Neem oil modulates rumen fermentation properties in a continuous cultures system

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Animal Feed Science and Technology 149 (2009) 78–88

Neem oil modulates rumen fermentation properties in a continuous cultures system夽 W.Z. Yang a,∗ , J. Laurain b , B.N. Ametaj c a

Research Centre, Agriculture and Agri-Food Canada, Box 3000, Lethbridge, Alberta, Canada T1J 4B1 b National Engineering School of Agronomy and Food Sciences, Nancy, France c Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta, Canada T6G 2P5 Received 3 December 2007; received in revised form 28 April 2008; accepted 9 May 2008

Abstract Neem oil is a commercialized product derived from fruits of the neem tree that has been shown to have antibacterial, antifungal and antiparasitic activities in various animal species. Our objective was to investigate effects of addition of neem oil to a feedlot finishing diet on rumen fermentation, ruminal digestibility and bacterial protein synthesis in a dual effluent continuous culture system. The experiment was designed as a replicated 3 × 3 Latin square with the treatments: control (no neem oil), low (20 g/kg of diet) and high (40 g/kg of diet, dry matter basis) amounts of neem oil. The experimental diet consisted of 872 g/kg of steam-rolled barley grain, 84 g/kg of whole crop barley silage and 44 g/kg of supplement (dry matter basis). Results indicate that concentrations of ruminal volatile fatty acid (VFA), as well as molar proportions of acetate and branch-chained FA, tended (P<0.10) to linearly decrease, whereas the proportion of butyrate tended (P<0.06) to be higher with increasing neem oil supplementation. Ruminal digestibilities of dry matter (0.79, 0.77 and 0.71), neutral detergent fibre (0.65, 0.64 and 0.56), starch (0.89, 0.85 and 0.82) and degradability of crude protein (0.78, 0.78 and 0.67) for control, low and high neem oil supplementation decreased linearly (P<0.01). The amount of bacterial N synthesized (g/day) tended (P=0.08) to increase linearly by 24% or 13%, respectively, for low and high neem oil supplementation compared with control. Bacterial N Abbreviations: ADF, acid detergent fibre; BCFA, branch-chained fatty acid; CC, continuous culture; CP, crude protein; DM, dry matter; aNDF, neutral detergent fibre with amylase and sodium sulfite in the procedure; NOIL, neem oil; OM, organic matter; VFA, volatile fatty acid. 夽 Contribution number: 38707070. ∗ Corresponding author. Tel.: +1 403 317 3427; fax: +1 403 382 3156. E-mail address: [email protected] (W.Z. Yang). 0377-8401/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.anifeedsci.2008.05.004

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efficiency (g N/kg of ruminal truly fermented organic matter) was improved (linear: P<0.01) with neem oil supplementation. Results indicate that supplementation with neem oil supplementation inhibited ruminal microbial activity, possibly due to the bioactive compounds contained in neem oil. However, the lower ruminal digestibility of starch with low neem oil supplementation might be used to alleviate acidosis without having detrimental digestion of fibre and protein in feedlot cattle fed high-grain diets. © 2008 Elsevier B.V. All rights reserved. Keywords: Neem oil; Fermentation; Digestibility; Feedlot diet; Continuous culture

