastrum and T. diversifolia respectively. The species with the lowest acid detergent fiber 173 content were SM, MP and GS with 171.18, 180.65 and 190.83 g kg-1 DM respectively. In vitro digestibility of 174 DM was highest for G. sepium (709.94 g kg-1 DM), T. diversifolia (704.03 g kg-1 DM), M. pruriens (700 g kg- 175 1 DM) and B. variegata (698.27 g kg-1 DM). A. pennatula had the lowest in vitro digestibility (447.44 g kg-1 176 DM). Gross energy content of the species ranged from 15.65 to 20.92 MJ kg-1 DM. CT were present in B. 177 variegata, C. obtusifolia, G. ulmifolia, S. mombin, A. pennatula, O. pyramidale, B. simaruba and M. pruriens 178 with 1.66, 13.27, 3.27, 0.99, 3.11, 3.95, 20.01 and 0.87% respectively. 179 In the phytochemical screening, presence of alkaloids was found in all species except for BS as shown in Table 180 3. Cyanogenic glycosides were found highly abundant only in A. pennatula. Saponins were found in G. sepium, 181 B. variegata, G. ulmifolia, E. goldmanii, A. pennatula and O. pyramidale with a low abundance (+) except for 182 G. sepium that was abundant (++). 183 In vitro gas fermentation and degradability 184 Maximum gas production (a), time at the inflection point (TIP), gas inflection point (GIP), maximum gas 185 production rate and Lag phase differed significantly (P < 0.05) among the forage species evaluated (Table 4). 186 Maximum in vitro gas production was obtained in B. alicastrum, T. diversifolia, M. pruriens, and S. mombin 187 with 256.7, 232.4, 225.7 and 216.5 ml. The lowest gas production was observed in B. simaruba with 118.03 188 ml. B. alicastrum, G. sepium, T. diversifolia, and B. variegata had the highest MGPR with 9.72, 8.10, 7.77 and 189 7.46 ml/h. 190 Degradability at different hours (12, 24, 48 and 72 h) differed significantly (P < 0.05) among species (Table 5). 191 The highest degradability at 12, 24, 48 and 72 h were observed for T. diversifolia with 56.78, 69.04, 78.11 and 192 78.71% respectively. Dry matter degradability at 72 h ranged from 35.34% (A. pennatula) to 78.71% (T. 193 diversifolia). 194 Methane production 195 Distribution and differences in CH4 produced at 48 h per degraded OM (mg/g-1) from forage is shown in Figure 196 2. CH production in mg/g-14 DOM (degraded OM) and mg/g-1 IOM (incubated OM) was different among 197 species (P <.0001). The lowest CH4 production at 48 h was observed in B. simaruba with 3.924 mg/g-1 IOM 198 and 9.077 mg/g-1 DOM. At 48 h B. alicastrum, G. sepium and M. pruriens had the highest CH4 production with 199 35.228, 20.713 and 19.977 mg/g-1 DOM respectively. Accumulated CH4 at 48 h from different species is shown 200 in Figure 3. Lowest CH4 production at 48 h were observed in B. simaruba, P. aculeata, S. mombin and A. 201 pennatula with 3.92, 4.10, 4.42 and 5.55 mg/g-1 IOM. 202 Discussion 203 The search for local resources with high nutritional value is important to improve profitability and productivity 204 of the systems as well as to reduce the impact of livestock on the environment (Valencia-Salazar et al. 2018). 205 However, there is not enough information on the use of these resources on specific animal production systems 206 or for the design of silvopastoral systems. Incorporation of a variety of forage species improves diet quality and 207 enhance milk production and weight gain. Some mitigation alternatives are associated with improved efficiency 208 of animal production given their advantages from the nutritional and environmental standpoints (Patra et al. 209 2012). In Southern Mexico, cattle producers have observed that during the dry season animals consume many 210 species present in native vegetation in the form of green fodder, dried pods and leaves from trees and shrubs 211 (López-Herrera et al. 2008, Albores-Moreno et al. 2018). Secondary vegetation in livestock systems has been 212 scarcely studied and is being displaced by introduced pastures for the establishment of extensive grazing 213 systems (Albores-Moreno et al., 2020). 