Livestock Research for Rural Development 26 (3) 2014 Guide for preparation of papers LRRD Newsletter

Citation of this paper

In vitro screening of some tropical goat feeds for low methane and high ammonia generating potential in the rumen

T Seresinhe, A N F Perera* and P K Lal

Dept. of Animal Science, Faculty of Agriculture, University of Ruhuna, Sri Lanka.
* Dept. of Animal Science, Faculty of Agriculture, University of Peradeniya, Sri Lanka.


The aim of this study was to determine the methane and ammonia generating potential of some common goat feeds used by small farmers in Sri Lanka. Eight treatments were examined in a randomized complete block design using four non legumes with low tannins and two shrub legumes with high tannins at a ratio of 3:1 (Terminalia catappa x Acacia auriculiformis (AA) [TC.AA], T. catappa x Calliandra calothyrsus (CC) [TC.CC], Symplocos splicata x A auriculiformis (AA) [SS.AA], S. splicata x CC [SS.CC], Mangifera indica x AA [MI.AA], M .indica x CC [MI.CC], Syzygium caryophyllatum x AA [SC.AA] and S. caryophyllatum x C C [SC.CC]. Plant samples were incubated with ruminal fluid /buffer mixture in four replicates for 48 h using Hohenheim gas test method.

Increasing the condensed tannin concentration in the fermentation substrate led to a decrease in the population of protozoa and a decrease in methane production. Protozoa population was positively related with methane production. The high positive correlation between crude protein content in the forage mixtures and ammona in the fermenmtation media indicated that tannins did not excessively inhibit feed protein degradation.

In conclusion, supplementing low-tannin non-leguminous forages with high tannin legume forage was found to be promising as an approach to the goal of improved nutrition of goats and simultaneous limited methane emissions.

Key words: buffer, protozoa, ruminal fluid, tannins


Goat farming is a livelihood activity which ensures food security for small and marginal farmers, landless labourers and rural folk in Sri Lanka. Goats are fed on diverse tree leaves which are virtually their sole food source in rural areas while in peri-urban areas goats are fed with diverse feeds in addition to limited supply of tree leaves. (Seresinhe and Marapana 2011).The poor growth performance of local goats is associated with low diet digestibility which may be due to the presence of condensed tannins (CT) in leaves fed to goats (Seresinhe et al 2003). Tannin concentrations higher than 5% in diets might negatively influence feed intake (Aerts et al 1999), due to reduced palatability. Several studies reported a negative influence of tannins on feed digestibility (Patra et al 2006; Grainger et al 2009; Jayanegara et al 2011). Protein degradation in the rumen is affected by tannins due to formation of tannin-protein-complexes (Mueller-Harvey 2006).

Researchers have reported that gas production is an indirect measure of substrate degradation (Liu et al 2002). The in vitro gas production is more efficient than the in sacco method in evaluating the effects of tannins (El-Waziry et al 2007). Effects of tannin from different sources in reducing ruminal gas production and ammonia levels have been reported. There are some indications that tannins in the diet might help to reduce ruminal methane production (Hess et al 2006).

Therefore, this study evaluated the suitability of several mixtures of low tanniniferous non-legume foliage mixed with highly tanniniferous legume foliage on in vitro gas production and rumen degradability characteristics.


Edible forage samples (leaves and tender stems) were hand harvested. Standard methods as described by AOAC (1990) were used for determination of dry matter, ash and crude protein. Fiber components (neutral detergent fiber, NDF; acid detergent fiber, ADF) were determined by methods of Van Soest (1967). Acid detergent residue was treated with 72% H2SO4 for lignin estimation.

Analyses of tannins

Tannins were analyzed by first weighing 200 mg of feed into a 50 ml conical flask. The feed sample was extracted with 70% aqueous acetone in an ultrasonic bath for 2 h and the contents were centrifuged for 20 min at 5,000g and the supernatant collected for tannin analyses. Total phenols were estimated by the Folin-Ciocalteu reaction (Makkar 2003a). For the condensed tannin (CT) fraction, the extract was treated with Butanol-HCl in the presence of ferric ammonium sulphate, and CT was expressed as leucocyanidin equivalent as weight of sample dry matter, following the procedure of Porter et al (1986).