1. Introduction Although use of antibiotics as feed additives has proven beneficial in improving nutrient utilization, feed conversion efficiency and growth rate of livestock (Tedeschi et al., 2003), there is increasing public concern due to potential development of microbial resistance to antibiotics used in human medicine. The growing interest in organic farming, and the need for herbal, naturopathic and homeopathic treatments, has stimulated the search for alternative feed additives. Accordingly, the scientific community and animal feed industry have been actively investigating plant extracts as growth promoters and rumen fermentation modifiers to improve health status and feed efficiency in cattle (Wallace, 2004). Neem (Azadirachta indica) has universally been accepted as a “wonder tree” because of its diverse utility. Various parts of the neem tree have been used as traditional Ayurvedic medicine in India (Brahmachari, 2004). The seed kernel of neem contains bitter compounds such as nimbin, nimbidin and azadurachtin, and is being used in human medicine (Brahmachari, 2004). Moreover, neem cake has been recommended as a feed supplement for ruminants after water processing (Nath et al., 1983; Agrawal et al., 1987), as the water extract from neem seed kernel cake was reported to stimulate in vitro enzyme activity in rumen bacteria related to fibre degradation (Agrawal et al., 1991). Neem oil (NOIL) is a vegetable oil derived from seeds or fruits of the neem tree through pressing or solvent extraction. It contains mostly glycerides (0.80–0.95) and free fatty acids (0.04–0.20), as well as a number of bioactive compounds such as nimbin, nimbidin and nimbinin (Rukmini, 1987; Goss´e et al., 2005), that have been demonstrated to have antibacterial, antifungal, antimalarial, antiparasitic, anti-inflammatory as well as immunomodulatory properties in different animal species (Biswas et al., 2002; Brahmachari, 2004; Goss´e et al., 2005). For example, Rukmini (1987) reported improved feed intake and growth performance in rats fed a diet containing 100 g/kg NOIL compared with the same amount of groundnut oil. In contrast, an in vitro study by Patra et al. (2006) showed that adding water extracts of neem seeds decreased total ruminal volatile fatty acid (VFA) concentrations, the ratio of acetate to propionate and ruminal feed digestibility. Interestingly, Patra et al. (2006) also reported antiprotozoal activity of the water extract of neem kernels. Although several studies have investigated effects of water extracts of neem leaves or seeds on ruminal digestibility, there are no reports of effects of NOIL in ruminants. We hypothesized that supplementating NOIL to a feedlot finishing diet could selectively affect microbial activity in the rumen, and thus modulate rumen fermentation and feed digestibility.

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Therefore, the objective of this study was to investigate effects of NOIL supplementation in a feedlot finishing diet on ruminal fermentation and protein synthesis, as well as nutrient digestibility in a dual-flow continuous culture (CC) system.

2. Materials and methods 2.1. Experimental diet and source of NOIL The experimental diet was a total mixed ration fed to feedlot finishing cattle at the Agriculture and Agri-Food Canada Research Center (Lethbridge, AB, Canada), and was typical of rations used in commercial feedlots in western Canada. It was approximately 872 g/kg steam-rolled barley grain, 84 g/kg whole crop barley silage and 44 g/kg supplement (dry matter [DM] basis) (Table 1), and formulated to meet or exceed current nutrient recommendations for finishing beef cattle (NRC, 1996). Prior to use in the fermenters, the diet was dried at 55 ◦ C for 48 h and ground through a 2.0-mm screen (standard model 4, Arthur Thomas Co., Philadelphia, PA, USA). Table 1 Ingredients and chemical composition of the dieta Ingredientsb (g/kg DM) Whole crop barley silagec Barley grain, steam-rolled Canola oil Canola meal, solvent extracted Calcium carbonate Dried molasses Salt Feedlot premixd Urea Flavoure

83.8 871.5 1.1 16.3 17.6 1.8 5.4 0.8 1.4 0.04

Chemical composition (g/kg DM) Organic matter Neutral detergent fibre (aNDF) Acid detergent fibre Starch Crude protein Fat Ca P

918.6 252.3 109.9 466.0 139.7 24.0 6.2 3.9

a

Diet was prepared from one source for entire experiment and one representative sample was analyzed for determining chemical composition. b All ingredients pelleted excluding steam-rolled barley and silage. c Composition (g/kg DM) was 629 moisture, 123 crude protein, 458 neutral detergent fibre, 283 acid detergent fibre and 48 lignin based on one sample when prepared the diet. d Supplied per head: 137 mg/d Cu, 579 mg/d Zn, 248 mg/d Mn, 6 mg/d I, 1.8 mg/d Co, 2.7 mg/d Se, 600,000 IU/d vitamin A, 56,000 IU/d vitamin D, and 120 IU/d vitamin E. e Anise 420 Power, Canadian Biosystems Inc., Calgary, Alberta, Canada.

