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Effect of Leucaena leucocephala on methane production of Lucerna heifers fed a diet based on Cynodon plectostachyus
Author’s Accepted Manuscript Effect of Leucaena leucocephala on methane production of Lucerna heifers fed a diet based on Cynodon plectostachyus I.C. Molina, E.A. Angarita, O.L. Mayorga, J. Chará, R. Barahona-Rosales www.elsevier.com/locate/livsci
PII: DOI: Reference:
S1871-1413(16)30009-9 http://dx.doi.org/10.1016/j.livsci.2016.01.009 LIVSCI2927
To appear in: Livestock Science Received date: 25 April 2015 Revised date: 6 October 2015 Accepted date: 12 January 2016 Cite this article as: I.C. Molina, E.A. Angarita, O.L. Mayorga, J. Chará and R. Barahona-Rosales, Effect of Leucaena leucocephala on methane production of Lucerna heifers fed a diet based on Cynodon plectostachyus, Livestock Science, http://dx.doi.org/10.1016/j.livsci.2016.01.009 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Effect of Leucaena leucocephala on methane production of Lucerna heifers fed a diet based on Cynodon plectostachyus
I. C. Molina a, E. A. Angarita b, O. L. Mayorga c, J. Chará d and R. Barahona-Rosales a
a
Department of Animal Production, Faculty of Agricultural Sciences, Universidad
Nacional de Colombia, Medellin, Colombia. b
Department of Animal Science, Faculty of Veterinary Medicine and Animal Sciences,
National University of Colombia, Bogota, Colombia. c
Colombian Agricultural Research Corporation, CORPOICA, Mosquera,
Cundinamarca, Colombia. d
Centro para la Investigación en Sistemas Sostenibles de Producción Agropecuaria,
CIPAV, Cali, Colombia.
Corresponding author: Rolando Barahona Rosales. Email: [email protected]. Tel: +5744309109
Short title: Leucaena and bovine enteric methane emissions
Abstract A rapid growth of the meat production industry is necessary to satisfy increased demand for this commodity, which might have negative impacts on the environment. The objective of this study was to assess enteric methane (CH4) emissions when a forage legume is introduced in the diet of animals consuming a tropical grass. Eight 1
Lucerna heifers, 218 ± 18 kg live weight with an average age of 19 ± 3 months were used in two experiments following a changeover design. The diets evaluated were 100% star grass (Cynodon plectostachyus, S) or 76% star grass plus 24% leucaena (Leucaena leucocephala, S+L). Throughout the experiment, animals were housed in two chambers, in which the diet was offered four times daily. Each chamber had a small wind tunnel, which housed a fan set to a constant speed of extraction. Air samples were obtained every hour during 24 hours both inside and outside (ambient) the tunnel. Methane concentration in these samples was determined by gas chromatography. Temperature and relative humidity both inside and outside the tunnel were recorded using a thermo-hygrometer. The S+L diet had greater protein content whereas the S diet had greater content of neutral detergent fiber. Average intake (kg/d) of fresh forage and dry matter (DM) was significantly greater (23.7 and 5.6) for the S+L than for the S diet (18.9 and 4.7), respectively (P < 0.05). The maximum recorded temperature and humidity inside the chamber was 35.5 °C and 99%, respectively, but the minimum values were 19.1 °C and 38%, respectively. Methane production (L/kg DMI) was 37.7 for the S+L treatment and 43. 6 for the S treatment. The energy loss in the form of methane emitted was 8.0% for S+L and 9.4% for the star grass based diet (P = 0.32). These results suggest that while increasing animal productivity by increasing dry matter intake, the inclusion of leucaena does not increase methane emission per animal, thus significantly decreasing methane emissions per kg of meat or milk produced.
Keywords: Bovines; enteric methane emissions; forage intake; intensive silvopastoral systems; polytunnel technique. 2
Introduction Global meat production will double by 2050, based on the 229 million tons produced in 2000, and milk production will rise from 722 million tons in 2010 to 1043 million tons in 2050 (FAO, 2009b). These production gains are largely expected to come from increases in livestock numbers and system productivity (FAO, 2009a). Cattle population is expected to increase from 350 to 483 million heads between 1999 and 2030 (Bruinsma, 2003). These changes can have negative environmental effects, as about 6–10% of the total gross energy consumed by bovines is converted to methane (Eckard et al., 2010). Rumen methane results from the action of different anaerobic microorganisms (Morgavi et al., 2010) when hydrolysing proteins, starches and cell wall components of the diet (Moss et al., 2000). Methane is produced by microorganisms of the Archaea domain, which mainly use hydrogen, formate, acetate, methanol and methylalanine groups as substrates (Liu and Whitman, 2008). The main factor affecting enteric methane emissions is diet composition, particularly protein, lipid and carbohydrate content (Ulyatt and Lassey, 2001). Other factors include the forage species, the stage of maturity, the use of preservation methods, chemical or physical treatments (Tan et al., 2011) and the presence of secondary metabolites (Huang et al., 2011). Intensive Silvopastoral Systems (ISS) are livestock production systems that are more efficient and productive than traditional systems (Murgueitio et al., 2014), but their environmental impacts, in terms of methane emissions, have not been yet comprehensively studied. Thus, the aim of this study was to evaluate in vivo the effect 3
of the inclusion of Leucaena leucocephala, a forage legume typical of intensive silvopastoral systems, on methane emissions and energy loss associated with the emission of this GHG.
Material and methods Location The research was carried out in El Hatico Natural Reserve, located in the municipality of Cerrito, Valle del Cauca Department, Colombia. Its ecological classification according to Holdridge corresponds to tropical dry forest (bs-T). The reserve is located 1000 meters above sea level and its average temperature, relative humidity and annual precipitation are 24°C, 75%, and 750 mm, respectively.
Description of animals To carry out this study, eight Lucerna breed heifers (4 for each cycle) with a live weight of 218 ± 18 kg and an average age of 19 ± 3 months were selected. Two independent cycles (runs), a month apart, were conducted with these animals. Two polytunnels were used in each experiment (two animals per polytunnel).
Diets used The diets evaluated were 100% star grass (S; Cynodon plectostachyus K. Schum) and 76% star grass with 24% leucaena (S+L; Leucaena leucocephala (Lam.) De Wit (leucaena) cv. Cunningham) on a fresh basis. Mineral salt and water were offered ad libitum. During the first period, diet x was assigned to the first group and diet y to the second group. During the second experimental period, diets were exchanged 4
between animals. In a second experiment, another four animals were used, divided also in two groups and diets were assigned for two periods, according to the procedure followed during the first experiment. The time allotted for adaptation to the diet in each of the four periods was seven days. The forages were harvested at an average re-growth age of 45 days of an ISS with L. leucocephala (10.000 bushes/ha) and C. plectostachyus. The star grass used in the two treatments was harvested from different paddocks with and without the presence of legumes. The ration was fed four times daily at 08:00, 11:00, 14:30, and 18:00 hours. Diet intake was determined as the difference between forage offered and refused, which were weighed daily during the stay of the animals in the polytunnel.
