Evaluation of several tropical tree leaves for methane production potential, degradability and rumen fermentation in vitro

Author’s Accepted Manuscript Evaluation of several tropical tree leaves for methane production potential, degradability and rumen fermentation in vitro K. Pal, A.K. Patra, A. Sahoo, P.K. Kumawat www.elsevier.com/locate/livsci

PII: DOI: Reference:

S1871-1413(15)00349-2 http://dx.doi.org/10.1016/j.livsci.2015.07.011 LIVSCI2796

To appear in: Livestock Science Received date: 19 March 2015 Revised date: 14 July 2015 Accepted date: 20 July 2015 Cite this article as: K. Pal, A.K. Patra, A. Sahoo and P.K. Kumawat, Evaluation of several tropical tree leaves for methane production potential, degradability and rumen fermentation in vitro, Livestock Science, http://dx.doi.org/10.1016/j.livsci.2015.07.011 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.

Evaluation of several tropical tree leaves for methane production potential, degradability and rumen fermentation in vitro

K. Pal1,3, A.K. Patra1*, A. Sahoo2, P.K. Kumawat2

1

Department of Animal Nutrition, Faculty of Veterinary and Animal Sciences, West Bengal University of Animal and Fishery

Sciences, 37 K. B. Sarani, Belgachia, Kolkata, 700037, India 2

Central Sheep and Wool Research Institute, Division of Animal Nutrition, Avikanagar, Rajasthan

3

Present address: Krishi Vigyan Kendra, West Bengal University of Animal and Fishery Sciences, Ashokenagar, North 24 Parganas,

743223, India ____________________________________________________________________ *Correspondence A.K. Patra, Department of Animal Nutrition West Bengal University of Animal and Fishery Sciences 37, K. B. Sarani, Belgachia, Kolkata, 700037, India Tel.: +91-33-25569234; Fax: +91-33-25571986 E-mail: [email protected] 1

Running title: Methane production potential of tropical tree leaves

2

Abstract The objective of this study was to investigate 18 tree leaves for methane production potential, degradability and rumen fermentation characteristics in vitro. The higher concentrations (P<0.01) of total phenolics were noted in Prosopis cineraria (99.7 g kg-1), Acacia tortilis (89.4 g kg-1) and Psidium guajava (89.3 g kg-1), non-tannin phenolics in A. tortilis (36.6 g kg-1) followed by Syzygium cumini (26.0 g kg-1), total tannins in P. cineraria (82.1 g kg-1) and P. guajava (74.3 g kg-1), and condensed tannins in A. tortilis (47.2 g kg-1), P. guajava (46.6 g kg-1) and P. cineraria (43.2 g kg-1). Among the 18 leaves, methane production expressed as ml g-1 degradable organic matter (OM) was lower (P<0.01) for Acacia nilotica (12.6 ml), P. cineraria (12.9 ml), Ficus religiosa (13.9 ml), S. cumini (13.8 ml) and Azadirachta indica (13.7 ml) than other tree leaves. Total volatile fatty acid (VFA) concentration was greater (P<0.01) for Tamarindus indica, followed by Acacia nilotica and lowest for S. cumini. Degradability of dry matter (DM) was higher (P<0.01) for M. oleifera, Acacia senegal, Acacia excelsa, Morus alba, A. indica and F. religiosa (77 to 83%), and lowest for Bambusa sp. (45%) and Ficus benghalensis (52%). Microbial biomass production was lowest for Bambusa sp. leaves and higher (P<0.01) for S. cumini, A. tortilis, A. nilotica, P. guajava than other leaves. Overall, the leaves of S. cumini, A. indica, F. religiosa and A. nilotica not only produced less methane per unit of degradable OM, but also had generally greater OM degradability and favoured production of microbial biomass compared with other leaves. These leaves could be explored for decreasing methane production in small ruminant production systems of tropical developing countries. Keywords: Degradability, methane production, rumen fermentation, tree leaves

3

1. Introduction Livestock production systems contribute greenhouse gas emissions (GHG) considerably to the atmosphere, and are thus accountable for one of the causes for climate changes and global warming (Gerber et al., 2013). Globally about 96 million tonnes of methane from enteric fermentation and 18 million tonnes of methane from livestock manure are released into the atmosphere (Patra, 2014). Methane emission from enteric fermentation and manure management is one of the major shares of total livestock GHG emission estimated by life cycle assessment analysis, and corresponds to 44% of total anthropogenic methane emissions (Gerber et al., 2013). Besides, methane emission from enteric fermentation represents a significant loss of feed energy. Several studies have been conducted to screen various feed additives, plant extracts, plant secondary compounds (Patra et al., 2006; Durmic et al., 2014) and tannin-containing legumes and tree leaves (Jayanagara et al., 2011; Bhatta et al., 2012) for inhibition of methane production. Various phytochemicals have been shown to modulate rumen fermentation favourably, and to inhibit methane production in the rumen (Patra and Saxena, 2010, 2011; Seradj et al., 2014). Tree leaves, which contain tannins and saponins in varying amounts, may be incorporated in diets to mitigate enteric methane emissions (Patra and Saxena, 2010). Leaves from trees and browses are important feed resources for small ruminant production in tropical countries especially for landless and marginal farmers (Devendra, 1990). In these regions, feeds from conventional resources are limited and often too expensive for the low input-output livestock production system. The multipurpose tree leaves contain moderate levels of crude protein (CP), minerals and vitamins (Topps, 1992; Patra, 2009) that are deficient in many low-quality roughages. Thus, the multipurpose tree leaves and shrubs have been

4

proclaimed as a solution to feeding of ruminants in the tropical areas, especially as supplementary feeds to low-quality forages containing low levels of CP and fermentable energy (Topps, 1992; Patra, 2009; 2010). In vitro gas production technique, which is less expensive, convenient and fast allowing large number of samples to be handled in a short time, has been widely used for evaluation of feeds for ruminants (Menke and Seingass, 1988; Blummel et al., 1997). Several rumen fermentation variables including methane production, feed degradability and volatile fatty acid (VFA) production are determined employing in vitro gas production technique. The screening of tree leaves employing in vitro gas production technique, which have low methane production potential and no adverse effect on nutrient digestibility, would be advantageous in formulating low methane producing diets for ruminants, particularly for small ruminants in tropical regions. Therefore, this study was undertaken to investigate several tree leaves for in vitro methane production, degradability and rumen fermentation.