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Neem oil used in our experiment is a commercially available product and was purchased from India (Universal Neem Co. Ltd., Tamil Nadu, India). The fatty acid content of NOIL is 178 g/kg palmitic acid, 144 g/kg stearic acid, 513 g/kg oleic acid, 147 g/kg linoleic acid and 16 g/kg arachidic acid (Rukmini, 1987). 2.2. Continuous culture conditions A six-unit dual effluent CC system similar to that described by Hoover et al. (1989) was used with inoculums obtained from two ruminally fistulated steers 1 h after feeding a diet similar with that used in the CC system. Following sampling, mixed rumen contents were immediately homogenized in a Waring blender (Waring Products Division, New Hartford, CT, USA), for 1 min under CO2 , and squeezed through two layers of cheesecloth. The processing aimed to detach bacteria from feed particles for preparation of a ruminal fluid containing mixed rumen bacteria. One liter of the filtrate plus 200 ml of warmed buffer solution were added to each of the six fermenters. Fermenters were manually provided with 60 g/d of diet (i.e., 57 g DM) in two equal portions. Temperature was maintained constant at 39 ± 0.5 ◦ C. The pH of the fermenters ranged between 5.9 and 6.3 by continuous infusion of a buffer solution as described by Slyter et al. (1966), and monitored by a pH meter (Model 4505, Chemtrix, Inc., Hillsboro, OR, USA). The infusion of buffer solution was constant per fermenter per time regardless of treatment. Therefore, the variation of fermenter pH among treatments would reflect that of acid production. Infusion of buffer solution and flows of filtrated liquid were set equally across fermenters to maintain a solid and liquid dilution rates at approximately 0.045 h−1 and 0.100 h−1 , respectively, using an infusion pump (Model MTP® , #1001, Medical Technology Products, Inc. Huntington Station, NY, USA). Fermenters also were constantly purged with CO2 (20 ml/min) to preserve anaerobiosis. Bacteria in the fermenters were labeled using 15 N by mixing ammonium sulfate enriched with 15 N [(15 NH4 )2 SO4 , 106 atoms 15 N per 1000 total N atoms; Isotec, Miamisburg, OH, USA] into the infused buffer solution. The daily amount of 15 N provided into each fermenter was about 1.5 mg (i.e., 60 mg 15 N (15 NH4 )2 SO4 ). 2.3. Experimental design and sampling The experiment was a replicated 3 × 3 Latin square with three 8 d periods including 6 d of stabilization followed by 2 d of sample collection. Treatments were control (no NOIL), low (20 g/kg of diet) and high (40 g/kg of diet, DM basis) amounts of NOIL. The NOIL was added and thoroughly mixed with the ground samples. During the last 2 d of each experimental period, the collection vessels were immersed in a cold (4 ◦ C) water bath and 1 ml of 12.3 M formaldehyde per 100 ml of effluent was added to the plastic jugs used to collect effluents to inhibit microbial activity. During each day of the sampling period, solids and liquid effluent were combined and homogenized, and a 250 ml sample was centrifuged at 27,000 × g for 40 min at 4 ◦ C to determine effluent DM (i.e., indigestible feed plus microbial materials); a second 700 ml sample was removed and composited by the fermenter during each period. The composite samples were centrifuged at 27,000 × g for 40 min at 4 ◦ C and sediments were freeze-dried, ground through a 1 mm screen (standard model 4 as described previously) and stored for later analyses. Fresh filtrates were sampled directly from filtrate