Feed analysis Pools were made with feed samples collected from each experimental group during the last three days of each measurement period, which were sent to the Nutrition laboratory of the Universidad Nacional de Colombia, Medellin where they were dried to constant weight. Forages were milled to pass through a 1 mm sieve using a Romer mill (Romer Labs, México). Samples were analyzed for crude protein content (CP) by the Kjeldahl method based on NTC 4657; neutral detergent (NDF) and acid detergent fiber (ADF) according to Van Soest et al. (1991) and ether extract (EE) by Soxhlet immersion (NTC 668). Ash was measured by direct incineration to 550 °C in a muffle (AOAC 942.05), the calcium (Ca) and phosphorus (P) contents were determined by UV-VIS AA spectrophotometry (NTC 5151 and 4981, respectively) and energy values were determined by calorimetry according to the ISO 9831 method.
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In vivo methane measurements These experiments followed Murray’s et al. (2001) recommendations. Polytunnels used were 7 m long, 5 m wide and 2.6 m high for a total volume of 83.5 m 3. The structure had an inlet for animals and staff and on the opposite side there was a 12” fume hood set at an extraction rate of 0.9 m3/s to allow sampling of gases. To adapt animals to the conditions in the polytunnel, during a 48 hour period whilst the animals were housed in the polytunnel, the door was closed for an hour and the then opened for the next two. Methane measurements were conducted at hourly intervals during a 24 hour period. To collect gas samples, a three-way valve was used; during the first 10 s after the start of extraction the first outlet, connected to a 12 mL plastic syringe was used; the second outlet was attached to a hypodermic needle and 7 mL vacuum tubes. Ambient air (outside of the tunnel) was sampled simultaneously. The collected gas samples were stored in a cool dry place and transported to the laboratory for analysis by gas chromatography. Methane production was calculated according to the ideal gas law (Lopez and Newbold, 2007) using the gas concentration measured by chromatography, the temperature inside the polytunnel when taking the methane samples and the atmospheric pressure at the site. Total methane production was calculated by multiplying by the total volume of the polytunnel.
Measurement of polytunnel temperature and relative humidity Before collecting the methane samples, the temperature and the relative humidity were recorded both inside and outside the polytunnel using a thermo-hygrometer. From these data, the temperature and humidity index (THI) was calculated to 6
evaluate the level of heat stress of experimental animals. To do this, the equation of Kibler (1964) was used: THI = 1.8 * T – (1 - (RH / 100)) * (T - 14.3) + 32
(Equation 1)
where THI denotes the temperature-relative humidity index, T is temperature in °C and RH is relative humidity in %. For the interpretation of this index, the safety index of the Livestock Conservation Institute was used based on THI values as follows: 74 or less: normal; 75-78: alert; 79-83: danger, and 83 or more: emergency
Gas chromatography The methane concentration in the samples was determined at the International Center for Tropical Agriculture (CIAT), Palmira, Valle del Cauca, Colombia. Determinations were made on a Shimadzu gas chromatograph (Shimadzu, Japan) equipped with a flame ionization detector and an electron capture detector. The chromatographic conditions were as follows: N Hayesep column 3 m in length; mobile phase: high purity nitrogen at a flow of 35 mL/min. Oven, injector and detector temperatures were 250, 100, and 325 °C, respectively. The standard used was Scott methane standard.
Experimental Design and Statistical Analysis To determine the effect of treatments on the production of methane the PROC MIXED procedure with SAS 9.1 software (SAS Institute, 2003). Separation of means was made using the Tukey test with an alpha of 0.05. The model is described below Yij = μ + δi + Rj + (δ *R)ij + ҽijk, 7
where Yij is observations of the subject in the diet i and period j ; μ: is the overall mean of the population; δi: is the effect of the i-th diet; Rj: is the effect of j-th run; (δ *R)ij is the interaction between the i-th diet and the j-th run; ҽijk: is the experimental error.
Results Nutritional quality The greatest difference between diets was related to the protein content, which in the S+L diet was almost 14% and only 10.8% in the grass diet (S; Table 1). The gross energy content (MJ/kg) of the S+L diet was 2% greater (18.3) than that of the S diet (17.9). In turn, NDF content was greater in the S diet (74.6%) than in the S+L diet (64.8%).
[Insert Table 1 around here]
Forage intake In the four periods, the average daily forage intake (DM basis) was 5.6 kg for the S+L diet and 4.7 kg for the S diet (P < 0.05; Table 2). In a dry matter basis, the proportion of leucaena in the S+L diet was 26% and animals consumed 2.47% of DM as a percentage of their body weight, compared with only 2.02% when they were offered the S diet.
[Insert Table 2 around here]
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Intake (g of DM per kg of metabolic weight) was 17% greater in the S+L treatment than in the S treatment (95.9 vs. 78.9, P < 0.05). As a result of greater DMI, nutrient and energy intake was higher (P < 0.05) in heifers receiving the S+L diet (Table 3) especially in reference to the intake of protein, calcium and gross energy which were at least 22% greater in the S+L diet than those observed in the S diet.
[Insert Table 3 around here]
Temperature and relative humidity During the 24 hour period in which methane measurements took place, the greatest temperature within the polytunnels (31.9 °C) occurred at 14:30, and the minimum temperature (20.1 °C) was observed at 06:30 h. Differences (P < 0.05) between temperatures inside and outside the polytunnels were only observed at 02:30, 06:30, 11:30, and, 23:30 h but were not greater than 0.8 °C. Contrary to temperature, the relative humidity (RH) within polytunnels was only equal to the outside RH in eight of the 24 hours during the methane collection period. Significant differences in RH inside and outside the polytunnels were observed at all other times (P < 0.05). The lowest RH value (40%) was measured at 14:30 h outside the polytunnel (P < 0.05). In the early morning hours (between 01:30 and 06:30 h) the RH inside the polytunnels had values close to 90%, whilst the outside relative humidity averaged 83.1%. According to the THI, 95% of the time, animals inside the polytunnels experienced a THI between "normal" and "alert". Only around 14:30 h, the THI suggested that the animals were in heat stress (Fig 1).
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[Insert Fig 1 around here]
Methane emissions Average methane emissions over the 4 experimental periods were 211.5 L/d for animal receiving the S+L diet, and 205.2 for those receiving the S diet (P = 0.78). This 6.5 L difference represent on average 3% less methane produced when animals were fed the S diet compared to when they received the S+L diet. Animals receiving the S diet, produced 144.9 g CH4/d compared with 149.4 g CH4/d for animal when they received the S+L diet (Table 4).
[Insert Table 4 around here]
To calculate methane emissions per kg of fermented dry matter (FDM), dry matter digestibility estimates were obtained from the studies conducted by Gaviria et al. (2015), Gaviria-Uribe et al. (2015) and Molina et al. (2013), who reported an average digestibility of 46.9% for star grass and of 60.1% for a diet consisting of 70% grass and 30% leucaena. Estimated methane production (% of fermented DM) for the S+L diet, typical of an ISS was 4.1 and 6.3 for the S diet (P = 0.04; Fig. 2).