214 Nutritional quality of forage species especially CP content is of great relevance due to protein deficiencies in 215 pastures used in tropical extensive livestock systems. In this experiment, CP contents were all above 120 g kg- 216 1 DM with the exception of B. simaruba (99.07 g kg-1 DM) and B. aliscastrum (116.21 g kg-1 DM). However, 217 these values can meet the requirements of CP for moderate levels of production of ruminants in tropical regions. 218 Crude protein values of C. obtusifolia (187.48 g kg-1 DM), G. ulmifolia (150.72 g kg-1 DM) and S. mombin 219 (126.95 g kg-1 DM) were similar to those obtained by López-Herrera el at. (2008) and Rodriguez et al. (2019); 220 C. obtusifolia (165.4 g kg-1 DM), G. ulmifolia (137.8 g kg-1 DM) and S. mombin (148.0 g kg-1 DM). T. 221 diversifolia and G. sepium had the highest content of CP and also the highest digestibility. Neutral detergent 222 fiber content of G. sepium was similar to the value reported by Molina-Botero et al (2019) (575.4 g/kg DM), 223 however, CP from G. sepium was much higher in the present trial. The content of CP and EE of S. mombin was 224 similar to those obtained by Yusuf et al (2020); 145.1 and 60.0 g kg-1 DM, respectively. The greatest DM 225 degradability was for the species that contained lower or no concentrations of secondary metabolites (G. sepium, 226 T. diversifolia and B. alicastrum), these were more fermentable and susceptible to bacterial attack. The majority 227 of secondary metabolites that have been studied for the reduction in CH4 synthesis in the rumen have been 228 shown to have antimicrobial properties (Patra et al. 2012) which constitutes one of the strategies to decrease 229 CH4 production. 230 Low CH4 production in B. simaruba and A. pennatula can be explained by their content of CT (20.01 and 231 3.11%) which is related to ruminal degradability and CH4 production (Soltan et al., 2012). Condensed and 232 hydrolyzed tannins play an important role in mitigation of CH4 emissions (Melesse et al. 2019, Rira et al. 2019). 233 No methane reduction per day was reported by Molina-Botero et al (2019) when G. sepium with 45.9 mg/g of 234 CT was included in different levels mixed with pods of Enterolobium cyclocarpum in cattle rations. Piñeiro- 235 Vasquez et al. (2017) found 28% reduction on CH4 when included 30% of B. simaruba with 13.3% of CT and 236 no reduction with A. pennatula with 18.9% of CT in sheep rations. However, although ruminant species have 237 similar CH4-synthesis pathways, they differ considerably in their level of feed intake, rumen morphology, and 238 rumen physiology so results with the use of the same strategy to reduce methane production may vary depending 239 on ruminant species (van Gastelen et al., 2019). 240 In general, CH4 emissions from plant species that had some content of CT were below average (8.88 mg per 241 incubated OM), except for M. pruriens with 11.26 mg CH4/IOM and 0.87% CT; the lowest concentration. 