Experimental design

Eight treatments were examined in a randomized complete block design using four non-legumes with high tannin content and two shrub legumes with low tannin contant at a ratio of 3:1. Treatment combinations of high-tannin non-legume and low-tannin legumes used for the experiment are given in Table 2.

In vitro gas production

In vitro gas production was determined as described by Menke and Steingass (1988). Rumen fluid was collected before feeding in the morning from two fistulated donor bulls at the experimental farm of the Faculty of Agriculture. Rumen fluid was strained through four layers of gauze into a pre-warmed, insulated bottle. All laboratory handling of rumen fluid was carried out under a continuous flow of CO2. Samples (200 mg) consisting of 150 mg high tannin non-legume + 50 mg low tannin legume) of the oven-dry feedstuffs were accurately weighed into 100-ml glass syringes fitted with plungers. Syringes were filled with 30 ml of medium consisting of 10 ml of rumen fluid and 20 ml of buffer solution. Two blank samples containing 30 ml of medium only were included. The syringes were placed in an incubator (39 oC) and the syringes rotated during first 4 h. Gas production was recorded after 4, 8, 12, 24 and 48 h of incubation. In all experiments, each incubation was repeated on three different days so that each treatment was conducted in triplicate.

In vitro dry matter digestibility

At the end of the fermentation period, the fermented residues were filtered into pre-weighed filter crucibles and dried for 24 h at 105º. In vitro dry matter digestibility (IVDMD) was calculated using the standard formula.

Ammonia production

Ammonium concentration in fermentation liquid was determined using th Kjeldhal method (AOAC 1990). Only distillation and titration steps were followed.

Methane production

Methane (CH4) was analyzed in the Department of Animal Science laboratory of ETH, Zurich using Hewlett Packard Gas Chromatograph (Model 5890, Series II, Avondale, PA, USA).

Protozoa and bacteria counts

Protozoal and bacterial were counted with Bürker counting chambers (0.1 and 0.02 mm depth, respectively; Blau Brandw, Wertheim, Germany) following the procedure of Soliva et al (2008).

Statistical analysis

Analysis of variance (ANOVA) was performed on chemical composition, in vitro digestibility and gas production data. The statistical significance of the differences between means was tested using the Duncan Multiple Range Test (DMRT). Regression coefficients were calculated using MS EXCEL version 2007.

Results and discussion

Recent studies conducted particularly with tree forages show that anti-nutritional factors (ANF) like tannins, can affect animal nutrition in rather diverse ways (Singh et al 2003; Singh and Sahoo 2004). The condensed tannin content of non legume and legume combinations used in the present study ranged between 1.73% (S. splicata x CC [SS.CC]) to 3.61% (S. caryophyllatum x CC [SC.CC].). Combining high and low tannin forages indicated that mixing of two forages can reduce the effect of tannins to a certain extent (Table 1). Several studies reported a negative influence of tannins on feed digestibility (e.g. Patra et al 2006; Grainger et al 2009; Jayanegara et al 2011). Makkar (2003b) found a strong negative correlation (r = -0.92) between tannins and in vitro rumen protein degradability. Moreover, Seresinhe and Iben (2003) and Kamalak et al (2005) reported the existence of a correlation between IVDMD and CP content. The CP content ranged from 9.4 to 14.6 in forage mixtures (Table 1). The high relationship (r2=0.73) between crude protein content and ammonia production (Figure 2) confirms that tannins did not affect feed protein degradation. Generally, tannins lower the rate of protein degradation and deamination in the rumen and therefore lower ruminal NH3-N (Woodward 1989). Similarly, Thadei et al (2001) observed an inverse relationship between tannin concentration and CP degradability. But this was not the case in this study. Sahoo et al (2010) reported that  for Bauhinia variegata and Dendrocalamus hamiltonii (20% CP in DM)  the substrate degradation was minimal; thus there was less microbial uptake and increased un-utilized NH3-N in the supernatant after 24h incubation.