[Insert Fig. 2 around here]
Discussion Diet and DMI
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In this study, although both diets had high NDF contents (Table 1), that of the S treatment can be limiting for animal production, as voluntary intake was diminished (Table 2), probably due to physical factors of the diet. Gaviria-Uribe et al. (2015) reported contents of 61.7% NDF for a diet containing 22.6% leucaena and 77.4% C. plectostachyus, and similar contents of ash, ADF and calorific value (11.9%, 42.6% y 17.7 MJ/kg), respectively than those found in this study. Observed DMI in the S+L diet is similar to that estimated by Gaviria-Uribe et al. (2015) with the n-alkane technique for animals grazing in a Leucaena ISS, both in quantity (2.5% of BW) and in composition (22.6% leucaena and 77.4% C. plectostachyus). Thus, from the standpoint of diet, the results of the current experiment, are applicable to animals grazing in ISS pastures. The Cornell Net Protein and Carbohydrate System (Fox et al., 2003) model was used to verify the suitability of the two diets evaluated in this investigation. In the S+L diet, after satisfying maintenance requirements, additional nutrients allowed for an estimated weight gain of 640 g/d as daily intake of metabolizable protein (MP) and metabolizable energy (ME) were 525 g and 48.8 MJ, respectively. In the case of the S diet, the estimated daily intake of energy and protein (34.7 MJ ME and 331 g MP) only satisfied maintenance requirements.
Thermal comfort Although the THI suggested that animals were in heat stress one hour per day when housed in the polytunnel (Fig 1), other experimental data suggest that this was not the case. For example, determination of heifer weight changes during their stay in the polytunnels showed an average daily gain of 500 g/d in both experimental periods. 11
This growth rate and the adequate feed intake observed, suggest that animals were not affected by heat stress. This is in part explained by the genetic and phenotypic adaptations of Lucerna breed to tropical conditions (pigmented skin with short, fine, and dense hair). Methane emissions In this study, average emissions were 147 g CH4/d in animals of 218 kg of liveweight. Using the polytunnel technique, Molina et al. (2015) reported emissions of 159 g CH4/d in animals of 280 kg of liveweight consuming a Leucaena-star grass diet. In turn, Lockyer (1997) reported emissions of 74.5 g CH4/d for calves with an average live weight of 170 kg and these lower emissions were due to the low DMI observed in that experiment. A positive correlation of r = 0.37 to 0.77 between dry matter intake and methane emissions per animal per day has been reported (Ulyatt and Lassey, 2001; PinaresPatiño, 2006). The results of the current study suggest that while there is a positive relationship between dry matter intake and methane emissions per animal per day, it depends on the diet consumed. In this study although the animals on diet S+L consumed 19% more of DMI/d (Table 2), they only produced 3% more methane per animal per day (Table 4) than animals fed the S diet. In terms of structural carbohydrate content (NDF), the greatest contribution came from the star grass. Many authors have reported that increases in NDF concentrations lead to increased methane emissions (Archimède et al., 2011). However, Meale et al. (2012) found no relationship between NDF content and methane production when comparing leguminous trees and grasses.
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A factor that should receive increased attention with regards to methane emissions is the presence of condensed tannins in species such as leucaena (Barahona et al., 2003), which can reduce methane production by inhibiting the growth of cellulolytic and proteolytic bacteria (McSweeney et al., 2001). This effect depends on the molecular weight and chemical structure of the phenol (Huang et al., 2011), and the amount of tannin per kilogram of DM (Archimède et al., 2011). When the ability of condensed tannins purified from tropical forage legumes to inhibit the in vitro degradability or the activity of several hydrolytic enzymes was evaluated, the lowest reductions in these parameters occurred in the presence of tannins from L. leucocephala (Barahona et al., 2006). Notably, tannins from L. leucocephala showed the low molecular weights, having on average eight anthocyanidins (ie pelargonidin, delphinidin, cyanidin, etc.), whereas tannins from other tropical legumes had between 11 and 15 such anthocyanidins (Barahona 1999). Due to its lower molecular weight, L. leucocephala tannins are not as efficient in binding to and precipitating protein and other nutrients and making them unavailable to the microorganisms in the rumen of cattle. The percentage of gross energy lost in the form of methane (Ym) was 9.4 for the S diet and 8.0 for the S+L diet. These Ym estimates are similar to those reported by Molina et al. (2015) with heavier animals but eating similar diets to the ones used in this experiment. Likewise, in studios in vitro, Rivera et al. (2015) reported an Ym of 9.10 and 6.93 for a diet of 100% star grass and another with 73% of star grass and 27% of L. leucocephala. Reports of Ym values are varied and the difference can be largely explained by the digestibility of the diet, which in turn explains differences in intake. Estimates of Ym found in this study are higher than those previously reported. 13
For example, the IPCC (2006) established a Ym of 6.5% for pastoral systems, whereas nearly two decades ago, Johnson and Ward (1996) set Ym values between 5.5 and 9% for dairy cattle and 3.5 to 6.5% for cattle in confinement. It is generally accepted that the most productive animals are the ones with greatest enteric methane emissions (IPCC, 2007), as increased productivity is in most of the cases associated with increased DMI. In the present study, however, increased DMI in the L. leucocephala diet was not accompanied by increased methane emissions, which corroborates the findings that Ym value decreases per each additional kg in DMI (Cantet el al., 2015). Interestingly, the implementation of ISS based on L. leucocephala generally leads to increments in animal productivity (Naranjo et al., 2012; Tarazona et al., 2013), due both to greater DMI and to better balance of dietary nutrients. Steers grazing in ISS have at least 1.5x the average daily weight of steers grazing in grass-only pastures, whereas cows in ISS produce at least two times the amount of milk produced by cows grazing in grass-only pastures. Thus, in a kg of weight gain basis, steers in ISS emit at least 33% less methane than steers in grassonly pastures, whereas emissions per liter of milk should only be 50% in ISS. This is in agreement with the reports of Blaxter and Clapperton (1965), who stated that the higher the digestibility of forages, the lower the methane production per unit consumed. Moreover, it is expected that the increased supply of nutrients from leucaena (Archimède et al., 2011), especially degradable protein and soluble carbohydrates, will lead to a decrease in the acetic acid: propionic acid ratio. If this ratio reaches 0.5, methane loss is minimal (Johnson and Johnson, 1995) because the lowering ruminal pH inhibits the growth of methanogenic microorganisms (Van Kessel and Rusell, 1996). 14
Conclusions The inclusion of Leucaena leucocephala in a diet based on star grass resulted in a greater dietary content of crude protein, calcium and gross energy and in a reduction in NDF content in the diet consumed. These nutritional benefits contribute to the greater animal productivity frequently reported in intensive silvopastoral systems. The inclusion of leucaena in the diet resulted in a 19% increase in DMI, but it was not associated with increased methane emissions from growing Lucerna heifers. On the contrary, the energy loss in the form of methane emitted per kilogram of fermented dry matter decreased by 53% with the inclusion of Leucaena leucocephala. This suggests that adopting and implementing ISS is a viable alternative to reduce the carbon footprint of beef products under tropical conditions.
Acknowledgements The present study is part of the "Nitrogen usage by Colombian Criollo Cattle in Intensive Silvopastoral Systems with Leucaena leucocephala in dry tropical forest conditions" project, funded by COLCIENCIAS through the Francisco José de Caldas National Fund for Science, Technology and Innovation, and executed by CIPAV and the National University of Colombia at Medellín in collaboration with CORPOICATibaitatá. The authors thank the Molina Durán family and the "Natural Reserve El Hatico" for the cooperation received during the project.