2. Materials and methods 2.1. Collection of leave samples Fresh leave samples from eighteen types of trees [Albizia lebbeck (siras), Morus alba (mulberry), Acacia tortilis (Isreali babool), Ailanthus excelsa (ardu), Prosopis cineraria (khejri), Acacia nilotica (babool or gum arabic), Acacia senegal (gum acacia), Azadirachta indica (neem), Leucaena leucocephala (leucaena), Ficus religiosa (peepal), Artocarpus heterophyllus (jackfruit), Bambusa sp. (bamboo), Moringa oleifera (moringa), Ficus benghalensis (banyan), Mangifera indica (mango), Tamarindus indica (tamarind), Syzygium cumini (jamun) and Psidium guajava (guava)] were harvested from several trees and pooled for each species of 5

leaves during winter months (December and January). These tree leaves grew in arid and semi-arid climate zone of Rajasthan and tropical wet climate zone of West Bengal, India and they are commonly available, and some of them are usually fed to small ruminants in India. The descriptions of the climatic conditions and seasons of leave sample collection together with height of the trees are presented in Table 1. The leaves were dried in a hot air oven maintained at 60°C, ground to pass 1 mm screen, and stored in airtight bags for experimental use.

2.2. Experimental procedures The in vitro gas production was carried out using 100 ml calibrated glass syringes (häberle LABORTECHNIK, Lonsee-Ettlenschieß, Germany) in duplicates for each leaves, a standard of wheat straw and blank, and the study was completed in three batch incubations (n=3). The anaerobic culture medium (rumen fluid as inocula: buffer in the ratio of 1:2) was prepared as described by Menke and Steingass (1988). Rumen fluid was collected from three male Malpura sheep (2.5 to 3 years of age and an average body weight of 54±2.52 kg) though stomach tube before morning feeding into a pre-warmed carbon dioxide filled thermos, and immediately carried to the laboratory. Rumen fluid was pooled in equal proportion, and filtered through four layers of muslin cloth under continuous flushing of carbon dioxide gas to maintain the anaerobic condition. Sheep were fed a roughage-based maintenance diet containing cenchrus straw [containing 70.6 g crude protein (CP), 714 g neutral detergent fiber (NDF) and 454 g acid detergent fiber (ADF) per kg dry matter (DM)] and a concentrate mixture (consisting of maize, barley, mustard oil cake, ground nut cake, mineral mixture and salt, and containing 127 g CP and 252 g NDF per kg) in a ratio of 60:40. 6

Accurately weighed ground substrates (200 mg for gas production kinetics and 400 mg for determination of degradability of leaves) were transferred into the syringes, and anaerobic culture medium (30 ml for gas production and 40 ml for determination of degradability) was dispensed to each syringe. Syringes were incubated in an incubator maintained at 39°C and shaken manually every two h for initial 12 h, and then at 6 h intervals. The gas production was recorded at 2, 4, 8, 12, 24, 36, 48, 72 and 96 h of incubation taking the reading from the graduated syringes. The fermentation was terminated at 96 h of incubation for determination of gas production kinetics. Net gas production was calculated by subtracting the volume of gas produced in blank from the volume of gas produced from incubated feeds. Incubations were terminated at 24 h for determination of degradability, methane production and fermentation variables. The volumes of gas produced were recorded, and gas samples were collected and immediately injected onto a gas chromatograph to determine methane concentrations. The liquid contents of the syringes were strained through four layers of muslin cloth, and samples were preserved at -20°C for determination of ammonia nitrogen and volatile fatty acid (VFA) concentrations. True OM degradability (TOMD) of leaves was measured following the procedure of Blummel et al. (1997). Briefly, the contents of the syringes were transferred into the Berzelius beaker by repeated washings with 50 ml of neutral detergent solution (double strength), refluxed for 1 h, filtered through silica crucibles (Grade - 1), and then residues were burnt in a muffle furnace at 600°C for 3 h. The partitioning factor (PF) was calculated as the ratio of truly degraded organic matter (OM) (mg) to the volume of gas (ml) produced per mg of OM. The microbial biomass production (MBP) was calculated by using the truly degraded OM, gas volume and stoichiometrical factor (Blummel et al., 1997): MBP (mg/400 mg) = truly degraded OM (mg/400 mg) – (gas volume (ml/400 mg) × stoichiometrical factor), 7

where the stoichiometrical factor was 2.2. The samples from each syringe including that of blank were collected for determination of ammonia-N (NH3-N), trichloroacetate precipitable-N (TCA-N) and total volatile fatty acid (VFA) concentrations.