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lines at 0 (before feed addition), 1, 3, 5 and 7 h after the morning feed provision. Five milliliters of filtrate were preserved by adding 1 ml of 3 M HPO3 to determine VFA, and 5 ml of filtrate were preserved by adding 1 ml of 0.18 M H2 SO4 to determine ammonia N. The samples were subsequently stored at −20 ◦ C until analyses. The pH of the fermenter solution was manually recorded every hour after morning feed provision. 2.4. Bacterial collection Bacterial pellets were isolated from the contents of the fermenter on the last day of each period. The contents were blended using a Waring blender (Waring Products Division) for 1 min and squeezed through four layers of cheesecloth. The filtrate was centrifuged at 800 × g for 15 min at 4 ◦ C and then the supernatant fraction was centrifuged at 27,000 × g for 30 min at 4 ◦ C to obtain a bacterial pellet. The pellet was freeze-dried and ground using a ball mill (Mixer Mill MM2000, Retsch, Haan, Germany) to a fine powder for determination of N and 15 N enrichment. 2.5. Chemical analyses Analytical DM content of the samples was determined by drying at 135 ◦ C for 2 h (ID 930.15; AOAC, 1990), and organic matter (OM) was calculated as the difference between DM and ash content. The latter was determined by combustion at 550 ◦ C for 5 h. Methods described by Van Soest et al. (1991) were used in the analyses of neutral detergent fibre (aNDF) and acid detergent fibre (ADF) with amylase and sodium sulfite used in the NDF procedure. Contents of aNDF or ADF are expressed with ash. Starch was determined by enzymatic hydrolysis of ␣-linked glucose polymers as described by Rode et al. (1999). Fat was determined by extraction with ether using a Soxlec system HT6 apparatus (Tecator, Fisher Scientific, Montreal, QC, Canada) according to the Method 920.39 (AOAC, 2002). Effluent VFA were separated and quantified by gas chromatography (Hewlett Packard 5890; Agilent Technologies, Mississauga, ON, Canada) using a 30 m (0.32 mm i.d.) fused silica column (Nukol column; Sigma–Aldrich Canada Ltd., Oakville, ON, Canada). Ammonia N content of effluent samples was determined according to the technique of Weatherburn (1967). Content of N in the samples and enrichment of 15 N in the effluent DM and the bacteria isolated from the fermenter contents were determined by flash combustion (Model 1500; Carlo Erba Instruments, Milan, Italy) with isotope ratio mass spectrometry (VG Isotech, Middlewich, England). 2.6. Calculations and statistical analyses Total N input was calculated as the sum of N in the feed provided plus the N provided in the 15 N infusate which was added into the buffer solution to label the microbial portion. The flow of protein N was determined by measuring N in the effluent particulate matter retained by centrifugation (i.e., 27,000 × g as previous). This fraction of N was considered to be true protein and include microbial and non-digested feed N. Bacterial production was estimated from the ratio of 15 N flow in the effluent to 15 N enrichment of the bacterial pellet. Thus, non-digested feed N was the difference between protein N and bacterial N.

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The true DM digestibility coefficients were calculated as (DM provided − [effluent DM − bacterial DM]) DM provided where, DM provided = grams feed/24 h × feed DM content and effluent DM = grams effluent/24 h × effluent DM content. True digestibility coefficients of OM or CP were calculated using a similar approach. For each experimental period, means for individual fermenters were calculated for all variables. Data were analyzed using the mixed model procedure of SAS (2006). The model included treatment as a fixed effect with period and fermenter as random effects. The restricted maximum likelihood method was used to estimate the variance components and the Kenwardroger method was used to approximate the degrees of freedom. Variables that were repeated in time were analyzed using the same mixed model but with sampling time added to the model, and a repeated statement included. Effects of the factors were declared significant if P<0.05 and trends were accepted if P<0.10.