242 Condensed tannins are the polyphenolic compounds most abundant in plants that can effectively decrease CH4 243 directly by inhibiting methanogenic archaea and indirectly by reducing hydrogen production as a result of 244 decreased fiber digestion and protozoan population in the rumen (Patra et al 2017). Methane mitigation with 245 the use of CT can be very variable between species and experimental methods. A few in vivo experiments have 246 shown that the effect of CT on methane production and animal response is dependent on the metabolite source, 247 type, dose and molecular weight (Aboagye and Beauchemin, 2019). 248 Cyanogenic glycosides from A. pennatula (++++) have the capacity to reduce methane synthesis as shown in 249 the present study. Cyanogenic glycosides are nitrogen compounds that, when hydrolyzed, produce hydrogen 250 cyanide that stops cellular respiration (Hassan, 2011) and inhibits cytochromes present in methanogens (Smith 251 et al 1985). In many species of the Acacia genus, the high presence of cyanogenic glycosides is common, 252 however, there is not much information on the use of this metabolite as an enteric methane mitigation strategy. 253 Saponin content was found abundant in G. sepium and in low abundance in B. variegata, G. ulmifolia, E. 254 goldmanii, S. mombin and O. pyramidale. Lopez-Herrera et al. (2008) observed low abundance of saponins in 255 O. pyramidale and Molina-Botero reported 17.0 mg/g of saponins leaves of G. sepium. CH4 production of the 256 plant species in relation to their saponin content was not consistent. Saponins can form complexes with the lipid 257 membrane of the bacteria, which increases its permeability generating an imbalance and consequent lysis of the 258 microorganism, most of the saponins also have a similar effect on protozoa population (Makkar et al. 1995). 259 The effect of any of the secondary metabolites on CH4 synthesis varies considerably depending on the 260 characteristics of the metabolite, as well as the type of diet (Patra et al. 2017). In addition, long-term studies on 261 animal performance must be carried out to verify the effect of secondary metabolites on CH4 synthesis in the 262 long term. Secondary metabolite biosynthesis and accumulation depend on genetic, ontogenic, morphogenic 263 and environmental factors such as light irradiation, temperature, soil water, soil fertility and salinity (Yang et 264 al., 2018). Likewise, nutritional composition of a forage is also dependent on environmental factors, plant 265 phenological stage and management methods. Plant species collected in this study have not undergone a genetic 266 selection so differences in chemical composition, secondary metabolite content and effect on CH4 synthesis 267 may vary in comparison with other studies. 268 The potential of forage trees, shrubs, and herbaceous plants lies in the wide biodiversity and traditional 269 knowledge that exists in their agroecosystems (Jiménez-Ferrer et al. 2008). Native vegetation must be studied 270 in order to be incorporated in the establishment of systems in association with grasses or management of the 271 secondary succession to allow the increase and conservation of biodiversity (Rosales and Gil, 1997) and the 272 existence of edible biomass in greater quantity and quality that remains during the critical seasons in grazing 273 areas. Also, native species and their biodiversity have a great potential in the reduction of ruminant methane 274 emissions in smallholds in tropical regions as demonstrated in this and other studies (Yusuf et al 2020, Albores- 275 Moreno et al 2018). However, for the implementation of CH4 mitigation strategies several aspects must be taken 276 into consideration such as: effects on animal performance, safety for the ruminant and the consumer alike, and 277 economic viability (Martin et al. 2010). Additionally, socioeconomic, cultural and agroecological aspects are 278 also determinants in the selection of an appropriate methane mitigation strategy. 