2.3. Analytical procedures The concentrations of DM, OM, CP (N × 6.25) and ether extract (EE) of leave samples were determined following the AOAC (1995) procedures. The contents of NDF, ADF and ADL in leaves were analyzed according to the methods described by Van Soest et al. (1991). Both NDF and ADF contents were measured exclusive of ash, and NDF was determined without α-amylase and sodium sulfite. The content of ADL was determined by solubilisation of cellulose with sulphuric acid in the ADF residue (Van Soest et al., 1991). The concentrations of cellulose and hemicelluloses in leaves were calculated as the difference between NDF and ADF, and between ADF and ADL, respectively. The concentrations of total phenolics (TP) and total tannins (TT) using Folin-Ciocalteu method of Makkar et al. (1993), condensed tannins (CT) as pro-anthocyanidins equivalent following the method of Porter et al. (1986) and non-tannin phenolics (as the difference between total phenolics and total tannins; NTP) of leaves were determined. For determination of methane concentration, the gas produced during fermentation of feeds in the syringes was collected by puncturing the silicon tube fitted with the syringes, and 50 ml of gas sample was injected in the gas chromatograph (GC-1000, Dani, Milan, Italy) equipped with a flame ionization detector and packed column (Chromatopak; length- 2 m, dia.- 1/8 inch., liquid- 10% SP-1000, solid- Ch-W/HP1). The concentration of methane in the standard was 999.98 ml L-1 (Sigma-Aldrich; Missouri, United

8

States). Temperatures of injector oven, column oven and detector were 120, 50 and 120°C, respectively. Nitrogen was used as a carrier gas. Concentration of ammonia nitrogen in fermentation solution was determined according to the modified Wetherburn method (Chaney and Marbach, 1962). The concentration of TCA-N was determined by Kjeldahl procedure (AOAC, 1995) after treating the rumen fluid with 200 g L-1 TCA. Total VFA concentrations in the fermented incubation media were determined using Markham apparatus following the procedure of Barnett and Reid (1956). The VFA in an acidic medium is vapourized by distillation, which is titrated against a standard alkali to obtain VFA concentrations (Barnett and Reid, 1956).

2.4. Statistical analyses All data were analyzed using one-way ANOVA through General Linear Model approach of SPSS (1996) in a completely randomized design. When F-test was significant (P<0.05), Tukey test was utilized to compare significant differences (P<0.05) among the leaves. Pearson correlation coefficients between chemical composition and fermentation variables were determined using SPSS (1996). The net gas production data were fitted to the following model to determine fermentation kinetics: Y = B × (1-e –c * t) where ‘y’ is the cumulative volume (ml) of gas produced at time ‘ t’ (h), ‘ B’ the asymptotic gas volume (ml) and ‘ c’ the rate constant of gas production (h-1). The parameters B and c were determined using the nonlinear procedure of SAS (2001). Substrate specific

9

times were defined by the half time (t1/2) of asymptotic gas production. Halftime (h) of gas production (t1/2) [i.e., the time (h) when half of the asymptotic gas volume (b; ml) was produced] was calculated as: t1/2 = ln2/c.

3. Results 3.1. Chemical composition The concentrations of CP ranged from 96 g kg-1 in P. guajava to 296 g kg-1 in M. oleifera leaves (Table 2). Leaves from A. lebbeck, M. alba and L. leucocaephala contained CP of approximately 180 g kg-1. The CP concentrations in other leaves generally ranged from 100 to 160 g kg-1. Highest amount of EE was found in M. oleifera and lowest in S. cumini and Bambusa sp. The EE percentages in other leaves were typically in the range of 40 to 60 g kg-1. Most of the tree leaves contained total ash of 90 to 140 g kg-1. The concentration of NDF was highest in Bambusa sp. and lowest in M. alba; while NDF contents in other leaves were in the range of 300 to 430 g kg-1. The concentrations of total phenolics, non-tannin phenolics, total tannins and condensed tannins were lower for Bambusa sp. and A. senegal than other leaves. The higher concentrations of total phenolics were noted in P. cineraria, A. tortilis and P. guajava, non-tannin phenolics in A. tortilis followed by S. cumini, total tannins in P. cineraria and P. guajava, and condensed tannins in A. tortilis, P. guajava and P. cineraria.

3.2. Gas production kinetics

10

Gas production was higher for T. indica and A. excelsa leaves at 24 h and lower for S. cumini and P. cineraria leaves than other leaves, and after 96 h of incubation, gas production was higher for T. indica, A. excelsa and F. religiosa and lower for S. cumini and P. cineraria (data not shown). Thus, potential gas production was estimated to be greater for T. indica, F. religiosa, and A. excelsa leaves, and lower for S. cumini, P. cineraria and P. guajava leaves (Table 3). Rate constant of gas production was higher for A. excelsa, followed by M. indica, A. senegal, T. indica and M. oleifera, and was lower for Bambusa sp., followed by P. cineraria, S. cumini and A. tortilis leaves. Accordingly, t1/2 of gas production was highest for Bambusa sp., followed by P. cineraria, and lowest for A. excelsa leaves (Table 2).

3.3. Methane production Methane production varied greatly (7.93 to 16.4 ml g-1 DM) among the leaves. Lowest methane production was noted for Bambusa sp., P. cineraria and F. benghalensis leaves, and highest methane for T. indica, M. oleifera and A. excelsa leaves when methane production was expressed as ml g-1 DM. However, methane production in terms of ml g-1 degradable OM was lowest for A. nilotica, F. religiosa, A. indica, S. cumini and P. cineraria leaves; however it was highest for T. indica, A. heterophyllus and M. oleifera leaves (Table 4).

3.4. Degradability, microbial biomass production and partitioning factor

11

True OM degradability was highest for A. excelsa, A. senegal and M. oleifera leaves with degradability of 81 to 83% (Table 5). True OM degradability was lowest for Bambusa sp., F. benghalensis and L. leucocephala leaves. Microbial biomass production ranged from 95 mg per 400 mg for Bambusa sp. leaves to 285 mg per 400 mg for A. tortilis. The partitioning factor was greatest for A. tortilis, and was lowest for L. leucocephala.

3.5. Concentrations of ammonia and VFA Concentration of VFA in the incubation media was highest for T. indica leaves, and was lowest for S. cumini leaves. Concentration of ammonia increased due to inclusion of M. oleifera leaves compared with other leaves. The ammonia concentration was lowest for F. religiosa leaves. The concentration of TCA-N was greatest for A. lebbeck leaves and lowest for M. alba, A. tortilis and T. indica.

