Effect of plant extracts on in vitro methanogenesis, enzyme activities and fermentation of feed in rumen liquor of buffalo

Animal Feed Science and Technology 128 (2006) 276–291

Effect of plant extracts on in vitro methanogenesis, enzyme activities and fermentation of feed in rumen liquor of buffalo A.K. Patra, D.N. Kamra ∗ , Neeta Agarwal Rumen Microbiology Laboratory, Centre of Advanced Studies in Animal Nutrition, Indian Veterinary Research Institute, Izatnagar, Bareilly 243122, India Received 8 April 2005; received in revised form 13 October 2005; accepted 1 November 2005

Abstract The extracts of pods of Acacia concinna (Shikakai), seed pulp of Terminalia chebula (harad), Terminalia belerica (bahera), Emblica officinalis (amla) and seed kernel of Azadirachta indica (neem seed) in different solvents (ethanol, methanol and water) were evaluated for their effect on methane production, enzymes activities and rumen fermentation in in vitro gas production test. Gas production per gram dry matter (DM) of substrate (wheat straw and concentrate mixture in 1:1 ratio) was significantly (P<0.05) higher with extracts of A. concinna, E. officinalis and T. belerica as compared to control. Among the extracts tested only methanol extract of T. chebula suppressed in vitro methane production significantly (P<0.05). Specific activities of carboxymethylcellulase (CMCase) and xylanase were similar (P>0.05) among the extracts at both the levels tested, whereas, the activity of acetylesterase was reduced significantly (P<0.05). Total volatile fatty acids (TVFA) were significantly (P<0.05) decreased with extracts of T. chebula and A. indica. There was a decrease (P<0.05) in acetate to propionate ratio due to addition of the extracts of A. concinna and A. indica in incubation medium as compared to control. In vitro dry matter and organic matter degradabilities of feed (g/g) were decreased significantly (P<0.05) with all the extracts compared to that of control. The extracts of A. concinna and A. indica at 0.25 and 0.50 ml levels and extracts of T. chebula at 0.50 ml level reduced

Abbreviations: ADF, acid detergent fiber; CMCase, carboxymethylcellulase; CP, crude protein; DM, dry matter; EE, ether extract; IVDMD, in vitro dry matter degradability; IVOMD, in vitro organic matter degradability; mIU, milli international units; mM, milli molar; NDF, neutral detergent fiber; OM, organic matter; S.E.M., standard error of means; TVFA, total volatile fatty acids ∗ Corresponding author. Tel.: +91 581 2301318; fax: +91 581 2303284. E-mail address: [email protected] (D.N. Kamra). 0377-8401/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.anifeedsci.2005.11.001

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total protozoa counts significantly. Similar trends were observed for small and large entodiniomorphid protozoa counts. However, no effect (P>0.05) was found on holotrich protozoa numbers. The results indicated that methane emission is not essentially associated with protozoa activity since among three seed pulps with high antiprotozoal activity, only T. chebula (methanol extract) had antimethanogenic activity. The methanol extract of seed pulp of T. chebula appears to have a potential to be used for mitigation of enteric methane production, but the level of feeding will have to be evaluated so that there is minimum adverse effect on the degradability of feed in the rumen. © 2005 Elsevier B.V. All rights reserved. Keywords: Plant extracts; Methane; Rumen fermentation; Fibrolytic enzymes; Protozoa; Acacia concinna; Terminalia chebula; Terminalia belerica; Emblica officinalis; Azadirachta indica