279 Conclusion 280 The use of native plant species from tropical regions in small farms may be considered as a viable, resource 281 conservation and low-cost strategy accessible to producers to mitigate methane emissions and improve protein 282 intake of ruminants grazing low-quality pastures. Species hereby described hold potential to be used in cattle 283 farms as live-fences and the forage can be directly browsed or cut by hand for the animals. Future research 284 should be aimed at the evaluation of biomass yields at different times of the year, propagation of species, yields 285 in holistic systems, in vivo evaluations that include voluntary intake, bioeconomic parameters and methane 286 production measurements in long-term studies with techniques such as SF6 or respiration chambers. 287 288 Funding: This study is part of the LivestockPlus project funded by the CGIAR Research Program (CRP) on 289 Climate Change, Agriculture and Food Security (CCAFS). In addition, this work was also done as part of the 290 Livestock CRP. Thanks to National Science and Technology Council (CONACYT, Mexico) for the funding of 291 the project “Quantification of enteric methane and nitrous oxide emissions in cattle grazing and the design of 292 strategies for its mitigation in Southern Mexico” (SEP-CONACYT CB 2014 No. 242541). 293 294 Acknowledgments: We thank all donors that globally support the work of the CRP programs through their 295 contributions to the CGIAR system. A special thanks to the University of the Jungle, Ocosingo for facilitating 296 their spaces for the handling of the samples. To the anthropologist Lorenzo Hernandez for his collaboration in 297 field work and translations in Tzeltal. 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(2020) Chemical characterization and in vitro methane 451 production of selected agroforestry plants as dry season feeding of ruminants livestock. Agroforest Syst. 452 https://doi.org/10.1007/s10457-019-00480-7 453 454 455 456 457 458 459 460 461 462 Figure 1. Geographic location of the study area 463 464 465 466 Table 1. Forage species from the Lacandon Rainforest region of Chiapas, Mexico Growth Family habit Scientific name Common name Uses MASL Fabaceae Tr Gliricidia sepium Cocoite, matarratón 1, 2, 3, 5 854 - 1014 Fabaceae Tr Bauhinia variegata Pata de vaca 2, 3, 4 877 - 911 Moraceae Tr Cecropia obtusifolia Guarumbo 1, 3, 4, 6, 7, 8 859 - 1047 Sterculiaceae Tr Guazuma ulmifolia Wasil, wasim, pixoy, caulote 1, ,3, 4, 5, 6, 7 878 - 897 Fabaceae Tr Erythrina goldmanii Mote, Colorín, Pichoco 1, 4, 5, 7 826 - 888 Anacardiaceae Tr Spondias mombin Jobo, lulúy, jujup 1, 3, 5, 7 866 - 912 Fabaceae Tr Acacia pennatula Acacia, chimay 6 881 - 891 Bignoniaceae Tr Parmentiera aculeata Cuajilote 1, 3, 4, 5, 6, 7 683 - 685 Asteraceae Sh Tithonia diversifolia Árnica 1, 3, 4, 5, 6, 7 882 - 1025 Asteraceae Sh Liabum glabrum Tsuy 5 869 - 1058 Fabaceae Tr Platymiscium dimorphandrum Judío, hormiguillo 3, 9 878 - 887 Malvaceae Tr Ochroma pyramidale Corcho, balso 3 854 - 888 Moraceae Tr Brosimum alicastrum Ash, ramón, ox 1, 3, 4, 5, 6, 7, 9 683 - 890 Burseraceae Tr Bursera simaruba Mulato, chakah 1, 3, 4, 6, 7 794 - 895 Fabaceae He Mucuna pruriens Nescafé 5 865 - 1059 Tr: Tree; Sh: Shrub; He: Herbaceous; Uses: 1: Live fences, 2: Wood, 3: Construction, 4: Firewood, 5: Edible, 6: Medicinal, 7: Shadow, 8: Wooden posts, 9: elaboration of tools/instruments. 