3.6. Correlations between chemical composition and fermentation parameters Methane production (ml g-1 DM) was only negatively correlated with ADL content of leaves, but methane production expressed as ml g-1 degradable OM was not associated with any of the chemical composition (Table 6). Concentration of ammonia in fermentation media was positively correlated with CP and negatively with ADL content. Total gas production was negatively correlated with phenolic fractions and ADL and potential gas production was negatively correlated with phenolic fractions. However, MBP was positively correlated with phenolic fractions, but was negatively correlated with NDF. The true OM degradability and rate constant of gas production were negatively correlated with NDF, ADF and ADL in this study. 12

4. Discussion Chemical composition ranged widely among the leaves, which was within the reported ranges. This suggests that the quality of tree leaves remarkably varies among leaves. In the study of Jayanegara et al. (2011), CP and NDF contents (g kg-1) ranged from 79 to 386 and 155 to 581, respectively. The concentrations of total phenolics, non-tannin phenolics, total tannins and condensed tannins were relatively within a narrow range as compared with the results of Jayanegara et al. (2011). Degradability of leaves and concentrations of VFA produced in the incubations varied considerably, which is generally resulted from inherent characteristics of chemical composition of leaves. Digestion of forages is greatly dependent on the relative proportion of cell wall types present in the plant tissues and the existence of factors restricting microbial access to cell walls. True OM degradability was negatively (P < 0.01) related to the NDF (r = 0.66), ADF (r = 0.67) and ADL (r = 0.73) content in this study. However, OM degradability was not related cellulose and hemicellulose concentrations. Therefore, lignin content in leaves was most important factor determining the degradability. Tannins have been implicated for their inhibitory effect on feed digestion, microbial population and enzyme activity in many experiments (Patra and Saxena, 2011). In this study, phenolics and tannins, however, were not correlated to true OM degradability and total VFA concentrations. In another study, OM degradability was negatively associated with concentrations of total tannins, condensed tannins and hydrolysable tannins (Jayanegara et al., 2011). The concentration of phenolic compounds in fermentation media was perhaps low to affect rumen cellulolytic bacteria. Total gas production and potential gas production were negatively, whereas MBP was positively associated with concentrations of total 13

phenolics and tannins in this study. The gas produced in vitro is inversely correlated to MBP as opposed to VFA production (Blümmel et al., 1997). It is likely that relatively low concentration of phenolic compounds in leaves may favour microbial protein synthesis. Many phenolic compounds have antioxidant (oxygen radical scavenger) properties, which might be involved in stimulation of microbial growth (Campos et al., 2003; Alberto et al., 2012). For example, bacterial populations were found to positively correlate with concentrations of total tannins and condensed tannins in leaves (Jayanagara et al., 2011). Several tropical tree leaves or their extracts have been evaluated for lowering methane production in the rumen. Bhatta et al. (2012) reported that tree leaves containing tannins such as Autocarpus integrifolia, Jatropha curcus and Sesbania grandiflora have the potential to suppress methanogenesis. Similar to this study, Chatterjee et al. (2014) noted that P. guajava leaves had low methane production potential in vitro. Patra et al. (2008) observed that methanol extract of P. deltoides leaves decreased methane production without impacting rumen fermentation in vitro. Differences in methane production may be attributed to the presence of various phytochemicals, mainly tannins. Pearson correlation coefficients between phenolic compounds and methane production of all leaves were not significant in this study, but were significant in another study (Jayanagara et al., 2011). However, the leaves of S. cumin, P. cineraria, P. guajava, F. religiosa and A. nilotica caused a greater inhibition of methane production and these leaves contained high concentrations of total tannins, total phenolics and condensed tannins. Different sources of tannins extracts have been shown to decrease methane production both in vitro and in vivo condition depending upon doses. Addition of acacia, quebracho, chestnut and valonea tannins suppressed methane production in vitro up to 40% (Hassanat and Benchaar, 2013), in sheep by 13% (Carulla et al., 2005) and in cattle up to 30% (Grainger et al., 2009). Animut et al. (2008) found that feeding of condensed tannin-containing forages 14

(Lespedeza cuneata) linearly decreased methane production in goats. Bhatta et al. (2009) reported that quebracho tannins decreased methane production linearly (13 to 45%) with increasing doses of tannins (5 to 25% of substrates). However, Beauchemin et al. (2007) did not find any effect on methanogenesis when a quebracho tannin extract (10 to 20 g kg-1 DM intake) was fed to beef cattle, which may be due to the inclusion of lower dosages of tannins compared with the doses used by Bhatta et al. (2009). It has been suggested that the action of tannins on methanogenesis may be attributed to the direct inhibitory effects on methanogens depending upon the chemical structure of tannins and also indirectly by decreasing fiber degradation (Patra and Saxena, 2010). All tannin-containing leaves were not equally effective in decreasing methane production (Bhatta et al., 2012). For example, M. indica leaves contained an appreciable amount of tannins, but methane production potential was high compared with other leaves tested in this study. The decrease in methane production by A. indica leaves could be attributed to the presence of limonoid compounds because these leaves contained low concentrations of phenolic compounds. A. indica leaves contain limonoids such as nimbin and azadirachtin (Govindachari, 1992), which may be responsible for methane inhibition. The extracts of seeds of A. indica, which contain limonoid compounds, also lowered methane production in vitro (Patra et al., 2006). Many additives feed additives have been explored for decreasing methane production in the rumen, but most of them exert adverse effect on feed degradability and rumen fermentation (Patra, 2012; Durmic et al., 2014). Inclusion of methane suppressing agents in diets, thus, should be aimed at direct inhibition of methane production without negative effects on feed degradability. In this study, the leaves of S. cumini, F. religiosa, A. indica and A. nilotica not only produced less methane per unit of degradable OM, but also generally had greater OM degradability (Figure 1) and produced higher microbial biomass compared with other leaves. Kumar et 15

al. (2011) also reported that the extracts of M. indica lowered methanogenesis by 9 to 36% without affecting degradability of feeds in vitro while other extracts of leaves though lowered methane but decreased feed degradability. In this study, leaves of M. indica decreased methane production to a less extent than the leaves of S. cumini, P. cineraria, F. religiosa and A. nilotica. The decrease in methane production (ml g-1 DM) for Bambusa leaves could be resulted from low degradability of OM due to presence of higher concentrations of less degradable fiber components. The low methane producing leaves identified in this study may be useful to decrease methane production, as tree leaves are usually a component of ration of small ruminants in the tropical developing countries (Devendra, 1990; Topps, 1992; Patra et al., 2003).