1. Introduction Manipulation of rumen microbial ecosystem for enhancing fibrous feed digestibility, reducing methane emission and nitrogen excretion by ruminants to improve their performance are some of the most important goals for animal nutritionists. Plant extracts with high concentration of secondary metabolites are good candidates for achieving one or more of these objectives (Teferedegne, 2000). The tropical plants containing saponins have been found to suppress or eliminate protozoa from the rumen and reduce methane and ammonia production (Kamra et al., 2000; Sliwinski et al., 2002; Lila et al., 2003). Similarly, some plant extracts having high content of flavonoids decrease methane production and induce extensive stimulation of microbial metabolism which increases both degradability of crude protein and cell wall constituents and the efficiency and yield of microbial biomass production (Broudiscou et al., 2000, 2002). Tannins have also been found to reduce methane production (Woodward et al., 2001). However, effectiveness of plants or plant extracts having high content of saponins, flavonoids and tannins varied depending upon the source, type and level of secondary metabolite present in it. Therefore in the present study, the plants were selected on the basis of the presence of secondary metabolites. The seed pods of Acacia concinna (shikakai) contain saponins and Terminalia belerica (Bahera), Terminalia chebula (harad) and Emblica officinalis (amla) are rich in phenolics (Chevallier, 1996; Sinha, 1996). The seed kernel of Azadirachta indica (neem) contains bitter principles like nimbin, nimbidin, azadirachtin, etc. and is being used in medicines and its cake has been recommended for the feeding of ruminants after water washing (Nath et al., 1983; Agrawal et al., 1987). The water extract of neem seed kernel cake was also found to stimulate fibre degrading enzymes of rumen when tested in in vitro (Agarwal et al., 1991b). All these plants are also used in ayurvedic (an ancient Indian system) medicines (Chevallier, 1996; Rastogi and Mehrotra, 1993; Sivarajan and Balachandran, 1996). This experiment was conducted to evaluate parts of these plants for their antiprotozoal and antimethanogenic activities. The fermentation pattern was also studied in in vitro gas production test so that selection could be made for the most potent antimethanogenic compound with least effect on rumen fermentation.

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2. Materials and methods 2.1. Preparation of extracts The plant materials [pods of A. concinna (Shikakai), seed pulp of T. chebula (harad), T. belerica (bahera), E. officinalis (amla) and seed kernel of A. indica (neem seed)] were dried at 55–60 ◦ C and ground. Plant extracts were prepared in three solvents [water, ethanol (95/100 ml) and methanol (98/100 ml) at 20 g/100 ml of solvent]. For the preparation of water extract, plant materials were boiled for 5 min on a low flame. The flasks of all the solvents were stoppered and incubated at 39 ◦ C on a rotory shaker for 24 h and filtered through Whatman filter paper no. 1. The filtrates were collected and stored at 4 ◦ C for further use. The soluble sugar content in plant extracts was estimated as per method of Dubois et al. (1956). 2.2. Experimental design Five plants A. concinna, T. belerica, T. chebula, E. officinalis and A. indica extracted in three solvents (E, ethanol; M, methanol and W, water), were tested at three levels (0, 0.25 and 0.5 ml) in five replicates for each treatment. Each set of syringes comprised of a single syringe for each plant product and a control without any product (total six) × solvent × level combination, two syringes as blanks (without substrate) and two syringes as standard (maize hay as substrate). A mixture of wheat straw and concentrate mixture (same as fed to the animals) in the ratio of 1:1 was used as substrate. 2.3. Preparation of inoculum Rumen liquor was collected from two fistulated buffaloes fed on a diet of wheat straw and concentrate mixture in 1:1 ratio. The wheat straw had organic matter (OM), 909 g; crude protein (CP), 31 g; ether extract (EE), 15 g; neutral detergent fibre (NDF), 835 g and acid detergent fibre (ADF), 535 g/kg and the concentrate mixture (consisting of maize, 32; solvent extracted soybean meal, 20; wheat bran, 45; mineral mixture, 2 and salt, 1 kg/100 kg) had OM, 895 g; CP, 195 g; EE, 40 g; NDF, 385 g and ADF, 164 g/kg on DM basis. The animals were fed at the rate of 2.5 kg dry matter/100 kg of body weight. The rumen liquor was sampled just before feeding (0 h) from two animals and transported in insulated flasks under anaerobic conditions to the laboratory, pooled in equal proportions and used as a source of inoculum. 2.4. In vitro gas production test The substrate (wheat straw and concentrate mixture in 1:1 ratio) was milled to pass through 1 mm sieve and 200 ± 10 mg was weighed in glass syringes of 100 ml capacity. The incubation medium was prepared as described by Menke and Steingass (1988) and 30 ml was dispensed anaerobically in each syringe. Syringes were incubated at 39 ◦ C for 24 h.