467 468 469 Table 2. Chemical composition, condensed tannin content and in vitro digestibility of multipurpose forage species from the Lacandon Rainforest region, Chiapas, Mexico g kg -1 DM MJ Species DM OM NDF ADF CP EE IVDDM kg-1 %CT DM Gliricidia sepium 242.83 890.78 431.56 190.83 261.41 30.12 709.94 19.48 0.00 Bauhinia variegata 384.04 854.41 275.19 194.57 146.45 28.72 698.27 17.92 1.66 Cecropia obtusifolia 295.95 897.12 437.00 214.00 187.48 35.10 498.94 19.12 13.27 Guazuma ulmifolia 398.35 886.42 480.45 260.29 150.72 32.02 516.88 19.06 3.27 Erytrhina goldmanii 297.68 888.16 417.38 271.05 206.78 41.40 595.97 19.06 0.00 Spondias mombin 259.75 852.29 307.77 171.18 126.95 44.78 638.59 16.25 0.99 Acacia pennatula 505.42 924.85 492.56 210.34 192.69 39.25 447.44 20.92 3.11 Parmentiera aculeata 308.95 874.47 614.35 268.81 183.17 13.85 548.37 18.04 0.00 Tithonia diversifolia 190.50 853.53 305.88 337.81 289.94 21.25 704.03 18.31 0.00 Liabum glabrum 182.26 868.04 443.00 334.97 240.16 53.30 531.11 19.01 0.00 Platymiscium dimorphandrum 279.54 931.01 539.15 324.83 236.32 18.72 616.47 20.45 0.00 Ochroma pyramidale 217.03 882.84 369.39 232.65 195.85 21.70 565.31 18.36 3.95 Brosimum alicastrum 489.18 821.28 298.17 269.22 116.21 29.92 686.38 15.65 0.00 Bursera simaruba 356.71 900.82 354.37 249.23 99.07 25.05 471.37 18.92 20.01 Mucuna pruriens 248.35 934.51 386.68 180.65 228.31 29.90 700.19 19.44 0.87 DM: Dry matter; OM: Organic matter; NDF: Neutral detergent fiber; ADF: Acid detergent fiber; CP: Crude protein; EE: Ether extract; IVDDM: In vitro digestibility of dry matter; CT: Condensed tannins 470 471 472 Table 3. Phytochemical screening of multipurpose forage species from Southern Mexico Alkaloids Cyanogenic Species Saponins Mayer Draggendorff Wagner glycosides Gliricidia sepium + +++ + - ++ Bauhinia variegata - +++ ++ - + Cecropia obtusifolia - +++ + - - Guazuma ulmifolia + + ++ - + Erythrina goldmanii +++ + ++ - + Spondias mombin - + ++ - - Acacia pennatula - ++ ++ ++++ + Parmentiera aculeata ++++ ++++ ++++ - - Tithonia diversifolia +++ ++++ ++++ - - Liabum glabrum ++ ++++ ++++ - - Platymiscium dimorphandrum ++ ++++ - - - Ochroma pyramidale - +++ - - + Brosimum alicastrum +++ + - - - Bursera simaruba - - - - - Mucuna pruriens ++ ++ - - - - (No presence); + (low abundance); ++ (abundant); +++ (moderately abundant); ++++ (very abundant) 473 474 475 Table 4. Gompertz model parameters for in vitro gas production measured in forage species from Southern Mexico Parameters TIP GIP MGPR Species a b c (h) (ml) (ml/h) LP GS 200.078(f, g, e, d, c) 1.070(a) 0.111(a) 9.903(d, e) 73.590(f, g, e, d, c) 8.1033(b, a) 0.204(a) BV 201.269(f, b, e, d, c) 0.865(b, a) 0.102(b, a) 17.287(d, e) 74.027(f, b, e, d, c) 7.4633(b, a) -1.957(b, d, a, c) CO 214.405(b, e, d, c) 0.837(b, a) 0.048(d, e) 8.613(b) 78.860(b, e, d, c) 3.8167(e, c, d) -3.363(e, b, d, a, c) GU 188.402(f, g, e, d) 0.742(b) 0.051(d, e) 14.573(c, b) 69.297(f, g, e, d) 3.5533(e, c, d) -5.123(e, d, f, c) EG 168.493(g, h) 0.668(b) 0.063(b, d, e, c) 10.637(c, d, e) 61.970(g, h) 3.8967(e, c, d) -5.290(e, d, f) SM 216.597(b, d, c) 0.836(b, a) 0.058(d, e, c) 14.573(c, b) 79.663(b, d, c) 4.5800(e, c, d) -2.833(e, b, d, a, c) AP 146.883(i, h) 0.679(b) 0.056(d, e, c) 12.453(c, d) 54.023(i, h) 2.9933(e, d) -6.090(e, f) PA 184.457(f, g, e) 0.763(b) 0.028(e) 27.253(a) 67.843(f, g, e) 1.9033(e) -8.307(f) TD 232.433(b, a) 0.832(b, a) 0.092(b, d, a, c) 9.157(d, e) 85.490(b, a) 7.7733(b, a) -2.077(b, d, a, c) LG 174.429(f, g, h) 0.899(b, a) 0.088(b, d, a, c) 10.207(d, e) 64.153(f, g, h) 5.6667(b, c, d) -1.150(b, a) PD 185.037(f, g, e, d) 0.806(b, a) 