Effect of Water Extract of Plants Containing Tannin on in Vitro Methagonesis and Fermentation Characteristics of the Grass Pennisetum Purpureophoides

This experiment was conducted to evaluate the effect of extract of plants containing tannin on invitro CH4 production, fermentation characteristics and nutrient degradability. Six of plant leaves i.e.Gliricidia sepium, Acacia mangium, Leucaena leucocephala, Desmodium intortum, Camellia sinensis,Calliandra calothyrsus and seed of Areca catechu were extracted by using water. Experimentaltreatments consisted of P. purpureophoides (300±5 mg) incubated alone or added with 1.2 mL of plantextracts. The in vitro neutral detergent fibre (NDF) degradability was determined using the first stagetechnique of Tilley and Terry. The results showed that total tannin concentration of plant extract rangedfrom 34 to 95 g/kg DM, and was lowest in D. intortum and highest in A. mangium. Methane productionwas significantly (P<0.001) lower with addition of A. mangium, L. leucocephala, A. catechu, C. sinensisand C. calothyrsus extracts compared to control. Total tannin had a close relationship with CH4production (r=-0.79). There was strong correlation between CH4 production and NDF degradability(r=0.61). It was concluded that water extracts of A. mangium, L. leucocephala, A. catechu, C. sinensisand C. calothyrsus have potential to be used as rumen manipulator in order to reduce CH4 production inruminants.


INTRODUCTION
Methane (CH 4 ) is produced as a result of anaerobic fermentation of the soluble and structural carbohydrates by methanogens in the rumen of ruminant animals, which is released into the environment by eructation. The CH 4 emissions from ruminant animals range from 2 to 12% of the gross energy intake (Johnson and Johnson, 1995). Besides, it has been estimated that the world's population of ruminants produce about 15% of total atmospheric CH 4 emissions Extract of Plants Containing Tannin (B. Santoso et al.) (Moss, 1993). This means that CH 4 production from ruminant is not only represents a substantial loss in efficiency of animal production, but also contributes significantly to global warming as the greenhouse gas.
Recently, there is an increasing interest in research activities to evaluate the potential of secondary plant compound as feed additives instead of chemical compounds i.e. ionophores and antibiotics as manipulators of rumen fermentation including decrease CH 4 production. As previously stated by Russell and Rychlik (2001), there has been an increased perception that antibiotics and chemical compounds should not be routinely used as feed additives.
Tannin is a phenolic plant secondary compound and is widely distributed through plant kingdom especially legume and browse. Makkar et al. (1995) noted that secondary compound tannin is prevalent in many tropical fodder plants. Effect of tannin from some plants such Acacia mangium, Biophytum petersianum and Psidium guajava has been demonstrated as supplement to the tropical grass Pennisetum purpureum (Hariadi and Santoso, 2010). Plant extract containing tannin have been shown to decrease CH4 production (Śliwiński et al., 2002;Sirohi et al., 2009) andruminal NDF digestibility (McSweeney et al., 2001;Oliveira et al., 2007). Previous study with tropical plants, Jayanegara et al. (2011) concluded that total tannin had close relationship with CH 4 /digestible OM (r=-0.74). However, use of water as a solvent is more applicable and safe for animals than chemical solvents i.e. methanol, ethanol or acetone. The objective of this study was to evaluate the effect of water extracts from plant containing tannin on in vitro CH 4 production, fermentation characteristics, and nutrient degradability.

Samples and Extract Preparations
King grass (P. purpureophoides) was planted in a 6 m 2 plot without fertilizer at the Animal Research Station of State University of Papua in Manokwari. Grass was harvested after 50 days, chopped to 5 cm in length and oven-dried at 60°C for 48 h.
The leaves of G. sepium, A. mangium, L. leucocephala, C. sinensis, D. intortum and C. calothyrsus and seed of A. catechu were collected from the Manokwari Regency. The collected samples were then pooled and oven-dried at 60°C for 48 h. A commercial leaf of C. sinensis was used in this experiment. Samples of grass, leaves and seed of plants were ground to pass a 1 mm sieve in a Wiley mill.
Plants extract were prepared in water following a modified method of Patra et al. (2006). About 5 g of finely ground plants material were weighed into 100 ml beaker glass and added 50 ml of water. Plant materials were boiled for 10 min on a hotplate. The beakers were stoppered and incubated at 39°C on a shaker for 24 h and filtered through 2 layers of cheesecloth. The filtrates were stored at 4°C for further use.

Experimental Design and Treatments
The experiment was arranged in a completely randomized design with eight treatments and three replications. Experimental treatments consisted of P. purpureophoides incubated alone as control or added with plant extract at level of 1.2 mL/300 mg of substrate.