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McSweeney, C., Palmer, B., Bunch, R., Krause, D., 2001. Effect of the tropical forage calliandra on microbial protein synthesis and ecology in the rumen. J. Appl. Microbiol. 90, 78-88. Meale, S.J., Chaves, A.V., Baah, J., McAllister, T.A., 2012. Methane production of different forages in vitro ruminal fermentation. Asian-Australas. J. Anim. Sci. 25, 86-91. Molina, I.C., Cantet, J.M., Montoya, S., Correa, G., Barahona, R., 2013. Producción in vitro de metano de dos gramíneas tropicales solas y mezcladas con Leucaena leucocephala o Gliricidia sepium. CES Med. Vet. Zootec. 8, 15-31. Molina, I.C., Donney`s, G., Montoya, S., Rivera, J.E., Villegas, G., Chará, J., Barahona, R., 2015. The inclusion of Leucaena leucocephala reduces the methane production in Lucerne heifers receiving a Cynodon plectostachyus and Megathyrsus maximus diet. Livest. Res. Rural. Dev. 27, 96, http://www.lrrd.org Morgavi, D.P., Forano, E., Martin, C., Newbold, C.J., 2010. Microbial ecosystem and methanogenesis in ruminants. Anim. 40, 1024-1036. Moss, A.R., Jouany, J.P., Newbold, J.A., 2000. Methane production by ruminants: its contribution to global warming. Ann. Zootech. 49, 231–253. Murgueitio Restrepo, E., Chará Orozco, J.D., Barahona Rosales, R., Cuartas Cardona, C.A., Naranjo Ramirez, J.F., 2014. Intensive silvopastoral systems (ISPS), mitigation and adaptation tool to climate change. Tropic. Subtropic. Agroecosyst. 17(3), 501 – 507. Murray, P.J., Gill, E., Balsdon, S.L., Jarvis, S.C., 2001. A comparison of methane emissions from sheep grazing pastures with differing management intensities. Nutr. Cycl. Agroecosyst. 60, 93-97. Naranjo, J.F., Cuartas, C.A., Murgueitio, E., Chará, J., Barahona, R., 2012. Balance de gases de efecto invernadero en sistemas silvopastoriles intensivos con Leucaena leucocephala en Colombia. Livest. Res. Rural. Dev. 24, http://www.lrrd.org/lrrd24/8/nara24150.htm
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Pinares-Patiño, C.S., D’Hour, P., Jouany, J.P., Martin, C., 2006. Effects of stocking rate on methane and carbon dioxide emissions from grazing cattle. Agric. Ecosyst. Environ. 121, 30-46. Possenti, R.A., Franzolin, R., Schammas, E.A., Assumpção, J.J., Shiraishi, R.T., Lima, M.A., 2008. Efeitos de dietas contendo Leucaena leucocephala e Saccharomyces cerevisiae sobre a fermentação ruminal e a emissão de gás metano em bovinos. Rev. Bras. Zootec. 37, 1509-1516. Rivera, J.E., Molina, I. C., Donney´s, G., Villegas, G., Chará, J., Barahona, R., 2015. Dinamica de fermentación y producción de metano en dietas de sistemas silvopastoriles intensivos con L. leucocephala y sistemas convencionales orientados a la producción de leche. Livest. Res. Rural. Dev. 27(96). http://www.lrrd.org/lrrd27/4/rive270680.html SAS Institute, 2003. User's Guide: Statistics Version 9.1, ed. SAS Inst. Inc., Cary, NC. Tan, H.Y., Sieo, C.C., Abdullah, N., Liang, J.B., Huang, X.D., Ho, Y.W., 2011. Effects of condensed tannins from Leucaena on methane production, rumen fermentation and populations of methanogens and protozoa in vitro. Anim. Feed. Sci. Technol. 169, 185193. Tarazona Morales, A.M., Ceballos, A.M., Cuartas Cardona, C.A., Naranjo Ramirez, J.F., Murgueitio Restrepo, E., Barahona Rosales, R., 2013. The relationship between nutritional status and bovine welfare associated with adoption of intensive silvopastoral systems in tropical conditions. In: Enhancing animal welfare and farmer income through strategic animal feeding − Some case studies. FAO, Rome, Italy, p. 69–78. Ulyatt, M.J., Lassey, K.R., 2001. Methane emissions from pastoral systems: the situation in New Zealand. Arch. Latinoam. Prod. Anim. 9, 118-126. Van Kessel, J.A., Russell, J.B., 1996. The effect of pH on ruminal methanogenesis. FEMS Microbiol. Ecol. 20, 205-210.
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Van Soest, P.J., Robertson, J.B., Lewis, B.A., 1991. Methods for dietary fiber neutral detergent fiber and nonstarch polysaccharides in relation to animal nutrition. J. Dairy Sci. 74, 3583-3597.
Fig 1.
Temperature and humidity index, %
82
Danger
80 78
Alert 76
Normal
74
Polytunnel1 Polytunnel2
72
Ambient 70 68 66
Time, h
Fig 2.
21
Methane emissions, g kg-1 fermented dry matter
70 60 50 40 Star grass + Leucaena
30
Star grass
20 10 0 0630 0930 1230 1530 1830 2130 0030 0330 0630
Time, h
Table 1 Average composition of the forages evaluated in this study Diet1 Nutrient
S
S+L
10,8
13,9
NDF , %
74,6
64,9
ADF3, %
43,0
41,9
Ether extract, %
1,16
1,17
Gross energy, Mj·kg-1
17,9
18,3
Ash, %
10,2
9,6
Calcium, %
0,37
0,42
Phosphorus, %
0,33
0,33
Crude protein, % 2
1
Diet: S= Star grass 100%; S+L= Star grass 74% + Leucaena 26%.
2
NFD = Neutral Detergent Fibre.
22
3
ADF = Acid Detergent Fibre.
23
Table 2 Average fresh forage and dry matter intake of Lucerna heifers receiving a star grass (C. plectostachyus) diet with (S+L) or without (S) leucaena (L. leucocephala) S2
S+L1 Star
P-value
Star
grass
Leucaena
Total
grass
Diet
Run
Diet*Run
18.00
5.68
23.7a
18.9b
0.03
0.50
0.47
13
Dry matter, kg day
4.27
1.34
5.61a
4.71b
0.01
0.11
0.60
13
Dry matter, % body weight
1.88
0.59
2.47a
2.02b
0.01
0.36
0.49
0
73.0
22.9
95.9a
78.9b
0.01
0.64
0.47
12
Item -1
Fresh forage, kg day -1
1
Dry matter, g kg- of weight 0.75 1
S+L= Star grass 74% + Leucaena 26%.
2
S= Star grass 100%;
a,b
Means in the same column with different letters are statistically different according to Tukey's test.