3. Results 3.1. Fermentation characteristics The highest pH values were observed just before feed provision, while the lowest pH values were about 5 h after feed addition. However, diurnal fluctuations of fermenter pH did not differ among treatments, and no treatment by time interactions occurred. The mean, the maximum, and the minimum pH values of the fermenters ranged from 6.02 to 6.11, 6.35 to 6.37, and 5.85 to 5.92, respectively (Table 2). Total VFA concentrations, and molar proportions of acetate and branch-chained FA (BCFA) tended (P<0.10) to linearly Table 2 Effects of neem oil (NOIL) supplementation on fermentation characterization in continuous culture NOIL (g/kg DM) 0 pH Mean Minimum Maximum Volatile fatty acids (VFA) Total (mmol/l) Acetate (mmol/mol) Propionate (mmol/mol) Butyrate (mmol/mol) BCFAb (mmol/mol) Acetate:propionate NH3 N (mmol/l) a b

S.E. 20

Linear

40

6.02 5.90 6.35

6.07 5.85 6.35

6.11 5.92 6.37

0.129 0.138 0.131

NSa NS NS

84.3 451 436 63 28.5 1.10 0.83

83.6 437 442 69 26.3 1.05 1.14

78.5 400 466 95 21.4 0.87 0.78

3.02 22.3 32.3 11.3 2.41 0.128 0.308

0.10 0.09 NS 0.06 0.10 NS NS

NS: not significant (P>0.10). Branch-chained FA (isobutyrate + isovalerate).

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Table 3 Effects of neem oil (NOIL) supplementation on digestibility in continuous culture NOIL (g/kg DM)

DM (apparent) DM (true)a OM (apparent) OM (true)a aNDF Starch

0

20

40

0.57 0.79 0.60 0.79 0.65 0.89

0.49 0.77 0.53 0.76 0.64 0.85

0.43 0.71 0.47 0.70 0.56 0.82

S.E.

Linear

0.022 0.015 0.021 0.014 0.017 0.014

0.001 0.006 0.001 0.010 0.002 0.004

DM: dry matter; OM: organic matter; aNDF: neutral detergent fibre. a Corrected for microbial portion.

decrease, whereas the proportion of butyrate tended (P<0.06) to be higher with increasing NOIL. 3.2. Digestibility Apparent and true digestibilities of DM and OM, and all of its measured components, linearly decreased (P<0.03) with increased NOIL supplementation (Table 3). The actual decrease in the true digestibilities of DM and OM, as well as in the digestibility of aNDF, was marginal (i.e., 2.2–4.3%) when NOIL was 20 g/kg of diet. In contrast, the reduction was about 8% for the true digestibilities of DM and OM, 13% for the digestibility of aNDF and 8% for starch when NOIL was 40 g/kg of diet. 3.3. Nitrogen metabolism Flows of total N were similar among treatments because it was controlled (Table 4). However, flows of true protein N increased linearly (P<0.01) with NOIL supplementation. The degradability of CP was about 14% lower for the high NOIL treatment versus the control. The amount of bacterial N synthesized (g/day) was higher with low NOIL or tended (P<0.09) to be higher with high NOIL than for the control. Bacterial N efficiency (g N/kg of ruminal truly fermented OM) improved linearly (P<0.01) with NOIL supplementation. In addition, flows of residual N (i.e., N was not included in the true protein or NH3 N portions) declined linearly with increased NOIL level. 4. Discussion Supplementing NOIL at two concentrations to a CC system of ruminal fluid from feedlot finishing steers had no effect on ruminal pH, which is consistent with little difference in the concentrations of VFA (Table 2). Beauchemin et al. (2003) reported that low ruminal pH was due to accumulation of VFA rather than lactate in steers fed a feedlot finishing diet. Similarly, Yang et al. (2004) showed that concentrations of ruminal lactate (1.24–1.65 mmol/l) in a CC system was negligible relative to concentrations of total ruminal

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Table 4 Effects of neem oil (NOIL) supplementation on N digestion and microbial CP synthesis in continuous culture NOIL (g/kg DM) 0 Flow of Na (g/day) Total N True protein N Non-digested feed N Ammonia N Residual N Bacterial N g/day g N/kg RFOMc Degradability of CP Trued a

20

S.E.