0.085(b, d, a, c) 9.477(d, e) 68.057(f, g, e, d) 5.7900(b, c) -2.303(e, b, d, a, c) OP 196.539(f, g, e, d, c) 0.928(b, a) 0.056(d, e, c) 16.573(b) 72.287(f, g, e, d, c) 4.0433(e, c, d) -1.287(b, a, c) BA 256.729(a) 0.951(b, a) 0.103(b, a, c) 9.240(d, e) 94.427(a) 9.7200(a) -0.473(b, a) BS 118.030(i) 0.956(b, a) 0.081(b, d, a, c) 11.883(c, d) 43.410(i) 3.5067(e, c, d) -0.697(b, a) MP 225.745(b, a, c) 0.665(b) 0.089(b, d, a, c) 7.430(e) 83.030(b, a, c) 7.4267(b, a) -3.767(e, b, d, c) MSE 10.49507 0.09600 0.0149 1.3507 3.8597 0.8964 1.2962 P-Value <.0001 0.0004 <.0001 <.0001 <.0001 <.0001 <.0001 Species: Means in the same column with different superscript are significantly different (P<0.05) according to Tukey test; Gliricidia sepium (GP), Bauhinia variegata (BV), Cecropia obtusifolia (CO), Guazuma ulmifolia (GU), Erythrina goldmanii (EG), Spondias mombin (SM), Acacia pennatula (AP), Parmentiera aculeata (PA), Tithonia diversifolia (TD), Liabum glabrum (LG), Platymiscium dimorphandrum (PD), Ochroma pyramidale (OP), Brosimum alicastrum (BA), Bursera simaruba (BS), Mucuna pruriens (MP); TIP: time at the inflection point (hours); GIP: Gas inflection point (ml); MGPR: Maximum gas production rate (ml/h); LP: Lag phase 476 477 Table 5. DM degradability at different hours of incubation of forage species from Southern Mexico Species 12 h 24 h 48 h 72 h GS 40.402(c, d, e, f) 51.050(b, d, e, f, g) 58.043(a, f, g, h, i, j, k) 59.507(a, c, d) BV 37.797(e, h, k, n, o, p) 47.778(e, h, k, l, m, n, o) 54.433(e, i, n, r, w, z, 1, 2, 3 55.242(f, g, j, k, l) CO 29.013(u, x, 2, 6, 7, 8) 40.218(w, z, 3, 5, 7) 52.039(j, o, s, x, z, 3, 4, 5, 6) 53.913(h, j, m, n) GU 25.456(1, 5, 8, 10, 11) 35.377(1, 4, 6, 7, 8, 9) 46.263(6, 9, 11, 14, 17, 18) 49.436(r, s, u) EG 31.261(p, r, s, w, x, y, z, 1) 42.121(n, r, u, x, 2, 3, 4) 47.504(2, 4, 7, 10, 11, 12, 13) 49.893(p, q, s, t) SM 34.053(j, m, o, q, s, t, u, v) 42.221(m, q, t, x, y, z, 1) 46.876(5, 8, 10, 14, 15, 16) 46.730(t, u, v) AP 29.346(v, y, 3, 6, 9, 10) 30.770(9, 10) 34.546(20) 35.348 PA 25.472(z, 4, 7, 9, 11) 32.141(8, 10) 41.941(13, 16, 18, 19, 20) 45.256(v) TD 56.781(a) 69.045 78.111 78.719 LG 37.923(d, g, k, l, m) 46.919(g, j, l, p, t, u, v, w) 55.715(c, g, l, q, r, s, t) 56.705(d, e, g, h, i) PD 39.795(c, g, h, i, j) 47.585(f, i, k, p, q, r, s) 51.824(k, p, t, y, 1, 3, 7, 8, 9) 53.546(I, k, m, o, p) OP 30.867(t, w, 2, 3, 4, 5) 49.589(c, d, h, i, j) 55.607(d, h, m, q, w, x, y) 57.907(b, c, e, f) BA 52.917(a, b) 59.729(a) 60.907(a, b, c, d, e) 61.358(a, b) BS 36.873(f, i, l, n, q, r) 41.892(o, s, v, y, 2, 5, 6) 43.321(12, 15, 17, 19) 51.202(l, n, o, q, r) MP 47.550(b) 56.431(a, b, c) 56.951(b, f, l, m, n, o, p) 66.320(t, u, v) MSE 1.948 2.254 2.315 1.228 P-value <.0001 <.0001 <.0001 <.0001 Means in the same column with different superscript are significantly different (P<0.05). Gliricidia sepium (GS), Bauhinia variegata (BV), Cecropia obtusifolia (CO), Guazuma ulmifolia (GU), Erythrina goldmanii (EG), Spondias mombin (SM), Acacia pennatula (AP), Parmentiera aculeata (PA), Tithonia diversifolia (TD), Liabum glabrum (LG), Platymiscium dimorphandrum (PD), Ochroma pyramidale (OP), Brosimum alicastrum (BA), Bursera simaruba (BS), Mucuna pruriens (MP). 478 479 480 481 Figure 2. Distribution and differences of CH4 produced at 48 h per degraded OM (mg/g-1) from forage 482 species from Southern Mexico 483 484 140 GS BV 120 CO GU EG 100 SM AP 80 PA TD LG 60 OP BA 40 BS MP 20 0 0 5 10 15 20 25 30 35 40 45 50 Time (h) 485 486 Figure 3. Accumulated CH4 at 48 h from different plant species from Southern Mexico Methane accumulated (mg) / DOM (g)