Table 1 . Condensed tannins, crude protein and dry matter in the forage mixtures. Data are mean values of four replicates

CT in mixture

CP in mixture

DM in mixture

Terminalia catappa + Acacia auriculiformis

2.86a ±0.31



T.catappa + Calliandra calothyrsus

2.61a ±0.34



Symplocos splicata + A auriculiformis




S. splicata + C. Calothyrsus

1.73b ±0.14



Mangifera indica + A. auriculiformis

2.78a ±0.32



M. indica + C. calothyrsus

2.59a ±0.24



Syzygium caryophyllatum + A. auriculiformis

3.61a ±0.31



S. caryophyllatum + C. calothyrsus

3.41a± 0.34

14. 6a±2.03


Means within the same column with differing superscripts (a, b, c and d) are different at * p<0.05, *** p<0.001).

There was a steady increase in the gas production for over a period of 48 hours as well significant differences between forage mixtures in net gas volume (Table 2) .The highest net gas production was observed with mixtures containing low levels of tannins while the lowest values were observed in mixtures with higher levels of tannins. The findings are consistent with those of Getachew et al (2002) who found strong correlations between CT and gas production.

 Ahmed et al (2007) reported that a negative correlation of potential gas production with CT may be due to the reduction of microbial activity from increasingly adverse microbial condition McMahon et al (2000) concluded that tannins do not simply inhibit cellulose digestion by ruminal fluid in vitro and in vivo, but the inhibitory effects of tannins are involved the bacterial cells themselves.

The protozoa population was positively related with methane production (Figure 1). Increasing the condensed tannin concentration in the fermentation substrate led to a decrease in the population of protozoa (Figure 2) and a decrease in methane production (Figure 3).  The high positive relationship between crude protein content in the forage mixtures and ammona in the fermentation media (Figure 4) indicated that tannins did not excessively inhibit feed protein degradation.

 Soliva et al (2008) confirmed that plants known to contain secondary metabolites are able to suppress methanogenesis. Hess et al (2006) reported that inhibition of methanogenesis by tannins was probably the result of a suppression of fibre degradation. Similarly, Patra and Saxena (2010) also reported that, due to antimicrobial action of condensed tannins on rumen microbes, tannins may also decrease fiber degradation. Further, a decreased CH4 production rate by methanogens might be possible. In contrast, Seresinhe et al (2012) reported that methane production was not significantly affected by the presence of CT or different levels of CP in forage mixtures containing high tannin non legume and low tannin legumes.

Figure 1. Relationship between protozoal propulation and methane production Figure 2. Relationship between condensed tannins and protozoal population

Table 2 . Total gas production, methane and protozoa counts in the forage mixtures). Data are mean values of four replicates.


Gas production
(ml /200 mg DM)

CH4 production
(ml/ 200 mg/DM)

Protozoa x 10-3

NH 3
(ml/200 mg DM)