Linear

40

1.59 1.21 0.28 0.04 0.35

1.59 1.43 0.28 0.06 0.11

1.60 1.47 0.42 0.04 0.09

0.019 0.057 0.032 0.002 0.054

NSb 0.004 0.007 NS 0.001

0.93 22.4

1.15 29.5

1.05 29.1

0.062 1.52

0.083 0.005

0.78

0.78

0.67

0.025

0.007

feed + 15 N

added in the buffer solution; true protein N: measured in the solids Total N: N provided with separated by centrifugation of effluents at 27,000 × g for 30 min at 4 ◦ C; non-digested feed N: true protein N – microbial N; non-determined N: total N – (true protein N + ammonia N). b NS: not significant (P>0.10). c Organic matter truly fermented in the fermenter. d Corrected for microbial N.

VFA (135 mmol/l). The VFA profile with high NOIL, marked by a decrease in the proportion of acetate, and an increase in the proportion of butyrate without change in the proportion of propionate, suggests that the high dose of NOIL favored butyrate-producing bacteria and inhibited acetate-producing bacteria. Gram-positive bacteria are generally acetateand butyrate-producing, while Gram-negative bacteria are generally propionate-producing (Stewart, 1991). The trend to a linear reduction of VFA concentration with increasing of NOIL in the digesta is consistent with the linear decrease in the ruminal digestibilities of DM and OM. Furthermore, the linear reduction of the acetate proportion is also consistent with the linear depression of fibre digestion. These results agree with Patra et al. (2006), who reported a reduction of in vitro ruminal VFA concentration and ruminal digestibility of DM with supplementation of water extracts of neem seeds. The decrease in the concentration of total VFA probably reflects the lower fermentability of the diet, potentially related to presence of the antibacterial compounds in NOIL (Biswas et al., 2002) and phenolics (Goss´e et al., 2005). The similar ratios of acetate to propionate among treatments in our study may contrast with Patra et al. (2006), who reported a decrease in the acetate to propionate ratio due to addition of water extracts of neem seeds, possibly due to a decrease in counts of ruminal protozoa. This suggestion was supported by Mondal and Garg (2002) suggesting no effect of water washed neem kernel cake on the number of ruminal protozoa because of the solubility of its active compounds in water. Similarly, Koenig et al. (2007) showed a decrease of up to 88% in counts of protozoa 2 h after feeding sheep with Enterolobium cyclocarpum, a tropical multipurpose tree that contains saponins. A lower number of protozoa in the rumen have been reported to be associated with an increase in the amount of propionate and a

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decrease in the ratio of acetate to propionate (Hess et al., 2003). The discrepancies in the VFA profiles between our study and Patra et al. (2006) might be related to lack of protozoa in our CC system. The lower ruminal digestibility of OM components with NOIL may indicate inhibitory activity of NOIL on ruminal bacteria. Polyunsaturated fatty acids in ruminant diets are detrimental to ruminal cellulolytic microbial activity (Jenkins, 1993), and the reduction in ruminal digestibility of aNDF with NOIL in our present study could be partially due to the presence of these fatty acids in NOIL. However, the negative effect of NOIL on starch digestion might be related to the inhibitory activity of secondary metabolites in NOIL on the bacterial activity rather than to the possible presence of fatty acids per se (Zinn, 1988; Bock et al., 1991). Lowering digestion of ruminal starch with NOIL supplementation could be beneficial in treating acute ruminal acidosis in cattle fed high-grain diets. Lower ruminal degradability of CP with the high NOIL supplementation was consistent with the higher flows of non-digested feed N, as well as with a linear reduction in BCFA concentrations. Ruminal BCFA derived from fermentation of branched-chain amino acids (Russell and Sniffen, 1984). The present results are consistent with Anandan et al. (1999) that feeding neem kernel meal to goats depressed digestibility of CP compared to a diet with peanut meal supplementation. The reduction in digestibility of CP has been attributed to the negative effects of the phenolic compounds through formation of indigestible complexes with proteins (Singh and Bhat, 2001). In contrast, Patra et al. (2003) reported no differences in digestibility of CP in the total digestive tract of goats between diets with soybean meal or leaf meal mixture that included neem leaf. The lack of effect on digestibility of CP was probably due to moderate levels of tannin in the diet (Barry and McNabb, 1999), as suggested by Patra et al. (2003).

