Effects of ruminal inoculations with tannin-tolerant bacteria on fibre and nitrogen digestibility of lambs fed a high condensed tannin diet

Animal Feed Science and Technology 81 (1999) 69±80

Effects of ruminal inoculations with tannin-tolerant bacteria on fibre and nitrogen digestibility of lambs fed a high condensed tannin diet D.O. Molina, A.N. Pell*, D.E. Hogue Department of Animal Science, Cornell University, 329 Morrison Hall, Ithaca, NY 14853, USA Received 8 December 1998; received in revised form 22 April 1999; accepted 19 May 1999

Abstract The goal of this study was to evaluate the effects of dosing unadapted lambs with tannin-tolerant bacteria to improve the digestibility of a high condensed tannin (CT) diet. During the initial phase (metabolism study), a diet containing 30% peanut skins was fed to two groups of Suffolk  FinnDorset ram lambs that were about three months old and weighed an average of 24.2  1.4 kg. All animals received 150 ml of a culture (A600 of 1.0) of a Gram positive rod (a close relative of Eubacterium cellulosolvens) that was able to tolerate 0.5 g/l of purified CT from Desmodium ovalifolium. The control group (7 animals) was inoculated with autoclaved bacteria. The treatment group (6 animals) was inoculated with actively growing bacteria. Inoculations were made daily during a three-week period. Dry matter intake (DMI) was 55.4 and 64.9 g/kg0.75/day for the control and treatment group, respectively, (P = 0.13). Digestibility of DM, crude protein (CP) and neutral detergent fibre (NDF) was similar between treatments. Crude protein intake (P = 0.10) and CP retention (P = 0.07) were higher for animals inoculated with live bacteria. The CP retention/CP intake ratio was also higher for animals inoculated with the live bacteria (P = 0.07). To investigate carry-over effects on animal performance due to the bacterial inoculations after the metabolism study, the animals were kept in metabolism cages, but they received no supplemental bacteria. During a subsequent two-week period, the animals continued to receive the high CT diet. Dry matter and CP intake, as well as the feed : gain ratio, were similar between the groups of animals. Finally, for a second two-week period, the animals were fed a low CT (normal) diet without peanut skins. Dry matter intake was 92.9 and 88.6 g/kg0.75/day for the control and treatment groups, respectively (P = 0.14). Crude protein intake and feed : gain ratio were similar between the two groups of animals. # 1999 Elsevier Science B.V. All rights reserved. Keywords: Tannins; Peanut skins; Sheep; Inoculation *

Corresponding author. Tel.: +1-607-255-2876; fax: +1-607-255-9829 E-mail address: [email protected] (A.N. Pell) 0377-8401/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 7 - 8 4 0 1 ( 9 9 ) 0 0 0 8 3 - 8

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1. Introduction Animals use several mechanisms to counteract the negative effect of ingested tannins on digestibility. Ruminal microbes that are resistant to high levels of tannins, either singly or in a consortium, may constitute a unique part of this response (Miller et al., 1995). However, the ability of the rumen microbial population to withstand high concentrations of plant phenolics has received minor consideration (Lowry et al., 1996). Recent studies have indicated the presence of bacteria able to tolerate elevated levels of condensed tannins (CT) in the rumens of animals fed forages high in tannins (Brooker et al., 1994; Nelson et al., 1995; Miller et al., 1995). Introduction of tannin-tolerant microbes into the rumens of animals through one or more inoculations of cultures of these bacteria may be beneficial in developing systems to improve the productivity of ruminants eating high CT forages or diets, especially in the tropics. An anaerobic Gram-positive curved rod has been recently isolated in our laboratory from the ruminal contents of a Rocky Mountain elk (Cervus elaphus nelsoni) from Oregon (USA) (Nelson et al., 1998). This isolate grew in a medium that contained up to 20 mM of pyrogallol, phloroglucinol, p-coumaric acid, ferulic acid or gallic acid. The bacterium tolerated concentrations of purified CT from Desmodium ovalifolium and tannic acid as high as 0.5 and 2 g/l, respectively. When grown without tannins in pure cultures, the average cell size was 3.2 mm long by 1.0 mm wide (Nelson et al., 1998). The 16S rRNA sequence of this tannin-tolerant bacterium was not identical to any previously cultured and described microorganism, but it is closely related to Eubacterium cellulosolvens, a Gram-positive cellulolytic rod. Based on the phylogenetic reclassification proposed by Collins et al. (1994), this isolate is a member of the phenotypically diverse subcluster XIVa and, not surprisingly, its closest neighbour is cellulolytic (Nelson et al., 1998). In this study, we evaluated the potential of this novel tannin-tolerant bacterium to improve digestibility when inoculated into the rumens of sheep fed a high-CT diet. In addition, the ability of this isolate to improve N digestion was examined. In order to establish whether the inoculations altered animal performance, carry-over effects with, and without, dietary tannins were evaluated after daily inoculations had been discontinued. 2. Materials and methods 2.1. Experimental design The experiment consisted of two treatments: inoculation with dead (autoclaved) bacteria and inoculation with live (actively growing) bacteria into the rumens of sheep. Animals were housed in individual metabolism cages grouped by treatment, with 4 m separating the two groups. The metabolism cages of the two treatment groups were oriented towards opposite walls to reduce the likelihood of aerosol transmission. In view of limitations imposed by the availability of metabolism cages, the experiment was conducted in two separate experimental periods.