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2.5. Estimation of gas and methane production After 24 h incubation gas production was estimated by the displacement of piston during incubation. The gas produced due to fermentation of substrate was calculated by subtracting gas produced in blank syringe (containing no substrate, but only the inoculum and buffer) from total gas produced in the syringe containing substrate and inoculum. The gas produced in standard syringe (containing standard maize hay) was used to check day to day variation in the quality of inoculum. For methane estimation 100 ␮l gas was sampled from the headspace of syringe in an airtight syringe and injected into Nucon-5765 gas chromatograph equipped with flame ionization detector (FID) and stainless steel column packed with Porapak-Q. The gas flow rates for nitrogen, hydrogen and air were 30, 30 and 300 ml/min, respectively. Temperature of injector oven, column oven and detector were 40, 50 and 50 ◦ C, respectively. A 50/50 mixture of methane and carbon dioxide (Spancan; Spantech Products Ltd., England) was used as a standard. 2.6. Enzyme assay The whole content of the syringe after 24 h incubation was transferred to a 100 ml beaker and mixed with carbontetrachloride and lysozyme solution (0.4 g/100 ml phosphate buffer, 0.1 M, pH 6.8) at a rate of 5 ml each per 30 ml of syringe content for enzyme extraction (Hristov et al., 1999a; Agarwal et al., 2000). The contents were incubated at 40 ◦ C for 3 h followed by sonication at 4 ◦ C using a sonicator (Labsonic U model; B. Braun Biotech International). The sonicated samples were centrifuged at 24 000 × g for 20 min at 4 ◦ C and clear supernatant was used for the estimation of enzyme activities. The reaction mixture contained 1 ml phosphate buffer (0.1 M, pH 6.8), 0.5 ml carboxymethylcellulose (1.0 g/100 ml phosphate buffer) (Koch-Light Laboratories Ltd., Colnbrook Bucks, England) in 0.1 M phosphate buffer (pH 6.8) and 0.5 ml extracted supernatant for the estimation of CMCase. For xylanase activity, the reaction mixture contained 1 ml phosphate buffer, 0.5 ml oat spelt xylan (0.25 g/100 ml phosphate buffer) (Sigma Chemical Co., St. Louis, USA) and 0.5 ml extracted supernatant. The reaction mixtures were incubated for 60 min (cmcase) and 15 min (xylanase) at 39 ◦ C. The reducing sugars thus released were estimated according to Miller (1959) using glucose (Himedia Laboratories Limited, India) and xylose (Sigma Chemical Company, USA) as standards. The acetylesterase activity was estimated by the method of Huggins and Lapides (1947) with some modifications. The assay mixture contained 0.2 ml extracted supernatant, 0.9 ml phosphate buffer (0.1 M, pH 6) and 0.9 ml of 2 mM p-nitrophenyl acetate with 10 min incubation. The protein content of the enzyme samples was estimated as described by Lowry et al. (1951). The enzyme activities were expressed as ␮mol of reducing sugars or pnitrophenol released per min per ml under assay conditions and expressed as international units (IU). 2.7. Volatile fatty acid estimation At the end of incubation (24 h) 1 ml of the supernatant was collected in a microfuge tube containing 0.20 ml metaphosphoric acid (25 ml/100 ml). The mixture was allowed to stand