5. Conclusions Although increasing supplementation of a feedlot finishing diet with NOIL from 0 to 40 g/kg of diet had a minimal effect on concentrations and patterns of VFA accumulation, ruminal digestibilities of DM and OM linearly decreased. This reduction in the ruminal digestibility of OM with NOIL supplementation is due to depression of digestibilities of fibre and starch, as well as to a decrease in the degradability of CP. Results suggest that supplementation with NOIL inhibited bacterial activity, which could be beneficial in treating acute acidosis in feedlot cattle fed high-grain diets.

References Agrawal, N., Gark, A.K., Nath, K., 1987. The use of water washed neem (Azadirachta indica) seed kernel cake in the feeding of buffalo calves. J. Agric. Sci. (Camb.) 108, 497–499. Agrawal, N., Kewalramani, N., Kamra, D.N., Agrawal, D.K., Nath, K., 1991. Effects of water extracts of neem (Azadirachta indica) cake on the activity of hydrolytic enzymes of mixed rumen bacteria from buffalo. J. Sci. Food Agric. 57, 147–150. Anandan, S., Sastry, V.R.B., Katiyar, R.C., Agarwal, D.K., 1999. Processed neem kernel meal as a substitute for peanut meal protein in growing goat diets. Small Rumin. Res. 32, 125–128.