In vitro CH 4 Production and Nutrient Degradability Measurements
The in vitro gas production method was essentially according to Menke and Steingass (1988) as previously demonstrated by Hariadi and Santoso (2010). Oven-dried samples of about 300±5 mg were weighed into 100 mL glass syringes with pistons lubricated with Vaseline. Rumen liquor was obtained from two ruminally fistulated Holstein Frisian cross-bred cows fed elephant grass twice a day at maintenance level. About 30 mL of rumen liquor-buffer mixtures (1 : 2, v/v) was added into each syringe and then incubated in a water bath at 39°C for 48 h. The volume of gas released from each syringe was recorded before incubation (0 h) and 2, 4, 6, 12, 24 and 48 h of incubation.
Ten milliliter of gas was collected at 24 and 48 h of incubation from the silicon tube at the syringe tip using Terumo syringe and pooled to vacutainer tube for CH4 analysis. Methane was determined by injection 100 mL of gas into a chromatograph gas. The volume of CH 4 production was calculated by using formula: CH 4 production (ml) = total gas produced (ml) × % CH 4 in the sample.
At the end of the incubation period, about 10 ml of syringe contents were sampled. The pH of medium incubation was recorded using a digital pH meter. Subsequently, 0.2 mL of sub-samples were pipetted into 1.5 mL micro centrifuge tube containing 1 ml of 25 g/100 mL (w/v) metaphosphoric acid and centrifuged at 9000 x g for 10 min for volatile fatty acids (VFA) determination. Further on 2 mL of sub-samples were added to 2 mL of 20 g/l (w/v) NaCl for NH 3 -N (Chaney and Marbach, 1962).
The in vitro organic matter (OM) and NDF degradability was determined using the first-stage technique as proposed by Tilley and Terry (1963) as previously demonstrated by Hariadi and Santoso (2010).

Laboratory Analyses
Dried samples were used to determine DM, ash and crude protein (CP) according to procedure of AOAC (1990). Total tannin and condensed tannin contents were assayed using Folin-Ciocalteu and butanol-HCl methods respectively (Makkar, 2003). Concentrations of NDF and acid detergent fibre (ADF) were determined following Van Soest et al. (1991).

Statistical Analysis
Data was analyzed using the procedure GLM of SAS (version 6.12, SAS institute, Cary, NC, USA). Duncan's multiple range test was used to separate treatment means. Correlation analysis was done to establish the relationship between variables.

Chemical Composition of Plants
The chemical composition of experimental plants is presented in Table 1. The NDF and ADF content in plants containing tannin ranging from 269 to 596 and 115 to 381 g/kg DM, respectively. The ranking order of plant samples on the basis their total tannin content was A. mangium > C. calothyrsus < L. leucocephala < A. catechu > C. sinensis > G. sepium > D. intortum. However, on the basis of their condensed tannin concentration the plant could be ranked as

In vitro Gas and CH4 Productions
Effect of plant extract containing tannin on gas and CH 4 production are given in Table 2. Gas production was significantly (P<0.001) different among treatments at 6, 24 and 48 h of incubation. Addition of plant extracts in grass substrate decreased (P<0.001) gas production at 48 h of incubation compared to control. Methane production in substrate with addition of A. mangium, L. leucocephala, A. catechu, C. sinensis and C. calothyrsus extracts was significantly (P<0.001) lower as compared to control. The CH 4 produced at 48 h of incubation averaged 12.3% of total gas at 48 h of incubation.

Fermentation Characteristics
The pH value, concentrations of NH 3 -N and VFA in the medium incubation are present in Table 3. The pH value in medium incubation was not significantly (P>0.05) different among treatments. Concentration of NH 3 -N significantly (P<0.05) increased in grass substrate with addition of extract of L. leucocephala as compared to control. Addition of extract of G. sepium, A. mangium, A. catechu, C. sinensis and C. calothyrsus in grass substrate decreased (P<0.001) total VFA concentration. Extracts of G. sepium, A. catechu and C. sinensis increased (P<0.001) proportion of acetate compared to control, whereas A. mangium and C. sinensis extracts reduced (P<0.001) proportion of propionate. Acetate:propionate ratio was highest in A. mangium and lowest in A. catechu extracts.

In vitro Nutrient Degradability
In vitro OM and NDF degradability of grass substrate with addition of tannin extracts is presented Table 4. Addition of L. leucochepala, C. sinensis and C. calothyrsus extracts to grass substrate decreased (P<0.001) IVOMD, whereas IVNDFD was decreased (P<0.001) by addition of A. mangium, L. leucocephala, A.catechu, D.
intortum, C. sinensis and C. calothyrsus extracts as compared to control.

Relationships between Tannin Content and Fermentation Variables
The correlations between total tannin, condensed tannin and fermentation variables are in Table 5. There was a negative correlation between total tannin and the gas production at 6, 24 and 48 h of incubation and CH 4 production, indicating that as total tannin concentration increased, the gas and CH 4 production decreased. Total tannin concentration had closer correlation with CH4 and CH4/total gas production than condensed tannin concentration. There was negative correlation between total tannin content and both IVOMD and IVNDFD.