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for 2 h at room temperature and centrifuged at 5000 × g for 10 min. The clear supernatant was collected and stored at −20 ◦ C until analyzed. For VFA estimation, 1 ␮l supernatant was injected in a gas chromatograph (Nucon-5765) equipped with a double flame ionization detector (FID) and chromosorb glass column (4 ft length and 1.8 mm diameter) as described by Cottyn and Boucque (1968). The gas flows for nitrogen, hydrogen and air were 30, 30 and 320 ml/min, respectively. Temperature of injector oven, column oven and detector were 270, 172 and 270 ◦ C, respectively. 2.8. In vitro dry matter degradability In vitro degradability was determined following the first step of Tilley and Terry (1963). In vitro medium (40 ml) and 10 ml of strained rumen liquor of buffalo were dispensed anaerobically in 100 ml conical flasks containing 500 mg of feed material (wheat straw and concentrate mixture in 1:1 ratio). After gassing CO2 in the flask for 5 min, a cork fitted with Bunsen gas release valve was tightly placed over the flasks and were incubated at 39 ◦ C for 24 h. After termination of incubation, the contents were filtered through pre-weighed Gooch crucibles and residual dry matter was estimated. The per cent loss in weight was determined and presented as IVDMD. The dried feed sample and residue left above was ashed at 550 ◦ C for determination of IVOMD. 2.9. Protozoa counts After termination of incubation, the contents of the syringe were mixed properly and 1 ml sample was mixed with 1 ml methyl green formal saline solution. The stained sample was kept overnight and protozoa were counted microscopically following the procedure described by Kamra et al. (1991). 2.10. Proximate analyses The DM (ID number 930.15), OM and ash (942.05) and CP (N × 6.25, ID number 954.01) and EE (ID number 920.39) of substrates were determined by AOAC (1995) procedures. NDF (estimated without amylase and expressed inclusive of ash), ADF (also expressed as inclusive of ash) and ADL were analyzed according to the methods described by Van Soest et al. (1991). 2.11. Statistical analyses The data of in vitro gas production test and in vitro dry matter and organic matter degradability were analyzed using General Linear Model procedure of SPSS (1996) as a randomized block design with treatments in a 6 × 3 × 3 factorial arrangement. For comparison of extracts Generalized Linear model multivariate ANOVA procedures were used and means were compared using Tukey’s test. When product × solvent, product × level and product × solvent × level interactions were not significant, data were averaged for solvents, levels and both solvents and levels, respectively.

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Table 1 Effect of plant extracts on in vitro gas production (ml/g DM) in 24 h Products

Solvents

Control

Ethanol Methanol Water

Mean of levels A. concinna

T. chebula

149 E

191 206 216 203.1 BC

178 185 184

149 E

196 204 187 196 BCD

179 173 180 177 BCDE

170 165 165

149 E

181 174 167 174 CDE

155 141 196 164 DE

162 145 178

149 E

182 144 188 172 CDE

225 247 247 240 A

196 207 198

149 E

214 223 198 212 AB

161 162 174 166 DE

165 166 162

149 E

184 188 164 179 BCDE

Ethanol Methanol Water

Mean of levels Overall S.E.M.

163 167 149

Ethanol Methanol Water

Mean of levels A. indica

0.50 ml 176 179 149 168 CDE

Ethanol Methanol Water

Mean of levels E. officinalis

0.25 ml 164 174 149 163 DE

Ethanol Methanol Water

Mean of levels

Mean of solvents

0 ml

Ethanol Methanol Water

Mean of levels T. belerica

Level of extract

2.23

Different letters in a row differ significantly (P<0.05) among levels of all products irrespective of solvents.

3. Results and discussion 3.1. Effect on gas and methane production The results of gas production as affected by different plant extracts are presented in Table 1. The solvents included in the incubation medium did not show any effect (P>0.05) on gas production which indicated that the levels of solvents used in the experiment were not detrimental for rumen microbes. The gas production (ml/g DM) differed significantly (P<0.05) among the products and levels of extracts. Addition of extracts of A. concinna, E. officinalis and T. belerica resulted in a significantly (P<0.05) higher production of gas per gram dry matter as compared to control. The inclusion of the extracts of T. chebula and A. indica increased gas production but not significantly (P>0.05). This increase in gas production might be partly due to addition of soluble sugars in the reaction mixture through inclusion of the extracts. The ethanol, methanol and water extracts of A. concinna, T. beler-

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Table 2 Effect of plant extracts on in vitro methane production (ml/g DM) in 24 h Products

Solvents

Control

Ethanol Methanol Water

Mean of levels A. concinna

T. chebula

31 AB

43 38 37 41 A

41 a 37 ab 34 ab

31 AB

45 41 35 40 A

43 34 27 34 AB

38 ab 35 ab 29 ab

31 AB

41 40 28 36 A

19 00 29 16 C

27 b 11 c 31 ab

31 AB

30 03 33 22 BC

36 34 39 39 A

36 ab 39 ab 34 ab

31 AB

41 41 38 40 A

30 36 36 34 AB

36 ab 37 ab 34 ab

31 AB

41 43 34 39 A

Ethanol Methanol Water

Mean of levels Overall S.E.M.