(P < 0.05). SD = Standard Deviation
24
S
Table 3 Average daily intake of nutrients and energy of Lucerna heifers receiving a star grass (C. plectostachyus) diet with (S+L) or without (S) leucaena (L. leucocephala) Diet1 Item
S+L
S
Diet
Run
Diet*Run
SD
Protein, g
755a
504b
˂0.01
0.09
0.04
16.81
NDF2, Kg
3.72
3.51
0.40
0.38
0.71
13.53
ADF3, Kg
2.39
2.02
0.06
0.43
0.62
13.57
Fat, g
65.3
55.0
0.29
0.23
0.49
15.82
Ash, g
549.4a
477.8b
0.02
0.53
0.26
13.55
Calcium, g
23.0a
17.2b
<0.01
0.13
0.37
15.06
Phosphorus, g
18.7
15.6
0.09
0.21
0.36
14.33
102.9a
83.6b
0.02
0.33
0.47
13.50
Gross Energy, Mj 1
P-value
Dieta: S = Star 100; S+L = Star 76 + Leucaena 24%.
2
NFD = Neutral Detergent Fibre.
3
ADF = Acid Detergent Fibre.
a,b
Means in the same column and item with different letters are statistically different according to
Tukey's test (P < 0.05). SD = Standard Deviation
25
Table 4 Daily production of methane in Lucerna breed heifers eating 100 % star grass or a mixture of 74 % star grass and 26 % leucaena. S+ L1
S2
Diet
Run
Diet*Run
Methane, g animal d
149.4
144.9
0.78
0.06
0.27
Methane, % of DMI3
2.46
2.95
0.23
0.18
0.26
Methane, % of organic matter intake
2.72
3.27
0.36
0.23
0.48
Methane, % of fermented DM4
4.10b
6.29a
0.04
0.19
0.25
Energy lost as methane, Mj/ day/ animal
8.24
7.99
0.78
0.06
0.27
Energy lost as methane, % of gross energy intake
7.96
9.42
0.32
0.22
0.49
Item -1
-1
1
S+L= Star grass 74% + Leucaena 26%.
2
S= Star grass 100%;
3
DMI = Dry matter Intake
4
DM = Dry matter
a,b
Means in the same column and item with different letters are statistically different according to
Tukey's test (P < 0.05). SD = Standard Deviation
26
Figure 1 Polytunnel and ambient (outside) temperature and humidity index during the four experimental periods
Figure 2 Methane produced during 24 hours by animals fed 100% star grass or a mixture of leucaena and star grass.
Highlights We measured methane emissions by heifers fed grass-only or grass-Leucaena diets. Leucaena-fed heifers ate more DM (1.19x) and protein (1.50x) than grass-only heifers. Methane emissions (g CH4·animal-1·d-1) were similar between both animal groups. Methane emissions per kg of fermented DM were 1.5X greater in grass-only heifers. The inclusion of Leucaena can lead to reduced carbon footprint of animal products.
27
PII: DOI: Reference:
S1871-1413(16)30009-9 http://dx.doi.org/10.1016/j.livsci.2016.01.009 LIVSCI2927
To appear in: Livestock Science Received date: 25 April 2015 Revised date: 6 October 2015 Accepted date: 12 January 2016 Cite this article as: I.C. Molina, E.A. Angarita, O.L. Mayorga, J. Chará and R. Barahona-Rosales, Effect of Leucaena leucocephala on methane production of Lucerna heifers fed a diet based on Cynodon plectostachyus, Livestock Science, http://dx.doi.org/10.1016/j.livsci.2016.01.009 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Effect of Leucaena leucocephala on methane production of Lucerna heifers fed a diet based on Cynodon plectostachyus
I. C. Molina a, E. A. Angarita b, O. L. Mayorga c, J. Chará d and R. Barahona-Rosales a
a
Department of Animal Production, Faculty of Agricultural Sciences, Universidad
Nacional de Colombia, Medellin, Colombia. b
Department of Animal Science, Faculty of Veterinary Medicine and Animal Sciences,
National University of Colombia, Bogota, Colombia. c
Colombian Agricultural Research Corporation, CORPOICA, Mosquera,
Cundinamarca, Colombia. d
Centro para la Investigación en Sistemas Sostenibles de Producción Agropecuaria,
CIPAV, Cali, Colombia.
Corresponding author: Rolando Barahona Rosales. Email: [email protected]. Tel: +5744309109
Short title: Leucaena and bovine enteric methane emissions
Abstract A rapid growth of the meat production industry is necessary to satisfy increased demand for this commodity, which might have negative impacts on the environment. The objective of this study was to assess enteric methane (CH4) emissions when a forage legume is introduced in the diet of animals consuming a tropical grass. Eight 1
Lucerna heifers, 218 ± 18 kg live weight with an average age of 19 ± 3 months were used in two experiments following a changeover design. The diets evaluated were 100% star grass (Cynodon plectostachyus, S) or 76% star grass plus 24% leucaena (Leucaena leucocephala, S+L). Throughout the experiment, animals were housed in two chambers, in which the diet was offered four times daily. Each chamber had a small wind tunnel, which housed a fan set to a constant speed of extraction. Air samples were obtained every hour during 24 hours both inside and outside (ambient) the tunnel. Methane concentration in these samples was determined by gas chromatography. Temperature and relative humidity both inside and outside the tunnel were recorded using a thermo-hygrometer. The S+L diet had greater protein content whereas the S diet had greater content of neutral detergent fiber. Average intake (kg/d) of fresh forage and dry matter (DM) was significantly greater (23.7 and 5.6) for the S+L than for the S diet (18.9 and 4.7), respectively (P < 0.05). The maximum recorded temperature and humidity inside the chamber was 35.5 °C and 99%, respectively, but the minimum values were 19.1 °C and 38%, respectively. Methane production (L/kg DMI) was 37.7 for the S+L treatment and 43. 6 for the S treatment. The energy loss in the form of methane emitted was 8.0% for S+L and 9.4% for the star grass based diet (P = 0.32). These results suggest that while increasing animal productivity by increasing dry matter intake, the inclusion of leucaena does not increase methane emission per animal, thus significantly decreasing methane emissions per kg of meat or milk produced.
Keywords: Bovines; enteric methane emissions; forage intake; intensive silvopastoral systems; polytunnel technique. 2
Introduction Global meat production will double by 2050, based on the 229 million tons produced in 2000, and milk production will rise from 722 million tons in 2010 to 1043 million tons in 2050 (FAO, 2009b). These production gains are largely expected to come from increases in livestock numbers and system productivity (FAO, 2009a). Cattle population is expected to increase from 350 to 483 million heads between 1999 and 2030 (Bruinsma, 2003). These changes can have negative environmental effects, as about 6–10% of the total gross energy consumed by bovines is converted to methane (Eckard et al., 2010). Rumen methane results from the action of different anaerobic microorganisms (Morgavi et al., 2010) when hydrolysing proteins, starches and cell wall components of the diet (Moss et al., 2000). Methane is produced by microorganisms of the Archaea domain, which mainly use hydrogen, formate, acetate, methanol and methylalanine groups as substrates (Liu and Whitman, 2008). The main factor affecting enteric methane emissions is diet composition, particularly protein, lipid and carbohydrate content (Ulyatt and Lassey, 2001). Other factors include the forage species, the stage of maturity, the use of preservation methods, chemical or physical treatments (Tan et al., 2011) and the presence of secondary metabolites (Huang et al., 2011). Intensive Silvopastoral Systems (ISS) are livestock production systems that are more efficient and productive than traditional systems (Murgueitio et al., 2014), but their environmental impacts, in terms of methane emissions, have not been yet comprehensively studied. Thus, the aim of this study was to evaluate in vivo the effect 3
of the inclusion of Leucaena leucocephala, a forage legume typical of intensive silvopastoral systems, on methane emissions and energy loss associated with the emission of this GHG.