Terminalia catappa + Acacia auriculiformis

36.8 a ±6.95

5.07 C ±0.96

17.0 ab ±1.282

3.08a ±0.88

T.catappa + Calliandra calothyrsus

40.5 a ±5.45

6.07 bc ±0.73

21.5a ±4.621

2.77a ±0.47

Symplocos splicata + A auriculiformis

45.5 a ± 8.54

8.42 ab ± 1.37

44.4d ±2.220

3.29a ±0.54

S. splicata + C. calothyrsus

46.5 a ±3.42

9.56a ±1.53

46.6 d ±2.220

3.49a ±0.17

Mangifera indica + A. auriculiformis

40.3 a ±3.86

6.43 b c ±1.57

13.0 bc ±2.563

3.52a ±0.41

M. indica + C. calothyrsus

42.3 ah ±4.92

5.38 bc ±0.64

20.0 a ±5.874

3.53a ±0.26

Syzygium caryophyllatum + A. auriculiformis

30.5 b ±6.76

5.04 c ±0.54

11.0 bc ±3.391

3.76a ±0.23

S. caryophyllatum + C. calothyrsus

29.0 b ±4.36

5.15 c -=-±1.52

16.3ab ±5.587

4.07a ±0.41

Means within the same column with differing superscripts (a, b, c and d) are different at * p<0.05, *** p<0.001

Figure 3. Relationship between methane production and
condensed tannins in forage mixtures
Figure 4. Relationship between ammonia production and
crude protein content in forage mixtures



The authors greatly acknowledge the financial and laboratory assistance and guidance given by Prof. Michael Kreuzer and Dr. Carla Soliva in the Department of Agricultural and Food Science, ETH Zurich, Switzerland. Authors also appreciate the technical assistance of Messieurs D S Wijewardhana and S Karunathilaka.


Aerts R J, Barry T N and McNabb W C 1999 Polyphenols and agriculture: Beneficial effects of proanthocyanidins in forages. Agricultural . Ecosystems and Environment, 75: 1–12.

Ahmed G N, Abdel Nasir M A and Fadel E 2007 Chemical composition and in vitro gas production characteristics of six fodder trees leaves and seeds. Journal Agriculture and. Biological Science. 3:983-986

AOAC 1990 Official Methods of Analysis (14th. edition.), Association of Official Analytical Chemists, Arlington, VA.

El-Waziry A M, Nasser M E A, Sallam S M A, Abdallah A L and Bueno I C S 2007 Processing methods of soybean meal 2. Effect of autoclaving and quebracho tannin treated-soybean meal on gas production and rumen fermentation in vitro. Journal of Applied Science Research 3: 17-24.

Getachew G, Makkar H P S and Becker K 2002 Tropical browses: Contents of phenolic compounds in vitro gas production and stoichiometric relationship between short chain Fatty acid and in vitro gas production. Journal of Agricultural. Science 139: 341-352.

Grainger C, Clarke T, Auldist M J, Beauchemin K A, McGinn S M, Waghorn G C and Eckard R J 2009 . Potential use of Acacia mearnsii condensed tannins to reduce methane emissions and nitrogen excretion from grazing dairy cows. Canadian Journal of Animal Science 89: 241-251.

Hess H D, Monsalve l M, Lascano C E, Carulla C E, Diaz T E and Kreuzer M 2003 Supplementation of a tropical grass diet with forage legumes and Sapindus saponaria fruits: effects of in vitro ruminal nitrogen turnover and methanogenensis. Austalian Journal of Agricultural Research. 54: 703-713.

Hess H D, Tiemann T T, Noto F, Carulla J E and Kreuzer M 2006 Strategic use of tannins as means to limit methane emission from ruminant livestock International Congress Series 1293:164 – 167.

Jayanegara A, Leiber F and Kreuzer M 2011 . Meta-analysis of the relationship between dietary tannin level and methane formation in ruminants from in vivo and in vitro experiments. Journal of Animal Physiology and Animal Nutrition 96: 365–375.

Kamalak A, Canbolat O, Gurbuz Y, Ozzy O and Ozkose C O 2005 Chemical composition and its relationship to in vitro gas production of several tannin-containing trees and shrub leaves. Asian-Austalasian Journal of Animal Science 18:203-208

Liu J X, Susenbeth A and Südekum K H 2002 In vitro gas production measurements to evaluate interactions between untreated and chemically treated rice straws, grass hay, and mulberry leaves Journal of Animal Science. 2002 80:517-524.