38 ab 40 ab 31 ab

Ethanol Methanol Water

Mean of levels A. indica

0.50 ml 45 47 31 41 A

Ethanol Methanol Water

Mean of levels E. officinalis

0.25 ml 37 43 31 37 A

Ethanol Methanol Water

Mean of levels

Mean of solvents

0 ml

Ethanol Methanol Water

Mean of levels T. belerica

Level of extract

0.78

Different letters (A–C) in a row differ significantly (P<0.05) among levels of all products irrespective of solvents. Different letters (a–c) in a column differ significantly (P<0.05) among solvents of all products irrespective of levels.

ica, T. chebula, E. officinalis and A. indica contained 25.6, 64.2, 64.2; 8.3, 31.8, 31.7; 18.0, 50.2, 32.1; 25.3, 38.3, 52.4 and 4.1, 5.6, 3.6 mg/ml soluble sugars, respectively. The extracts of plants in methanol and water had more soluble sugars as compared to that in ethanol. The sugars extracted in all solvents were minimum from A. indica as compared to other plants. The plant extracts did not have any influence on in vitro methane emission with the exception of methanol extract of T. chebula (Table 2). There was 95% reduction in methane emission with the lower dose (0.25 ml/30 ml incubation medium) and the inhibition was almost complete at the double level of extract. The presence of tannins in T. chebula might be responsible for reduction in methane emission. Phenolic acids such as p-coumaric acids, ferulic acids, cinnamic acids and phloretic acids and some monomeric phenolics have been found to decrease methane, acetate and propionate production (Ushida et al., 1989; Asiegbu et al., 1995). Although T. belerica and E. officinalis also contain phenolics, they appeared not effective against methanogenesis in the present experiment. Waghorn

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Table 3 Effect of plant extracts on total protozoa count (×103 ml−1 ) Products

Solvents

Control

Ethanol Methanol Water

Level of extract 0 ml

Mean of levels A. concinna

T. belerica

113 97 103

28 14 21 21 D

64 45 53

103 A

60 18 34 38 CD

92 78 108 93 AB

98 95 102

103 A

98 105 95 100 AB

Ethanol Methanol Water 103 A

74 88 87 83 ABC

103 A

82 98 88 89 AB

103 A

66 61 73 67 ABCD

Ethanol Methanol Water

Mean of levels A. indica

123 90 103 105 A

Ethanol Methanol Water

Mean of levels E. officinalis

0.50 ml

113 98 103 105 A

103 A

Mean of levels T. chebula

0.25 ml

Ethanol Methanol Water

Mean of levels

Ethanol Methanol Water

Mean of levels Overall S.E.M.

Mean of solvents

61 64 89 71 ABC 64 120 98 94 AB 35 39 88 54 BCD

79 85 93 83 107 96 68 68 88

0.78

Different letters in a row differ significantly (P<0.05) among levels of all products irrespective of solvents.

and McNabb (2003) suggested that the effect of tannins depends on their chemical and physical structures and their concentration in the diet. 3.2. Effect on protozoa The effect of plant extracts on different protozoa counts in in vitro gas production test are presented in Table 3. Inclusion of A. concinna and A. indica extracts at 0.25 and 0.50 ml levels in the medium resulted in a significant reduction in protozoa count, whereas, extracts of T. chebula reduced the protozoa count only at 0.50 ml level. Other extracts had no influence on total protozoal counts. Similar trends were observed for small and large entodiniomorphid protozoa counts (Table 4), whereas, holotrichs (0.8–1.8 × 103 cells/ml) were not influenced by any of the extracts tested. This is probably the first report that extracts of the pods of A. concinna, a tropical plant could inhibit rumen protozoa. Pods of A. concinna have been found to contain triterpenoids and steroid saponins, acacidiol, acacic acid and sonunin