Material and methods Location The research was carried out in El Hatico Natural Reserve, located in the municipality of Cerrito, Valle del Cauca Department, Colombia. Its ecological classification according to Holdridge corresponds to tropical dry forest (bs-T). The reserve is located 1000 meters above sea level and its average temperature, relative humidity and annual precipitation are 24°C, 75%, and 750 mm, respectively.
Description of animals To carry out this study, eight Lucerna breed heifers (4 for each cycle) with a live weight of 218 ± 18 kg and an average age of 19 ± 3 months were selected. Two independent cycles (runs), a month apart, were conducted with these animals. Two polytunnels were used in each experiment (two animals per polytunnel).
Diets used The diets evaluated were 100% star grass (S; Cynodon plectostachyus K. Schum) and 76% star grass with 24% leucaena (S+L; Leucaena leucocephala (Lam.) De Wit (leucaena) cv. Cunningham) on a fresh basis. Mineral salt and water were offered ad libitum. During the first period, diet x was assigned to the first group and diet y to the second group. During the second experimental period, diets were exchanged 4
between animals. In a second experiment, another four animals were used, divided also in two groups and diets were assigned for two periods, according to the procedure followed during the first experiment. The time allotted for adaptation to the diet in each of the four periods was seven days. The forages were harvested at an average re-growth age of 45 days of an ISS with L. leucocephala (10.000 bushes/ha) and C. plectostachyus. The star grass used in the two treatments was harvested from different paddocks with and without the presence of legumes. The ration was fed four times daily at 08:00, 11:00, 14:30, and 18:00 hours. Diet intake was determined as the difference between forage offered and refused, which were weighed daily during the stay of the animals in the polytunnel.
Feed analysis Pools were made with feed samples collected from each experimental group during the last three days of each measurement period, which were sent to the Nutrition laboratory of the Universidad Nacional de Colombia, Medellin where they were dried to constant weight. Forages were milled to pass through a 1 mm sieve using a Romer mill (Romer Labs, México). Samples were analyzed for crude protein content (CP) by the Kjeldahl method based on NTC 4657; neutral detergent (NDF) and acid detergent fiber (ADF) according to Van Soest et al. (1991) and ether extract (EE) by Soxhlet immersion (NTC 668). Ash was measured by direct incineration to 550 °C in a muffle (AOAC 942.05), the calcium (Ca) and phosphorus (P) contents were determined by UV-VIS AA spectrophotometry (NTC 5151 and 4981, respectively) and energy values were determined by calorimetry according to the ISO 9831 method.
5
In vivo methane measurements These experiments followed Murray’s et al. (2001) recommendations. Polytunnels used were 7 m long, 5 m wide and 2.6 m high for a total volume of 83.5 m 3. The structure had an inlet for animals and staff and on the opposite side there was a 12” fume hood set at an extraction rate of 0.9 m3/s to allow sampling of gases. To adapt animals to the conditions in the polytunnel, during a 48 hour period whilst the animals were housed in the polytunnel, the door was closed for an hour and the then opened for the next two. Methane measurements were conducted at hourly intervals during a 24 hour period. To collect gas samples, a three-way valve was used; during the first 10 s after the start of extraction the first outlet, connected to a 12 mL plastic syringe was used; the second outlet was attached to a hypodermic needle and 7 mL vacuum tubes. Ambient air (outside of the tunnel) was sampled simultaneously. The collected gas samples were stored in a cool dry place and transported to the laboratory for analysis by gas chromatography. Methane production was calculated according to the ideal gas law (Lopez and Newbold, 2007) using the gas concentration measured by chromatography, the temperature inside the polytunnel when taking the methane samples and the atmospheric pressure at the site. Total methane production was calculated by multiplying by the total volume of the polytunnel.
Measurement of polytunnel temperature and relative humidity Before collecting the methane samples, the temperature and the relative humidity were recorded both inside and outside the polytunnel using a thermo-hygrometer. From these data, the temperature and humidity index (THI) was calculated to 6
evaluate the level of heat stress of experimental animals. To do this, the equation of Kibler (1964) was used: THI = 1.8 * T – (1 - (RH / 100)) * (T - 14.3) + 32
(Equation 1)
where THI denotes the temperature-relative humidity index, T is temperature in °C and RH is relative humidity in %. For the interpretation of this index, the safety index of the Livestock Conservation Institute was used based on THI values as follows: 74 or less: normal; 75-78: alert; 79-83: danger, and 83 or more: emergency
Gas chromatography The methane concentration in the samples was determined at the International Center for Tropical Agriculture (CIAT), Palmira, Valle del Cauca, Colombia. Determinations were made on a Shimadzu gas chromatograph (Shimadzu, Japan) equipped with a flame ionization detector and an electron capture detector. The chromatographic conditions were as follows: N Hayesep column 3 m in length; mobile phase: high purity nitrogen at a flow of 35 mL/min. Oven, injector and detector temperatures were 250, 100, and 325 °C, respectively. The standard used was Scott methane standard.
Experimental Design and Statistical Analysis To determine the effect of treatments on the production of methane the PROC MIXED procedure with SAS 9.1 software (SAS Institute, 2003). Separation of means was made using the Tukey test with an alpha of 0.05. The model is described below Yij = μ + δi + Rj + (δ *R)ij + ҽijk, 7
where Yij is observations of the subject in the diet i and period j ; μ: is the overall mean of the population; δi: is the effect of the i-th diet; Rj: is the effect of j-th run; (δ *R)ij is the interaction between the i-th diet and the j-th run; ҽijk: is the experimental error.
Results Nutritional quality The greatest difference between diets was related to the protein content, which in the S+L diet was almost 14% and only 10.8% in the grass diet (S; Table 1). The gross energy content (MJ/kg) of the S+L diet was 2% greater (18.3) than that of the S diet (17.9). In turn, NDF content was greater in the S diet (74.6%) than in the S+L diet (64.8%).
[Insert Table 1 around here]
Forage intake In the four periods, the average daily forage intake (DM basis) was 5.6 kg for the S+L diet and 4.7 kg for the S diet (P < 0.05; Table 2). In a dry matter basis, the proportion of leucaena in the S+L diet was 26% and animals consumed 2.47% of DM as a percentage of their body weight, compared with only 2.02% when they were offered the S diet.
[Insert Table 2 around here]
8
Intake (g of DM per kg of metabolic weight) was 17% greater in the S+L treatment than in the S treatment (95.9 vs. 78.9, P < 0.05). As a result of greater DMI, nutrient and energy intake was higher (P < 0.05) in heifers receiving the S+L diet (Table 3) especially in reference to the intake of protein, calcium and gross energy which were at least 22% greater in the S+L diet than those observed in the S diet.