5. Conclusions The leaves of S. cumini, F. religiosa, A. nilotica and A. indica had a low methane production potential expressed in terms of degradable OM, and also these leaves favoured the microbial biomass production and had higher OM degradability. The information of low methane production potential of these tree leaves could be practical for methane mitigation in small ruminant production systems for tropical developing countries, where tree leaves could be used as supplementary feeds for low-quality forages.

Acknowledgement Authors are grateful to the Director, Central Sheep and Wool Research Institute, Avikanagar for providing the facilities to conduct this research. This project was supported by young scientist scheme from the Indian Council of Agricultural Research, New Delhi. 16

References Animut, G., Puchala, R., Goetsch, A.L., Patra, A.K., Sahlu, T., Varel, V.H., Wells, J., 2008. Methane emission by goats consuming diets with different levels of condensed tannins from lespedeza. Anim. Feed Sci. Technol. 144, 212–227. Alberto, M.R., Manca de Nadra, M.C., Arena, M.E., 2012. Influence of phenolic compounds on the growth and arginine deiminase system in a wine lactic acid bacterium. Braz. J. Microbiol. 43, 167-176. AOAC. 1995. Association of Official Analytical Chemists, Official Methods of Analysis (16th Edn.), Washington, DC. Barnett, J.G.A., Reid, R.L., 1956. Studies on the production of volatile fatty acids from the grass by rumen liquor in an artificial rumen. J. Agric. Sci. 48, 315-321. Beauchemin, K.A., McGinn, S.M., Martinez, T.F., McAllister, T.A., 2007. Use of condensed tannin extract from quebracho trees to reduce methane emissions from cattle. J. Anim. Sci. 85, 1990–1996. Bhatta, R., Saravanan, M., Baruah, L., Sampath, K.T., 2012. Nutrient content, in vitro ruminal fermentation characteristics and methane reduction potential of tropical tannin-containing leaves. J. Sci. Food Agric. 92, 2929–2935. Bhatta, R., Uyeno, Y., Tajima, K., Takenaka, A., Yabumoto, Y., Nonaka, I., Enishi, O., Kurihara, M., 2009. Difference in the nature of tannins on in vitro ruminal methane and volatile fatty acid production and on methanogenic archaea and protozoal populations. J. Dairy Sci. 92, 5512–5522.

17

Blummel, M., Makkar, H.P.S., Becker, K., 1997. The relationship between in vitro gas production, in vitro microbial mass yield and 15

N incorporation and its implications for the prediction of voluntary feed intake of roughages. Br. J. Nutr. 77, 911-921.

Campos, F.M., Couto, J.A., Hogg, T.A., 2003. Influence of phenolic acids on growth and inactivation of Oenococcus oeni and Lactobacillus hilgardii. J Appl Microbiol. 94, 167-174. Carulla, J.E., Kreuzer, M., Machmüller, A., Hess, H.D., 2005. Supplementation of Acacia mearnsii tannins decreases methanogenesis and urinary nitrogen in forage-fed sheep. Aust. J. Agric. Res. 56, 961–970. Chaney, A.L., Marbach, E.P., 1962. Modified reagents for determination of urea and ammonia. Clin. Chem. 8, 130–136. Chatterjee, P.N., Kamra, D.N., Agarwal, N., Patra, A.K., 2014. Influence of supplementation of tropical plant feed additives on in vitro rumen fermentation and methanogenesis. Anim. Prod. Sci. 54, 1770-174. Devendra, C., 1990. The use of shrubs and tree fodders by ruminants, In: Devendra, C. (ed.), Shrubs and Tree Fodders for Farm Animals, International Development Research Centre, IDRC-276e, Ottawa, Canada, pp. 42–60. Durmic, Z., Moate, P.J., Eckard, R., Revell, D.K., Williams, R., Vercoe, P.E., 2014. In vitro screening of selected feed additives, plant essential oils and plant extracts for rumen methane mitigation. J. Sci. Food Agric. 94, 1191–1196. Gerber, P.J., Steinfeld, H., Henderson, B., Mottet, A., Opio, C., Dijkman, J., Falcucci, A., Tempio, G., 2013. Tackling climate change through livestock – A global assessment of emissions and mitigation opportunities. Food and Agriculture Organization of the United Nations (FAO), Rome.

18

Getachew, G., Robinson, P.H., DePeters, E.J., Taylor, S.J., 2004. Relationships between chemical composition, dry matter degradation and in vitro gas production of several ruminant feeds. Anim. Feed Sci. Technol. 111, 57–71. Gobindachari, T.R., 1992. The chemical and biological investigations of Azadirachta indica (the neem tree). Curr. Sci. 63, 117-122. Grainger, C., Clarke, T., Auldist, M.J., Beauchemin, K.A., McGinn, S.M., Waghorn, G.C., Eckard, R.J., 2009. Potential use of Acacia mearnsii condensed tannins to reduce methane emissions and nitrogen excretion from grazing dairy cows. Can. J. Anim. Sci. 89, 241-251. Hassanat, F., Benchaar, C., 2013. Assessment of the effect of condensed (acacia and quebracho) and hydrolysable (chestnut and valonea) tannins on rumen fermentation and methane production in vitro. J. Sci. Food Agric. 93, 332-339. Jayanegara, A., Wina, E., Soliva, C.R., Marquardt, S., Kreuzer, M., Leiber, F., 2011. Dependence of forage quality and methanogenic potential of tropical plants on their phenolic fractions as determined by principal component analysis. Anim. Feed Sci. Technol. 163, 231–243. Kumar, R., Kamra, D.N., Agarwal, N., Chaudhary, L.C., Zadbuke, S.S., 2011. Effect of tree leaves containing plant secondary metabolites on in vitro methanogenesis and fermentation of feed with buffalo rumen liquor. Anim. Nutr. Feed Technol. 11, 103114. Larbi, A., Smith, J.W., Kurdi, I.O., Adekunle, I.O., Raji, A.M., Ladipo, D.O., 1998. Chemical composition, rumen degradation, and gas production characteristics of some multipurpose fodder trees and shrubs during wet and dry seasons in the humid tropics. Anim. Feed Sci. Technol. 72, 81–96. 19