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Table 4 Effect of plant extracts on small and large entodiniomorphid populations (×103 ml−1 ) in the incubation medium Products

Small entodiniomorphs Control

Solvents

T. belerica

61 57 76 65 AB

70 74 79

85 A

64 80 75 73 AB

55 103 86 81 AB

71 92 82

85 A

72 86 73 77 AB

30 33 76 46 BCD

58 57 75

85 A

57 52 65 58 ABC

13.6 6.0 11.6 8.9 9.8 6.7

14.6 8.5 9.7 7.6 10.2 6.5

14.6 a 8.5 b 12.4 ab 10.9 ab 12.0 ab 9.8 ab

2.22

Large entodiniomorphs Control A. concinna T. belerica T. chebula E. officinalis A. indica

16.0

Mean Overall S.E.M.

72 69 92 78 AB

81 81 87

85 A

85 89 82 86 AB

Ethanol Methanol Water

Mean of levels Overall S.E.M.

23 11 17 17 D

52 37 44

85 A

48 15 29 31 CD

Ethanol Methanol Water

Mean of levels A. indica

106 76 85 89 A

96 82 85

85 A

97 86 85 89 A

Ethanol Methanol Water

Mean of levels E. officinalis

0.50 ml

Ethanol Methanol Water

Mean of levels T. chebula

0.25 ml

Ethanol Methanol Water

Mean of levels

Mean of solvents

0 ml Ethanol Methanol Water

Mean of levels A. concinna

Level of extract

16.0 A

9.4 B

8.6 B

0.94

Different letters (A–D) in a row differ significantly (P<0.05) among levels of all products irrespective of solvents within a parameter. Different letters (a and b) in a column differ significantly (P<0.05) among extracts.

(Chevallier, 1996). The inhibitory effect of these extracts on protozoa could be due to their saponin content. Decreased protozoal counts with supplementation of saponins rich extract (Hristov et al., 1999b; Kamra et al., 2000) or saponin rich forages (Newbold et al., 1997; Teferedegne et al., 1999) or fruits (Thalib et al., 1998; Hess et al., 2003) have

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been reported. Saponins possibly bind with sterol of cell membrane of protozoa and change the permeability of cell membrane. Adverse effects of extracts of A. indica on total, small and large entodiniomorphid protozoa might be due to the presence of bitter principles. Mondal and Garg (2002) also observed a significant (P<0.05) reduction in total protozoa and entodiniomorphid protozoa counts in crossbred cattle by feeding of A. indica cake, however, holotrich protozaoa were not affected (P>0.05). The number of entodiniomorphids was similar (P>0.05) to that of control when water washed A. indica cake was fed, which indicated that active principles are soluble in water. Methanol extract of T. chebula at 0.5 ml/30 ml of incubation medium reduced protozoal numbers in the present study. T. chebula contains a large amount of tannic acids, gallic acids, chebulinic acids and resins (Sivarajan and Balachandran, 1996) which might be responsible for antiprotozoal activity, especially tannic acids as reported earlier by Hristov et al. (2003). Since a reduction in methanogenesis was achieved only with methanol extract of T. chebula and not by extracts of A. concinna and A. indica which showed antiprotozoal activity, it is suggested that methanogenesis is not essentially related to the density of protozoa population in the rumen. According to Newbold et al. (1997) and Hess et al. (2003) only a small portion of total methane production is due to the presence of methanogens attached with the ciliate protozoa. Dohme et al. (1999) also reported inhibition of in vitro methane emission both in defaunated and faunated rumen liquor with coconut oil. Machm¨uller et al. (2003) demonstrated an increased number of methanogens in defaunated sheep, and suggested that association between protozoa and methanogens does not play an important role in methanogenesis in rumen. 3.3. Effect on VFA The TVFA concentration (mM/100 ml) was significantly (P<0.05) decreased when extracts of T. chebula and A. indica were added to the medium while other extracts were ineffective (Table 5). Concentration of propionate was significantly increased by extract of A. concinna. There was a decrease (P<0.05) in acetate to propionate ratio due to addition of the extracts of A. concinna and A. indica in incubation medium as compared to control. Concentrations (mM) and per cent of isobutyrate and butyrate were similar among the extracts tested (data not presented). Tannic acids have been found to reduce production of total volatile fatty acids (Hristov et al., 2003). Increase in propionate (per cent) and decrease in acetate (per cent) and consequently decrease in acetate and propionate ratio by extracts of A. concinna could be due to the presence of saponins and its inhibitory effect on protozoa, which is in agreement with previous studies (Wang et al., 2000; Ye et al., 2001; Lila et al., 2003). The reduced protozoa numbers is sometimes associated with increase in propionate (per cent) and decrease in A:P ratio (Hess et al., 2003; Machm¨uller et al., 2003). According to Jouany et al. (1988) changes in the VFA pattern due to reduction in protozoa population is not always consistent because nature of diet also plays an important role in VFA pattern. 3.4. Effect on enzyme profile The effect of plant extracts on enzyme activities are presented in Table 6. The specific activities of CMCase were not affected by any of the extracts tested, whereas specific activity