[Insert Table 3 around here]
Temperature and relative humidity During the 24 hour period in which methane measurements took place, the greatest temperature within the polytunnels (31.9 °C) occurred at 14:30, and the minimum temperature (20.1 °C) was observed at 06:30 h. Differences (P < 0.05) between temperatures inside and outside the polytunnels were only observed at 02:30, 06:30, 11:30, and, 23:30 h but were not greater than 0.8 °C. Contrary to temperature, the relative humidity (RH) within polytunnels was only equal to the outside RH in eight of the 24 hours during the methane collection period. Significant differences in RH inside and outside the polytunnels were observed at all other times (P < 0.05). The lowest RH value (40%) was measured at 14:30 h outside the polytunnel (P < 0.05). In the early morning hours (between 01:30 and 06:30 h) the RH inside the polytunnels had values close to 90%, whilst the outside relative humidity averaged 83.1%. According to the THI, 95% of the time, animals inside the polytunnels experienced a THI between "normal" and "alert". Only around 14:30 h, the THI suggested that the animals were in heat stress (Fig 1).
9
[Insert Fig 1 around here]
Methane emissions Average methane emissions over the 4 experimental periods were 211.5 L/d for animal receiving the S+L diet, and 205.2 for those receiving the S diet (P = 0.78). This 6.5 L difference represent on average 3% less methane produced when animals were fed the S diet compared to when they received the S+L diet. Animals receiving the S diet, produced 144.9 g CH4/d compared with 149.4 g CH4/d for animal when they received the S+L diet (Table 4).
[Insert Table 4 around here]
To calculate methane emissions per kg of fermented dry matter (FDM), dry matter digestibility estimates were obtained from the studies conducted by Gaviria et al. (2015), Gaviria-Uribe et al. (2015) and Molina et al. (2013), who reported an average digestibility of 46.9% for star grass and of 60.1% for a diet consisting of 70% grass and 30% leucaena. Estimated methane production (% of fermented DM) for the S+L diet, typical of an ISS was 4.1 and 6.3 for the S diet (P = 0.04; Fig. 2).
[Insert Fig. 2 around here]
Discussion Diet and DMI
10
In this study, although both diets had high NDF contents (Table 1), that of the S treatment can be limiting for animal production, as voluntary intake was diminished (Table 2), probably due to physical factors of the diet. Gaviria-Uribe et al. (2015) reported contents of 61.7% NDF for a diet containing 22.6% leucaena and 77.4% C. plectostachyus, and similar contents of ash, ADF and calorific value (11.9%, 42.6% y 17.7 MJ/kg), respectively than those found in this study. Observed DMI in the S+L diet is similar to that estimated by Gaviria-Uribe et al. (2015) with the n-alkane technique for animals grazing in a Leucaena ISS, both in quantity (2.5% of BW) and in composition (22.6% leucaena and 77.4% C. plectostachyus). Thus, from the standpoint of diet, the results of the current experiment, are applicable to animals grazing in ISS pastures. The Cornell Net Protein and Carbohydrate System (Fox et al., 2003) model was used to verify the suitability of the two diets evaluated in this investigation. In the S+L diet, after satisfying maintenance requirements, additional nutrients allowed for an estimated weight gain of 640 g/d as daily intake of metabolizable protein (MP) and metabolizable energy (ME) were 525 g and 48.8 MJ, respectively. In the case of the S diet, the estimated daily intake of energy and protein (34.7 MJ ME and 331 g MP) only satisfied maintenance requirements.
Thermal comfort Although the THI suggested that animals were in heat stress one hour per day when housed in the polytunnel (Fig 1), other experimental data suggest that this was not the case. For example, determination of heifer weight changes during their stay in the polytunnels showed an average daily gain of 500 g/d in both experimental periods. 11
This growth rate and the adequate feed intake observed, suggest that animals were not affected by heat stress. This is in part explained by the genetic and phenotypic adaptations of Lucerna breed to tropical conditions (pigmented skin with short, fine, and dense hair). Methane emissions In this study, average emissions were 147 g CH4/d in animals of 218 kg of liveweight. Using the polytunnel technique, Molina et al. (2015) reported emissions of 159 g CH4/d in animals of 280 kg of liveweight consuming a Leucaena-star grass diet. In turn, Lockyer (1997) reported emissions of 74.5 g CH4/d for calves with an average live weight of 170 kg and these lower emissions were due to the low DMI observed in that experiment. A positive correlation of r = 0.37 to 0.77 between dry matter intake and methane emissions per animal per day has been reported (Ulyatt and Lassey, 2001; PinaresPatiño, 2006). The results of the current study suggest that while there is a positive relationship between dry matter intake and methane emissions per animal per day, it depends on the diet consumed. In this study although the animals on diet S+L consumed 19% more of DMI/d (Table 2), they only produced 3% more methane per animal per day (Table 4) than animals fed the S diet. In terms of structural carbohydrate content (NDF), the greatest contribution came from the star grass. Many authors have reported that increases in NDF concentrations lead to increased methane emissions (Archimède et al., 2011). However, Meale et al. (2012) found no relationship between NDF content and methane production when comparing leguminous trees and grasses.
12
A factor that should receive increased attention with regards to methane emissions is the presence of condensed tannins in species such as leucaena (Barahona et al., 2003), which can reduce methane production by inhibiting the growth of cellulolytic and proteolytic bacteria (McSweeney et al., 2001). This effect depends on the molecular weight and chemical structure of the phenol (Huang et al., 2011), and the amount of tannin per kilogram of DM (Archimède et al., 2011). When the ability of condensed tannins purified from tropical forage legumes to inhibit the in vitro degradability or the activity of several hydrolytic enzymes was evaluated, the lowest reductions in these parameters occurred in the presence of tannins from L. leucocephala (Barahona et al., 2006). Notably, tannins from L. leucocephala showed the low molecular weights, having on average eight anthocyanidins (ie pelargonidin, delphinidin, cyanidin, etc.), whereas tannins from other tropical legumes had between 11 and 15 such anthocyanidins (Barahona 1999). Due to its lower molecular weight, L. leucocephala tannins are not as efficient in binding to and precipitating protein and other nutrients and making them unavailable to the microorganisms in the rumen of cattle. The percentage of gross energy lost in the form of methane (Ym) was 9.4 for the S diet and 8.0 for the S+L diet. These Ym estimates are similar to those reported by Molina et al. (2015) with heavier animals but eating similar diets to the ones used in this experiment. Likewise, in studios in vitro, Rivera et al. (2015) reported an Ym of 9.10 and 6.93 for a diet of 100% star grass and another with 73% of star grass and 27% of L. leucocephala. Reports of Ym values are varied and the difference can be largely explained by the digestibility of the diet, which in turn explains differences in intake. Estimates of Ym found in this study are higher than those previously reported. 13
For example, the IPCC (2006) established a Ym of 6.5% for pastoral systems, whereas nearly two decades ago, Johnson and Ward (1996) set Ym values between 5.5 and 9% for dairy cattle and 3.5 to 6.5% for cattle in confinement. It is generally accepted that the most productive animals are the ones with greatest enteric methane emissions (IPCC, 2007), as increased productivity is in most of the cases associated with increased DMI. In the present study, however, increased DMI in the L. leucocephala diet was not accompanied by increased methane emissions, which corroborates the findings that Ym value decreases per each additional kg in DMI (Cantet el al., 2015). Interestingly, the implementation of ISS based on L. leucocephala generally leads to increments in animal productivity (Naranjo et al., 2012; Tarazona et al., 2013), due both to greater DMI and to better balance of dietary nutrients. Steers grazing in ISS have at least 1.5x the average daily weight of steers grazing in grass-only pastures, whereas cows in ISS produce at least two times the amount of milk produced by cows grazing in grass-only pastures. Thus, in a kg of weight gain basis, steers in ISS emit at least 33% less methane than steers in grassonly pastures, whereas emissions per liter of milk should only be 50% in ISS. This is in agreement with the reports of Blaxter and Clapperton (1965), who stated that the higher the digestibility of forages, the lower the methane production per unit consumed. Moreover, it is expected that the increased supply of nutrients from leucaena (Archimède et al., 2011), especially degradable protein and soluble carbohydrates, will lead to a decrease in the acetic acid: propionic acid ratio. If this ratio reaches 0.5, methane loss is minimal (Johnson and Johnson, 1995) because the lowering ruminal pH inhibits the growth of methanogenic microorganisms (Van Kessel and Rusell, 1996). 14
Conclusions The inclusion of Leucaena leucocephala in a diet based on star grass resulted in a greater dietary content of crude protein, calcium and gross energy and in a reduction in NDF content in the diet consumed. These nutritional benefits contribute to the greater animal productivity frequently reported in intensive silvopastoral systems. The inclusion of leucaena in the diet resulted in a 19% increase in DMI, but it was not associated with increased methane emissions from growing Lucerna heifers. On the contrary, the energy loss in the form of methane emitted per kilogram of fermented dry matter decreased by 53% with the inclusion of Leucaena leucocephala. This suggests that adopting and implementing ISS is a viable alternative to reduce the carbon footprint of beef products under tropical conditions.