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Fig. 1. Schematic representation of the experimental periods. During the metabolism study, the animals received daily inoculations and were fed a high condensed tannin (CT) diet. After the metabolism study, the animals received no bacterial inoculations. For the subsequent two weeks the high-CT diet was offered. For the final two weeks, a low-tannin (normal) diet was fed to the lambs.

During each period, the animals had a two-week adaptation phase to the high CT diet before a five-day collection phase (metabolism study). During the metabolism study, a 30% peanut skin (high-CT) diet was fed to the animals, and they received bacterial inoculations according to the treatment to which they were allocated. After the metabolism study (Fig. 1), the animals were kept in the metabolism cages, but they received no supplemental bacteria. For two more weeks, the animals continued to receive the high-CT diet. Then, for the final two weeks, the animals were fed a low-CT (normal) diet, without peanut skins. During the study, two sheep on the live bacteria treatment were removed from the experiment, because their feed intake was drastically reduced due to respiratory problems. The first animal was removed during the metabolism study and the second was eliminated after the metabolism study, when the animals received no supplemental bacteria. 2.2. Metabolism study An N digestion and balance study was conducted using 13 Suffolk  Finn-Dorset ram lambs that were approximately three months old with an initial BW of 24.2  1.4 kg. Animals were randomly assigned to one of the two experimental treatments. Animals were fed twice daily at around 0800 and 1500 h. During the first two-week adaptation phase, the feed offered was adjusted daily based on the previous day's consumption, allowing orts (as fed basis) of 10±15%. During the third week, the amount of feed offered was fixed at 90% of the average consumption for each individual animal, based on the intake records of the two previous weeks. From Day 15 to 20, feed offered and refused was measured and sampled. The faeces and urine were collected daily for the last five days and aliquots (10%) were taken. Volatilization of ammonia-N was prevented by adding 10 ml of 50% HCl to the urine collection vessels. The samples were composited by animal across the five-day collection period, appropriately mixed and stored at ÿ208C prior to chemical analysis.

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2.3. In vitro cultured rumen inoculations The bacterial inoculum was prepared daily from lateÿlogarithmic (A600 of 1.0) cultures grown anaerobically in rumen fluid medium (Bryant and Burkey, 1953) containing 0.3% cellobiose. After autoclaving and while the medium was flushed with CO2, tannic acid (powder) was added to a final concentration of 1 g/l. Once the tannic acid was completely dissolved, 10 ml of the tannic acid-containing medium were transferred to a sterilised Balch tube (Bellco, Vineland, NJ), purged with oxygen-free CO2, and sealed with a butyl rubber-stopper. The medium was then inoculated with 50 ml inoculum/l of medium using overnight cultures grown anaerobically in rumen fluid medium. A 10-ml sample of the inoculated medium was transferred to a sterilised Balch tube (Bellco, Vineland, NJ), purged with oxygen-free CO2, and sealed with a butyl rubber-stopper. Optical density (A600) was monitored by comparing the blank tube with tannic acid containing medium against the tube with inoculated medium in a Spectronic 601 (Milton Roy, Rochester, NY). The inoculated medium was incubated anaerobically at 398C for 6±7 h. Upon reaching the appropriate optical density, the medium was poured from the flask into airtight plastic containers flushed with CO2, before being transported from the laboratory to the animal facility. The sheep were inoculated with the live cultures within 10 min of pouring the medium. For the dead bacteria treatment, the culture was grown to the desired optical density and autoclaved before inoculation. Animals were inoculated using a drenching gun with 150 ml of culture/animal/day prior to the afternoon feeding. To prevent unwanted cross-contamination, animals on the dead bacteria treatment were always drenched before those on the live bacteria treatment during the whole three week experimental period. After each use, the drenching gun was taken apart for cleaning, washed with detergent and warm water, and completely airdried. 2.4. Post-inoculation periods At the end of the metabolism study, the animals that then weighed 25.1  2.4 kg, were left in the metabolism crates, but no inoculations were performed. However, all the experimental activities were performed first on the lambs on the dead-bacteria treatment to prevent cross-contamination. For two weeks, the animals continued to receive the experimental diet that contained 7% CT. Then, for a second two-week period, the animals were offered a low-CT diet. During the post-inoculation periods, feed was offered ad libitum, allowing orts of 10±15% of the amount offered. Individual body weights were measured at the beginning, and at the end, of each two-week period. 2.5. Experimental diets Fresh peanut skins (IFN 403631), relatively free of foreign material, were obtained from a blanching plant in Culpepper, VA. Normal and high-CT diets, and their chemical compositions, are shown in Table 1. In the normal diet, soyabean hulls replaced peanut skins and the diets were formulated to be isonitrogenous. Crude protein in the diet was

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Table 1 Ration components and chemical composition of the experimental diets fed to sheep during the metabolism study and the post-inoculation periods Item