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Table 5 Effect of plant extracts on TVFA production and acetate propionate (A:P) ratio in the incubation medium Products

TVFA (mM/100 ml) Control A. concinna T. belerica T. chebula E. officinalis A. indica Mean Overall S.E.M. A:P ratio Control A. concinna T. belerica T. chebula E. officinalis A. indica

Level of extract

Mean of products

0 ml

0.25 ml

0.50 ml

56.6

54.0 54.7 53.6 48.6 54.9 49.9

55.6 51.6 51.2 48.2 55.6 49.8

56.6A

52.6 B

52.7 B

Methanol 3.76 3.43 3.84 3.59 3.78 3.64

Water 3.85 3.50 3.83 3.88 3.81 3.71

55.4 55.6 53.8 51.5 55.7 52.1

0.50 Ethanol 4.06 3.96 4.12 3.88 4.23 3.81

Mean

4.01 A

Overall S.E.M.

0.023

3.67 B

3.89 a 3.63 b 3.93 a 3.78 ab 3.94 a 3.72 b

3.76 B

Different letters (A and B) in a row differ significantly (P<0.05) among levels of all the products irrespective of solvents within a parameter. Different letters (a and b) in a column differ significantly (P<0.05) among the extracts within a parameter.

of acetylesterase was reduced significantly (P<0.05) by all the extracts. The numerically lower activities in the presence of different extracts on CMCase and xylanase might be due to its antiprotozoal activity, as it has been reported that about 38% of cellulase activity is associated with protozoa fraction of rumen liquor (Agarwal et al., 1991a). But the presence of different extracts influenced the activity of acetyl esterase differently, which might be due to the fact that this enzyme is mainly of fungal origin (Borneman et al., 1990). A decrease in CMCase and xylanase activity by addition of yucca and quillaja saponins have been observed by Hristov et al. (2003). Though the earlier reports indicate that tannins make a complex with the enzymes resulting in inhibition of their activity (Butler, 1992) but in the present study the extracts of tannin rich plants did not suppress fibre degrading enzyme activities, which might be due to different types and levels of tannins present in these plants. 3.5. Effect on degradability of feed IVDMD was significantly suppressed by the addition of extracts of T. chebula, E. officinalis and A. indica, whereas, IVOMD was suppressed (P<0.05) by all the extracts tested (Table 7). The suppression in degradability varied only between 6 and 7% in comparison to control, but all extracts of plants tested in this experiment reduced degradability of

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Table 6 Effect of plant extracts on specific activity (IU/mg protein) of enzymes in the incubation medium Product

Level of extract 0 ml

Carboxymethylcellulase Control A. concinna T. belerica T. chebula E. officinalis A. indica Mean Overall S.E.M. Xylanase Control A. concinna T. belerica T. chebula E. officinalis A. indica Mean Overall S.E.M. Acetylesterase Control A. concinna T. belerica T. chebula E. officinalis A. indica Mean Overall S.E.M.