Acknowledgements The present study is part of the "Nitrogen usage by Colombian Criollo Cattle in Intensive Silvopastoral Systems with Leucaena leucocephala in dry tropical forest conditions" project, funded by COLCIENCIAS through the Francisco José de Caldas National Fund for Science, Technology and Innovation, and executed by CIPAV and the National University of Colombia at Medellín in collaboration with CORPOICATibaitatá. The authors thank the Molina Durán family and the "Natural Reserve El Hatico" for the cooperation received during the project.
References
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Fig 1.
Temperature and humidity index, %
82
Danger
80 78
Alert 76
Normal
74
Polytunnel1 Polytunnel2
72
Ambient 70 68 66
Time, h
Fig 2.
21
Methane emissions, g kg-1 fermented dry matter
70 60 50 40 Star grass + Leucaena
30
Star grass
20 10 0 0630 0930 1230 1530 1830 2130 0030 0330 0630
Time, h
Table 1 Average composition of the forages evaluated in this study Diet1 Nutrient
S
S+L
10,8
13,9
NDF , %
74,6
64,9
ADF3, %
43,0
41,9
Ether extract, %
1,16
1,17
Gross energy, Mj·kg-1
17,9
18,3
Ash, %
10,2
9,6
Calcium, %
0,37
0,42
Phosphorus, %
0,33
0,33
Crude protein, % 2
1
Diet: S= Star grass 100%; S+L= Star grass 74% + Leucaena 26%.
2
NFD = Neutral Detergent Fibre.
22
3
ADF = Acid Detergent Fibre.
23
Table 2 Average fresh forage and dry matter intake of Lucerna heifers receiving a star grass (C. plectostachyus) diet with (S+L) or without (S) leucaena (L. leucocephala) S2
S+L1 Star
P-value
Star
grass
Leucaena
Total
grass
Diet
Run
Diet*Run
18.00
5.68
23.7a
18.9b
0.03
0.50
0.47
13
Dry matter, kg day
4.27
1.34
5.61a
4.71b
0.01
0.11
0.60
13
Dry matter, % body weight
1.88
0.59
2.47a
2.02b
0.01
0.36
0.49
0
73.0
22.9
95.9a
78.9b
0.01
0.64
0.47
12
Item -1
Fresh forage, kg day -1
1
Dry matter, g kg- of weight 0.75 1
S+L= Star grass 74% + Leucaena 26%.
2
S= Star grass 100%;
a,b
Means in the same column with different letters are statistically different according to Tukey's test.
(P < 0.05). SD = Standard Deviation
24
S
Table 3 Average daily intake of nutrients and energy of Lucerna heifers receiving a star grass (C. plectostachyus) diet with (S+L) or without (S) leucaena (L. leucocephala) Diet1 Item
S+L
S
Diet
Run
Diet*Run
SD
Protein, g
755a
504b
˂0.01
0.09
0.04
16.81
NDF2, Kg
3.72
3.51
0.40
0.38
0.71
13.53
ADF3, Kg
2.39
2.02
0.06
0.43
0.62
13.57
Fat, g
65.3
55.0
0.29
0.23
0.49
15.82
Ash, g
549.4a
477.8b
0.02
0.53
0.26
13.55
Calcium, g
23.0a
17.2b
<0.01
0.13
0.37
15.06
Phosphorus, g
18.7
15.6
0.09
0.21
0.36
14.33
102.9a
83.6b
0.02
0.33
0.47
13.50
Gross Energy, Mj 1
P-value
Dieta: S = Star 100; S+L = Star 76 + Leucaena 24%.
2
NFD = Neutral Detergent Fibre.
3
ADF = Acid Detergent Fibre.
a,b
Means in the same column and item with different letters are statistically different according to
Tukey's test (P < 0.05). SD = Standard Deviation
25
Table 4 Daily production of methane in Lucerna breed heifers eating 100 % star grass or a mixture of 74 % star grass and 26 % leucaena. S+ L1
S2
Diet
Run
Diet*Run
Methane, g animal d
149.4
144.9
0.78
0.06
0.27
Methane, % of DMI3
2.46
2.95
0.23
0.18
0.26
Methane, % of organic matter intake
2.72
3.27
0.36
0.23
0.48
Methane, % of fermented DM4
4.10b
6.29a
0.04
0.19
0.25
Energy lost as methane, Mj/ day/ animal
8.24
7.99
0.78
0.06
0.27
Energy lost as methane, % of gross energy intake
7.96
9.42
0.32
0.22
0.49
Item -1
-1
1
S+L= Star grass 74% + Leucaena 26%.
2
S= Star grass 100%;
3
DMI = Dry matter Intake
4
DM = Dry matter
a,b
Means in the same column and item with different letters are statistically different according to
Tukey's test (P < 0.05). SD = Standard Deviation
26
Figure 1 Polytunnel and ambient (outside) temperature and humidity index during the four experimental periods
Figure 2 Methane produced during 24 hours by animals fed 100% star grass or a mixture of leucaena and star grass.
Highlights We measured methane emissions by heifers fed grass-only or grass-Leucaena diets. Leucaena-fed heifers ate more DM (1.19x) and protein (1.50x) than grass-only heifers. Methane emissions (g CH4·animal-1·d-1) were similar between both animal groups. Methane emissions per kg of fermented DM were 1.5X greater in grass-only heifers. The inclusion of Leucaena can lead to reduced carbon footprint of animal products.
27