Makkar, H.P.S., Blummel, M., Borowy, N.K., Becker, K., 1993. Gravimetric determination of tannins and their correlations with chemical and protein precipitation methods. J. Sci. Food Agric. 61, 161–165. McCune, B., Mefford, M.J., 2011. PC-ORD. Multivariate Analysis of Ecological Data. Version 6. MjM Software, Gleneden Beach, Oregon, U.S.A. Menke, K.H., Steingass, H., 1988. Estimation of the energetic feed value obtained from chemical analysis and in vitro gas production using rumen fluid. Anim. Res. Dev. 27, 7-55. Patra, A.K., Saxena, J., 2010. A new perspective on the use of plant secondary metabolites to inhibit methanogenesis in the rumen. Phytochemistry 71, 1198-1222. Patra, A.K., Saxena, J., 2011. Exploitation of dietary tannins to improve rumen metabolism and ruminant nutrition. J. Sci. Food Agric. 91, 24-37. Patra, A.K., 2010. Effects of supplementing low quality roughages with tree foliages on digestibility, nitrogen utilization and rumen characteristics in sheep: a meta-analysis. J. Anim. Physiol. Anim. Nutr. 94, 338-353. Patra, A.K., Kamra, D.N., Agarwal, N., 2006. Effect of plant extracts on in vitro methanogenesis, enzyme activities and fermentation of feed in rumen liquor of buffalo. Anim. Feed Sci. Technol. 128, 276-291. Patra, A.K., Kamra, D.N., Agarwal, N., 2008. Effect of leaf extracts on in vitro fermentation of feed and methanogenesis with rumen liquor of buffalo. Indian J. Anim. Sci. 78, 91-96.

20

Patra, A.K., 2009. Responses of intake, digestibility and nitrogen utilisation in goats fed low-quality roughages supplemented with tree foliages. J. Sci. Food Agric. 89, 1462–1472. Patra, A.K., Sharma, K., Dutta, N., Pattanaik, A.K., 2003. Response of gravid does to partial replacement of dietary protein by a leaf meal mixture of Leucaena leucocephala, Morus alba and Azadirachta indica. Anim. Feed Sci. Technol. 109, 171-182. Patra, A.K., 2014. Trends and projected estimates of GHG emissions from Indian livestock in comparisons with GHG emissions from world and developing countries. Asian-Australas. J. Anim. Sci. 27, 592-599. Porter, L.J., Hrstich, L.N., Chan, B.G., 1986. The conversion of procyanidins and prodelphinidins to cyanidin and delphinidin. Phytochemistry 25, 223-230. SAS. 2001. SAS Institute Inc., SAS/STAT Software, Version 8.2. SAS Institute Inc., Cary, NC, USA. Seradj, A.R., Abecia, L., Crespo, J., Villalba, D., Fondevila, M., Balcells, J., 2014. The effect of Bioflavex® and its pure flavonoid components on in vitro fermentation parameters and methane production in rumen fluid from steers given high concentrate diets. Anim. Feed Sci. Technol. 197, 85-91. SPSS. 1997. Statistical Package for Social Sciences, Base Applications Guide 7.5. SPSS, Chicago, USA. Topps, J.H., 1992. Potential, composition and use of legume shrubs and trees as fodders for livestock in the tropics. J. Agric. Sci. (Camb.) 118, 1-8. 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. 21

22

Figure legend Figure 1. A plot showing relationship between organic matter (OM) degradability and methane production per g of degradable OM for different tree leaves. The horizontal dotted line separates leaves with respect to low (<14 ml g-1 degradable OM) and high (>14 ml g-1 degradable OM) methane production; whereas vertical dotted line separates leaves based on low (<70%) and high (>70%) OM degradability.

Highlights · Methane production (MP) potential of 18 tree leaves was investigated in vitro. · S. cumin, A. indica, F. religiosa and A. nilotica lowered MP substantially. · These tree leaves generally had higher dry matter degradability. · These leaves also usually favoured microbial biomass production. · These leaves may be explored for MP inhibition in tropical ruminant production.

Conflict of interest Authors declare that they do not have any conflict of interest.

23

Table 1. Climatic conditions of the regions from where leave samples taken along with season and description of sample collection Number of Approximate Climatic Maturity of Parts of Scientific name Common Season trees height of the environment leaves trees taken sampled trees name Young and Leaves with Arid and semi Winter Acacia nilotica Gum arabic 4 10-11 m mature small arid region (December) branches leaves Young and Leaves with Arid and semi Winter Acacia senegal Kheri 4 8-9 m mature small arid region (December) leaves branches Young and Leaves with Israeli Arid and semi Winter Acacia tortilis 4 mature small 6-7 m babool arid region (December) leaves branches Young and Arid and semi Winter Ailanthus excelsa Ardu 5 mature Leaves 18-20 m arid region (December) leaves Young and Arid and semi Winter Albizia lebbeck Siras 4 mature Leaves 21-22 m arid region (December) leaves 24

Artocarpus heterophyllus

Jackfruit

Tropical wet climate

Winter (January)

4

Neem

Tropical wet climate

Winter (January)

5

Bamboo

Tropical wet climate

Winter (January)