W.Z. Yang et al. / Animal Feed Science and Technology 149 (2009) 78–88

87

Association of Official Analytical, 1990. Official Methods of Analysis„ 15th ed. AOAC, Arlington, VA, USA. Association of Official Analytical, 2002. Official Methods of Analysis, Revision 1, vol. 1, 17th ed. AOAC, Arlington, VA, USA. Barry, T.N., McNabb, W.C., 1999. The implications of condensed tannins on the nutritive value of temperate forages fed to ruminants. Br. J. Nutr. 81, 263–272. Beauchemin, K.A., Yang, W.Z., Morgavi, D.P., Ghorbani, G.R., Kautz, B., 2003. Effects of bacterial direct-fed microbials and yeast on site and extent of digestion, blood chemistry, and subclinical ruminal acidosis in feedlot cattle. J. Anim. Sci. 81, 1628–1640. Biswas, K., Chattopadhyay, I., Banerjit, R.K., Bandyopadhyay, U., 2002. Biological activities and medicinal properties of neem (Azadirachta indica). Curr. Sci. 82, 1336–1344. Bock, B.J., Harmon, D.L., Brandt Jr., R.T., Schneider, J.E., 1991. Fat source and calcium level effects on finishing steer performance, digestion, and metabolism. J. Anim. Sci. 69, 2211–2224. Brahmachari, G., 2004. Neem—an omnipotent plant: a retrospection. Chem. Biol. Chem. 5, 408–421. Goss´e, B., Amissa, A.A., Adj´e, F.A., Niamk´e, F.B., Ollivier, D., Ito, Y., 2005. Analysis of components of neem (Azadirachta indica) oil by diverse chromatographis techniques. J. Liquid Chromatogr. Relat. Technol. 28, 2225–2233. Hess, H.D., Kreuzer, M., Diaz, T.E., Lascano, C.E., Carulla, J.E., Solvia, C.R., 2003. Saponin rich tropical fruits affect fermentation and methanogenesis in fated and defaunated fluid. Anim. Feed Sci. Technol. 109, 79–94. Hoover, W.H., Miller, T.K., Stokes, S.R., Thayne, W.V., 1989. Effects of fish meals on ruminal bacterial fermentation in continuous culture. J. Dairy Sci. 72, 2991–2998. Jenkins, T.C., 1993. Lipid metabolism in the rumen. Symposium: advances in ruminant lipid metabolism. J. Dairy Sci. 76, 3851–3863. Koenig, K.M., Ivan, M., Teferedegne, B.T., Morgavi, D.P., Rode, L.M., Ibrahim, I.M., 2007. Effect of dietary Enterolobium cyclocarpum on microbial protein flow and nutrient digestibility in sheep maintained fauna-free, with total mixed fauna or with Entodinium caudatum monofauna. Br. J. Nutr. 98, 504–516. Mondal, D.P., Garg, A.K., 2002. Effect of feeding untreated and water washed neem (Azadirachta indica a juss) seed kernel cake on rumen enzyme profile and fermentation pattern in crossbred calves. Anim. Nutr. Feed Technol. 2, 27–37. Nath, K., Rajagapol, S., Garg, A.K., 1983. Water washed neem (Azadirachta indica juss) seed kernel cake as a cattle feed. J. Agric. Sci. (Camb.) 101, 323–326. NRC, 1996. Nutrient Requirements of Beef Cattle, 7th rev. ed. Natl. Acad. Press, Washington, DC, USA. 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., 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. Rode, L.M., Yang, W.Z., Beauchemin, K.A., 1999. Fibrolytic enzyme supplements for dairy cows in early lactation. J. Dairy Sci. 82, 2121–2126. Rukmini, C., 1987. Chemical and nutritional evaluation of neem oil. Food Chem. 26, 119–124. Russell, J.B., Sniffen, C.J., 1984. Effects of varbon-4 and carbon-5 volatile fatty acids on growth of mixed rumen bacteria in vitro. J. Dairy Sci. 67, 987–994. S.A.S. Institute Inc., 2006. SAS OnlineDoc® 9.1.3. SAS Institute Inc., Cary, NC, USA. Singh, B., Bhat, T.K., 2001. Tannins revisited-changing perception of their effects on animal system. Anim. Nutr. Feed Technol. 1, 3–18. Slyter, L.L., Bryant, M.P., Wolin, M.J., 1966. Effect of pH on population and fermentation in a continuously cultured rumen ecosystem. Appl. Microbiol. 14, 573–578. Stewart, C.S., 1991. The rumen bacteria. In: Jouany, J.P. (Ed.), Rumen Microbial Metabolism and Ruminant Digestion. INRA Editions, Paris, France, pp. 15–26. Tedeschi, L.O., Fox, D.G., Tylutki, T.P., 2003. Potential environmental benefits of ionophores in ruminant diets. J. Environ. Qual. 32, 1591–1602. Van Soest, P.J., Robertson, J.B., Lewis, B.A., 1991. Methods for dietary fiber, neutral detergent fiber and non-starch polysaccharides in relation to animal nutrition. J. Dairy Sci. 74, 3583–3597. Wallace, R.J., 2004. Antimicrobial properties of plant secondary metabolites. Proc. Nutr. Soc. 63, 621–629.

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Weatherburn, M.W., 1967. Phenol–hypochlorite reaction for determination of ammonia. Anal. Chem. 39, 971–974. Yang, W.Z., Beauchemin, K.A., Vedres, D.D., Ghorbani, G.R., Colombatto, D., Morgavi, D.P., 2004. Effects of direct-fed microbial supplementation on fermentation, digestibility, and bacterial protein synthesis in continuous culture. Anim. Feed Sci. Technol. 114, 179–193. Zinn, R.A., 1988. Comparative feeding value of supplemental fat in finishing diets for feedlot steers supplemented with and without monensin. J. Anim. Sci. 66, 213–227.