Makkar H P S 2003a Quantification of tannins in tree and shrub foliage. A Laboratory Manual. Kluwer Academic Publishers, Amsterdam, The Netherlands

Makkar H P S 2003b Review. Effects and fate of tannins in ruminant animals, adaptation to tannins, and strategies to overcome detrimental effects of feeding tannin-rich feeds. Small Ruminant Research 49: 241–256.

McMahon L R, McAllister T A, Berg B P, Majak W, Acharya S N, Popp J D, Coulman B E, Wang Y and Cheng K J 2000 A review of the effects of forage condensed tannins on ruminal fermentation and bloat in grazing cattle. Canadian Journal of Plant Science 80: 469–485.

Menke K H and Steingass H 1988 Estimation of the energetic feed value obtained from chemical analysis and in vitro gas production using rumen fluid. Animal Research and Development 28: 7-55.

Mueller-Harvey I 2006 Unraveling the conundrum of tannins in animal nutrition and health. Journal of Science Food and Agriculture 86: 2010–2037.

Patra A K, Kamra D N, Agarwal N 2006 Effect of plant extract on in vitro methanogenesis, enzyme activities and fermentation of feed in rumen liquor of buffalo, Animal Feed Science and Technology 128: 276–291.

Patra A K and Saxena J 2010 A new perspective on the use of plant secondary metabolites to inhibit methanogenesis in the rumen. Phytochemistry, 71: 1198–1222.

Porter I P, Hrstich I N and Chan B G 1986 The conversion of procyanidins and prodelphinidins to cyanidin and delphinidin. Phytochemistry 25:223-230.

Sahoo A, Singh B and Bhat T K 2010 Effect of tannins on in vitro ruminal protein degradability of various tree forages. Livestock Research for Rural Development. Volume 22, Article #119.

Seresinhe T and Marapana R A U J 2011 Goat farming systems in the southern province of Sri Lanka: Feeding and management strategies. World Journal of Agricultural Science. 7: 383-390.

Seresinhe Y D J W, Seresinhe T and Pathirana K K. 2003 Assessment of the feeding value of five shrub legumes of local interest. In: Proceedings of the second academic session, University of Ruhuna, 23. Jan 2003, Matara, Sri Lanka. pp. 43-48.

Seresinhe T, Madushika S A C, Seresinhe Y, Lal P K and. Ørskov E R 2012 Effects of Tropical High Tannin Non Legume and Low Tannin Legume Browse Mixtures on Fermentation Parameters and Methanogenesis Using Gas Production Technique Asian-Australasina Journal of. Animal. Science Vol. 25,( 10 ): 1404 - 1410

Seresinhe T and Iben C 2003 In vitro quality assessment of two tropical shrub legumes in relation to their extractable tannins content. Journal of Animal Physiology and Animal Nutrition 87:109-115.

Singh B and Sahoo A 2004 Newer concepts, implications and approaches in the use of unconventional feeds to improve animal production. In: Proceedings of VANA Conferences, 24-26 Nov. 2004, NIANP, Bangalore, India. pp1-14.

Singh B, Bhat T K and Singh B 2003 Potential therapeutic applications of some anti-nutritional plant secondary metabolites. J. of Agriculture and Food Chemistry 51:5579-5597.

Soliva C R, Zeleke A B, Clement C, Hess H D, Fievez V and Kreuzer M 2008 In virto screening of various tropical foliages, seeds, fruits and medicinal plants for low methane and high ammonia generating potentials in the rumen. Animal Feed Science and Technology 147:53-71.

Thadei C, Kimambo A E and Mushi D E 2001 Relationship between tannins concentration in plant materials and their effects on protein degradation in the rumen. TSAP Proceedings Volume 28 pp.1-11.

Van Soest P J 1967 . Development of a comprehensive system of feed analysis and its application to forages. Journal of Animal Science 26: 119-128.

Woodward A and Reed J D 1989 The influence of polyphenolics on the nutritive value of browse: a summary of research conducted at ILCA. ILCA Bulletin 35:2-11.

Received 29 September 2013; Accepted 8 February 2014; Published 1 March 2014

Go to top