8

Banyan

Tropical wet climate, and arid and semi-arid region

Winter (DecemberJanuary)

Peepal

Tropical wet climate

Winter (January)

4

Leucaena

Arid and semi arid region

Winter (December)

6

Tropical wet climate, and arid and semi-arid region

Winter (DecemberJanuary) Winter (January)

6

Azadirachta indica Bambusa sp. Ficus benghalensis Ficus religiosa Leucaena leucocephala

Mangifera indica

Mango

4

5

Moringa oleifera

Moringa

Tropical wet climate

Morus alba

Mulberry

Arid and semi arid region

Winter (December)

4

Khejri

Arid and semi arid region

Winter (December)

5

Prosopis cineraria

25

Young and mature leaves Young and mature leaves Young and mature leaves Young and mature leaves Young and matured leaves Young and mature leaves Young and mature leaves Young and mature leaves Young and mature leaves Young and mature leaves

Leaves

12-16 m

Leaves with small 17-18 m branches Leaves

22-25 m

Leaves

16-20 m

Leaves

22-25 m

Leaves with 10-12 m small branches

Leaves

15-16 m

Leaves with small 9-10 m branches Leaves

3-4 m

Leaves with small 5-6 m branches

Psidium guajava

Syzygium cumini

Guava

Tropical wet climate

Winter (January)

Jamun

Tropical wet climate, and arid and semi-arid region

Winter (DecemberJanuary)

Tamarind

Tropical wet climate

Winter (January)

Tamarindus indica

26

6

Young and mature leaves

Leaves

5-6 m

5

Young and mature leaves

Leaves

10-12 m

4

Young and mature leaves

Leaves with 20-21 m small branches

Table 2. Chemical composition (g kg-1) of different tree leaves Leaves CP EE TA NDF ADF ADL TP NTP Acacia nilotica 159 43.3 69.3 336 214 116 57.9 13.6 Acacia senegal 141 49.2 97.1 310 168 71.4 6.80 4.23 Acacia tortilis 153 52.1 55.2 322 215 101 89.4 36.6 Ailanthus excelsa 146 58.9 90.4 369 219 64.9 29.5 10.4 Albizia lebbeck 184 55.5 131 320 202 92.9 18.5 12.6 Artocarpus heterophyllus 135 43.1 139 426 254 144 40.1 12.9 Azadirachta indica 149 60.7 122 327 234 122 32.9 11.8 Bambusa sp. 132 27.9 112 523 334 118 3.95 2.55 Ficus benghalensis 120 49.0 126 415 301 160 30.5 6.93 Ficus religiosa 125 43.7 126 402 291 72.6 18.8 11.2 Leucaena leucocephala 174 51.5 114 303 202 140 35.2 9.68 Mangifera indica 106 56.0 127 364 211 118 48.2 14.2 Moringa oleifera 296 74.6 131 266 124 27.5 13.9 8.58 Morus alba 184 65.1 125 285 159 52.4 28.2 8.60 Prosopis cineraria 134 60.0 112 431 282 135 99.7 17.6 Psidium guajava 95.9 49.5 78.6 327 201 115 89.3 15.0 Syzygium cumini 108 24.3 66.9 394 248 118 50.9 26.0 Tamarindus indica 119 66.6 67.9 43.1 308 147 49.4 11.9 CP, crude protein; EE, ether extract, TA, total ash; NDF, neutral detergent fiber; ADF, acid detergent fiber; lignin; TP, total phenolics, NTP, non-tannin phenolics; TT, total tannins; CT, condensed tannin.

27

TT CT 44.3 4.60 2.57 0.69 52.8 47.2 19.2 1.93 5.87 1.25 27.2 24.1 21.0 3.00 1.41 0.86 23.6 17.0 7.56 2.08 25.5 5.26 34.0 12.0 5.31 1.20 19.6 14.5 82.1 43.2 74.3 46.6 24.9 21.9 37.6 26.1 ADL, acid detergent

<0.001

B (ml g-1 DM) 178bc 151d 156d 127e 159d 175bc 113fgh 119ef 105gh 185b 152d 169c 117efe 154d 96.2hi 97.1hi 87.6i 201a 3.32

<0.001

c (h-1) 0.09a 0.05de 0.06cd 0.04fg 0.05de 0.07b 0.04g 0.02h 0.04efg 0.07bc 0.05def 0.06d 0.08b 0.07b 0.03g 0.05def 0.04g 0.07b 0.003

<0.001

t1/2 (h) 7.73j 13.2ef 11.9efgh 17.6c 13.2ef 10.3ghij 18.9c 30.1a 16.3cd 10.8fghi 14.3d 12.2efg 8.97ij 9.43hij 22.8a 14.2de 18.1c 9.60ghij 0.840

Table 3. Gas production kinetics of different tree leaves A. excelsa A. heterophyllus A. indica A. lebbeck A. nilotica A. senegal A. tortilis Bambusa sp. F. benghalensis F. religiosa L. leucocephala M. alba M. indica M. oleifera P. cineraria P. guajava S. cumini T. indica SEM P value

B, potential gas production; c, rate constant of gas production; t1/2, the half time of asymptotic gas production; SEM, standard error of mean Values followed by different letters (a to j) within parameter differ significantly (P<0.05).