Mean of products 0.25 ml

0.50 ml

259

260 145 197 245 210 189

242 144 211 190 177 180

259 A

207 AB

191 B

254 183 222 231 215 209

10.8 1529

1536 929 1153 1367 1211 1058

1480 963 1224 1070 1033 1196

1529 A

1209 B

1129 B

428

474 377 317 247 258 219

470 380 320 261 198 219

428 A

315 B

310 B

1515 1141 1302 1322 1258 1196

51 457 a 398 ab 355 ab 312 ab 295 b 288 b

16.1

Different letters (A and B) in a row differ significantly (P<0.05) among levels of all the products irrespective of solvents within a parameter. Different letters (a and b) in a column differ significantly (P<0.05) among the products within a parameter.

feed. It seems that these extracts had some secondary metabolite which might be detrimental to one or the other important rumen microbe. Lila et al. (2003) also observed that sarsaponin reduced IVDMD of hay plus concentrate after 24 h of incubation. A depression in feed degradability by extracts of T. chebula, T. belerica and E. officinalis could be due to phenolic compounds such as tannins, gallic acids, ellagic acids and tannic acids. Tannins have been implicated for their inhibitory effect on feed digestion, microbial population and enzymes activity in many experiments (Bae et al., 1993; Jones et al., 1994; Makkar et al., 1995; McSweeney et al., 2001; Hristov et al., 2003). Reduction in IVDMD and IVOMD by

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Table 7 Effect of plant extracts on the coefficient of in vitro dry matter (DM) and organic matter (OM) degradability of substrate Product

Dry matter degradability (g/g DM) Control A. concinna T. belerica T. chebula E. officinalis A. indica Mean

Level of extract 0 ml

0.25 ml

0.50 ml

0.467

0.459 0.446 0.447 0.423 0.445 0.438

0.456 0.453 0.432 0.401 0.424 0.431

0.467 A

0.443 B

0.432 B

0.462 0.444 0.446 0.420 0.443 0.438

0.460 0.428 0.429 0.404 0.423 0.431

0.443 B

0.429 C

Water 0.457 X 0.460 X

Ethanol 0.444 Y 0.447 XY

Overall S.E.M. 0.002 Organic matter degradability (g/g OM) Control 0.476 A. concinna T. belerica T. chebula E. officinalis A. indica Mean

0.476 A

Overall S.E.M.

0.002

Overall mean of solvents DMD (g/g DM) OMD (g/g OM)

Mean of products

0.460 a 0.453 a 0.449 ab 0.430 b 0.445 ab 0.445 ab

0.466 a 0.449 ab 0.451 ab 0.435 b 0.447 ab 0.449 ab

Methanol 0.440 Y 0.441 Y

Different letters (A–C) in a row differ significantly (P<0.05) among levels of all the products irrespective of solvents within a parameter. Different letters (a and b) in a column differ significantly (P<0.05) among the products within a parameter. Different letters (X and Y) in a row differ significantly (P<0.05) among solvents within a parameter.

extracts of A. indica could be due to different bitter principles which significantly reduced protozoa numbers. Moreover, limonoids and an aqueous extracts of bark of neem stick showed significant antibacterial activity against various Gram-positive and Gram-negative organisms (Siddiqui et al., 1992; Wolinsky et al., 1996).

4. Conclusion The results of this experiment indicate that the plant extracts appear to have a potential to manipulate rumen fermentation favourably. The methanol extract of the seed pulp of T. chebula has antimethanogenic activity and extracts of the pods of A. concinna and A. indica have defaunating properties. Since these extracts also suppressed IVDMD and IVOMD of feed, various levels of the extract should be tested to find out a suitable dose to get maximum inhibition in methane emission without adversely affecting feed degradability.

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