Discussion
The total tannin content in A. mangium was comparable to that value reported by Jayanegara et al. (2011), but condensed tannin content was lower than value of 42 g/kg DM as reported by Jayanegara et al.(2011) Concentration of total tannin in D. intortum was lower than in Desmodium intortum (Getachew et al., 2000). Concentration of condensed tannin in L. leucocephala and C. calothyrsus was lower than values of 76 and 240 g/kg DM, respectively (Tiemann et al., 2008). Different of plant nutrients content in this study as compared to previous study could be due to difference in location of sample source and plants maturity. Higher gas production with addition of plant extract could be due to the presence of higher of soluble sugar from these extract. In previous studies of Śliwiński et al. (2002); Patra et al. (2010), increasing gas production by plant extracts that contain phenol or saponin caused by increasing soluble sugar from these plant extract.
No differences in pH values obtained in the present study, consistent with previous studies of Śliwiński et al. (2002);Oliviera et al.(2007); Animut et al. (2008) who found that H was not changed by addition of tannin in both sheep and cattle rumens.
Tannin has beneficial effect on protection on dietary protein in the rumen and subsequently enhancement of amino acid absorption and utilization by the ruminant animal (Waghorn et al., 1994). McSweeney et al. (2001) revealed that tannin has ability to bind protein by forming hydrogen bonds between the phenolic sub-units of   Min et al. (2005) suggested that the action of condensed tannin in forages markedly reduced both growth and population of proteolytic bacterial. However, the extent of positive or negative effects of tannins may vary depending on the type and level of tannins in plants and their biological activity (Getachew et al., 2000). Relatively higher concentration ammonia-N in the medium incubation with addition of extract of G. sepium, A. mangium, L. leucochepala, A. catechu and D. intortum could be due to higher crude protein content in those plants or tannin concentration is lower than minimum concentration needed to produce maximum inhibition of proteolysis. The inhibition of methanogenesis has long been considered from nutritional aspects, and more recently from the perspectives on greenhouse gas emissions. In the previous studies by Śliwiński et al. (2002); Patra et al. (2006); Patra et al. (2010); Rodríguez et al. (2011) who reported that CH4 production reduced by addition of plant extract containing tannin. In contrast, Beauchemin et al. (2007) described that supplementing a forage-based diet with quebracho tannin extract failed to reduce CH4 production from growing cattle. The same result has been reported by Oliviera et al. (2007) that feeding sorghum silage containing high or low concentrations of tannin did not affect nutrient digestibility and CH 4 production in cattle. However, effect of tannin on methanogenesis depends on the source, type and dose of tannins (Patra et al., 2006). In this study, relative to control, addition 1.2 ml extract of A. mangium,L. leucocephala,A. catechu,C. sinensis or C. calothyrsus decreased CH 4 production by 43.6,30.9,30.9,36.3 and 31.8%, respectively. Decreased CH4 production in this study, however might be related to decreasing of fibre degradation which is shown by high correlation coefficient value between CH 4 production and NDF degradability (r =0.61; P<0.01) (result not shown). This result was supported by previous study of Estermann et al. (2002) that there was a strong relationship between CH4 production and digestible NDF for cows and calves. Bhatta et al. (2009) concluded that tannins suppress methanogenesis by reducing methanogenic populations in the rumen either directly or by reducing the protozoal population, thereby reducing methanogens symbiotically associated with the protozoal population. Tavendale et al. (2005) revealed that condensed tannin reduce CH 4 production could be due to indirect effect by reduced hydrogen production as result of reduced feed degradability, and by direct inhibitory effect on methanogens. A high correlation (r=-0.79, P<0.001) between total tannin concentration and CH4 production in this study (Table 5) was agreed with previous studies of Jayanegara et al. (2009Jayanegara et al. ( , 2011; Hariadi and Santoso (2010) that found r values -0.60, -0.66 and -0.76, respectively. A similar observation was found by Bhatta et al. (2009) that total tannin had close correlation with CH 4 output (r=-0.97). In our study, IVNDFD in substrate with addition of L. leucocephala, A. catechu, D. intortum, C. sinensis or C. calothyrsus extracts was significantly lower than control. This finding agrees with previous study of Bhatta et al. (2009) who found that tannin significantly suppressed bacteria population, through a direct effect (Koike and Kobayashi, 2009) and by reducing nutrient availability  (Sallam et al., 2010). In addition, tannin could reduce fibre digestion by complexing with lignocellulose and preventing microbial digestion or by directly inhibiting cellulolytic microorganism or both (McSweeney et al., 2001). In our results, IVOMD decreased ranged from 7.3 to 20.1% when addition of L. leucocephala, C. sinensis or C. calothyrsus extract to P. purpurophoides substrate. Similar result has been reported by Patra et al. (2006) that addition of extract of plants containing tannin reduced IVDMD and IVOMD by about 7% in comparison to control. Hess et al. (2003) also reported that tannins from C. calothyrsus may cause significant shifts in rumen microbial populations, especially a reduced total number of cellulolytic bacteria, which could have contributed to lower OM degradation.

CONCLUSION
Plant extracts from A. mangium, L. leucocephala, A. catechu, C. sinensis and C. calothyrsus have potential to be used as rumen manipulator in order to reduce CH 4 production in ruminants. Among plant extracts, A. mangium had the strongest effect on in vitro CH 4 production. The mode of action tannin on methanogenesis might be partially attributed to reduce fibre degradability.