28

Table 4. Effects of tree leaves on methane production, concentrations of different N fractions (mg L-1) and total VFA (mM) in the fermentation media Methane, ml Methane, ml Leaves NH3-N TCA-N Total VFA g-1 DM g-1 TDOM A. excelsa 15.3a 18.7bcd 160c 288bc 57.8e A. heterophyllus 13.5b 20.5ab 135de 296bc 65.8cd A. indica 10.5ef 13.7gh 121e 221cd 53.0f A. lebbeck 13.6b 18.3bcde 194b 410a 55.3ef A. nilotica 9.37fg 12.6h 122e 258c 70.3b A. senegal 13.2bc 16.2ef 126e 272bc 62.2d A. tortilis 11.1de 14.9fg 121e 217d 49.3fg Bambusa sp. 7.93h 17.2de 139de 296bc 44.8gh F. benghalensis 8.64gh 17.3de 124e 244cd 45.8gh F. religiosa 10.5ef 13.9gh 91.9f 253cd 59.2de L. leucocephala 10.2ef 18.3cde 113e 264bc 50.8fg M. alba 12.1cd 14.8fg 150cd 213d 67.2c M. indica 12.0cd 17.3de 115e 297bc 50.0fg M. oleifera 16.4a 19.7abc 222a 319b 63.7cd P. cineraria 8.4gh 12.9gh 129de 226cd 47.5g P. guajava 10.3ef 14.9fg 123e 299b 53.2f S. cumini 9.53fg 13.8gh 140d 268bc 42.8h T. indica 15.6a 21.7a 146cd 216d 73.3a SEM 0.454 0.755 4.83 13.86 1.39 P value <0.001 <0.001 <0.001 <0.001 <0.001 Values followed by different letters (a to k) within parameter differ significantly (P<0.05). DM, dry matter; TDOM, truly degradable organic matter; VFA, total volatile fatty acids; TCAN, trichloacetate precipitable-N; NH3-N, ammonia-N; SEM, standard error of mean

29

Table 5. Effects of tree leaves on total gas production (TGP; ml g-1 dry matter at 24 h), microbial biomass production (MBP; mg g-1 dry matter), partitioning factor (PF) and true dry matter (TDMD, %) and organic matter degradability (TOMD; %) Leaves TGP MBP PF TDMD TOMD A. excelsa 153a 410g 4.87g 82.8a 81.9a A. heterophyllus 111f 327i 5.14g 54.0j 66.3f A. indica 112f 428f 6.01f 78.4bc 76.8b A. lebbeck 89.1h 447e 7.22de 70.0fg 73.9cd A. nilotica 106g 555b 7.44d 74.4d 74.6c A. senegal 148b 410fg 4.98g 81.4a 81.5a A. tortilis 65.3i 708a 20.1a 74.8d 74.5c Bambusa sp. 65.8i 238k 6.49ef 45.4l 46.4h F. benghalensis 65.1i 295j 6.74e 52.0k 50.1g F. religiosa 122e 392g 5.42fg 77.5c 75.7bc L. leucocephala 125d 320i 3.97h 53.3jk 55.8g M. alba 128d 457de 5.79fg 79.4b 81.6a M. indica 88.6h 412fg 6.85de 70.6ef 69.4e M. oleifera 130d 265d 5.78fg 82.2a 83.1a P. cineraria 52.6k 264d 11.3c 66.3i 65.3f P. guajava 58.4j 507c 10.9c 68.2h 68.9e S. cumini 59.4j 570b 19.2b 69.1gh 69.0e T. indica 135c 375h 4.97g 71.8e 72.0d SEM 0.979 5.34 0.242 0.509 0.711 P value <0.001 <0.001 <0.001 <0.001 <0.001 Values followed by different letters (a to k) within parameter differ significantly (P<0.05). SEM, standard error of mean

30

25

Table 6. The correlation coefficients (r) between the chemical composition (g kg-1) and rumen fermentation variables of tree leaves

26

incubated with rumen fluid

27

CP NDF ADF ADL Hcel Cel TP NTP TT CT 0.47 -0.37 -0.46 -0.49* 0.06 -0.19 -0.30 -0.13 -0.32 -0.22 Methane (ml g-1 DM) -1 0.24 0.11 0.01 0.05 0.25 -0.03 -0.38 -0.33 -0.35 -0.18 Methane (ml g TDOM) -1 0.74** -0.29 -0.43 -0.50* 0.20 -0.15 -0.31 -0.15 -0.32 -0.23 Ammonia-N (mg L ) 0.26 -0.11 -0.26 -0.23 0.30 -0.15 -0.35 -0.22 -0.35 -0.35 TCA-N (mg L-1) 0.26 -0.21 -0.20 -0.22 -0.10 -0.08 -0.15 -0.25 -0.09 -0.16 Total VFA (mM) 0.39 -0.37 -0.38 -0.48* -0.12 -0.10 -0.56* -0.47* -0.52* -0.59** Total gas (ml g-1 DM) 0.38 -0.65** -0.66** -0.73** -0.21 -0.25 -0.03 0.16 -0.09 -0.13 TOMD (%) 0.10 -0.49* -0.42 -0.27 -0.31 -0.33 0.60** 0.83** 0.45 0.47* MBP (mg g-1 DM) -1 0.24 -0.15 -0.11 -0.34 -0.17 0.15 -0.49* -0.46 -0.44 -0.51* B (ml g DM) -1 0.34 -0.51* -0.53* -0.55* -0.15 -0.24 -0.32 -0.26 -0.31 -0.43 c (h ) DM, dry matter; TDOM, truly degradable organic matter; CP, crude protein; NDF, neutral detergent fiber; ADF, acid detergent fiber;

28

ADL, acid detergent lignin; Hcel, hemicelluloses; Cel, cellulose; TP, total phenolics, NTP, non-tannin phenolics; TT, total tannins;

29

CT, condensed tannin; TOMD, true OM degradability; VFA, volatile fatty acids; MBP, microbial biomass production; B, potential gas

30

production, c, rate constant of gas production.

31

*P<0.05; **P<0.01

32

7

Figure

22 T. indica A. heterophyllus

Methane, ml per g degradable OM

20

M. oleifera

A. excelsa L. leucocephala

18

A. lebbeck

F. benghalensis Bambusa sp.

M. indica A. senegal

16 P. guajava

A. tortilis

M. alba

F. religiosa

14

S. cumini P. cineraria

A. indica A. nilotica

12

10 40

45

50

55 60 65 70 75 Organic matter (OM) degradability (%)

80

85

90