Normal diet (%)

High-CTa diet (%)

Dietary ingredientsb Peanut skins Soyabean hulls Barley Soyabean meal Vegetable oil Calcium carbonate Trace minerals

0 60.3 28.6 5.9 2.3 2.3 0.7

29.5 30.4 28.9 5.9 2.3 2.3 0.7

Measured analysesc Dry matter Crude protein Neutral detergent fibre (NDF) Acid detergent fibre (ADF) Tannins (proanthocyanidins)

88.5 17.2 65.0 27.0 0.0

87.5 17.7 58.6 21.9 7.1

a b c

Condensed tannin. Ingredient percentages expressed on DM basis. Chemical analysis values, except DM, expressed on DM basis.

formulated to provide protein levels slightly below the requirement (NRC, 1985). This was to ensure that the negative effects of the tannins would not be counteracted by high levels of protein (McBrayer et al., 1983). 2.6. Sample preparation and analysis Samples of feed, orts and faeces (10% of the total) were dried in a forced-air oven at 608C to a constant weight, ground through a 1-mm screen in a Wiley mill (Model 4, Arthur H. Thomas, Philadelphia, PA). Frozen urine was thawed and shaken prior to taking samples for analysis. Dry matter (measured at 1058C), and organic matter (OM) were determined on the same sample (Goering and Van Soest, 1970). Neutral detergent fibre (NDF) and acid detergent fibre (ADF) were determined non-sequentially by the methods of Van Soest et al. (1991). The permanganate±lignin procedure was used (Goering and Van Soest, 1970). Crude protein (CP = N  6.25) was measured by the modified macro-Kjeldahl procedure (AOAC, 1990), using boric acid in the distillation process. The protein fractions of the diet were determined as described by Licitra et al. (1996). In vitro digestibility of the diets was estimated using the computerised gas monitoring system of Pell and Schofield (1993). Estimates of the DM digestibility were calculated from the measurement of DM disappearance after a 30-h fermentation. To determine the effect of the CT on the digestibility of the diets, a 100-mg sample of the diet was incubated in triplicate with, and without, the addition of polyethylene glycol (PEG) 8000 (Sigma, St Louis, MO), using 0.6 g PEG/0.5 g of sample (Makkar et al., 1995a).

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2.7. Isolation of condensed tannins and preparation of standards Purification of peanut skin tannins was performed using the method of Asquith and Butler (1985), as modified by Hagerman and Butler (1994). The fluffy tannin power was stored in a desiccator at 48C in the dark. The standard curve for the acid-butanol assay (Porter et al., 1986) was constructed as described by Giner-Chavez et al. (1997). Extraction of crude-tannin extracts was performed as described by Giner-Chavez et al. (1997) except that 2 g of fresh sample, instead of lyophilised material, was used for the extraction. Soluble condensed tannins were measured in crude plant extracts in triplicate by the acid butanol assay (Porter et al., 1986), using an internal standard generated for peanut skin tannins (Giner-Chavez et al., 1997). 2.8. Hydrolysable tannin determination The level of hydrolysable tannins was measured using the assay described by Inoue and Hagerman (1988) for gallotannin determination. 2.9. Statistical analysis All the analyses and statistical computations were conducted using the general linear model procedure of the SAS statistical analysis software program, version 6.03 (SAS Institute, 1985). Differences in DM and CP intake and digestibility of DM, CP and NDF between treatments were determined by a two-way analysis of variance, with treatment and period as source of variation in the model: Yij ˆ  ‡ Ti ‡ Pj ‡ …TP†ij ‡ Eij Yij is the dependent variable in the ith treatment in the jth period;  the common mean; Ti the effect of the ith treatment, i = 1 and 2; Pj the effect of the jth period, j = 1 and 2; (TP)ij the interaction of the ith treatment in the jth period, and Eij the random residual. 3. Results 3.1. Composition of peanut skins The chemical composition of the peanut skins used in the preparation of the high CT diet is shown in Table 2. Crude protein, NDF, and ADF contents of peanut skins were similar to values previously reported (Atuahene et al., 1989; West et al., 1993). Condensed-tannin content, however, was slightly higher. Purification of CT from peanut skins resulted in a yield of 3% of the initial material on dry matter basis. Hydrolyzable tannins, or gallic acid equivalents, as measured by the method of Inoue and Hagerman (1988), were absent from the peanut skin extracts. To evaluate whether tannins affected diet digestibility, samples of both, the normal and high-CT diets were incubated with, and without, PEG. Digestibility of the high-CT diet without PEG (63.6%) was significantly (P <0.05) lower than when PEG was included

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Table 2 Chemical composition of peanut skins Component

%a

Dry matter Ash Crude protein Neutral detergent fibre (NDF) Acid detergent fibre (ADF) Lignin Proanthocyanidins

92.0 2.7 20.5 39.6 20.8 7.6 24.0

Nitrogen fractions True protein Soluble protein CP-NDF CP-ADF

17.8 3.8 6.1 2.1

a

Chemical analysis values, except DM, expressed on DM basis.

(70.7%). Although the digestibility of the normal diet was 75.1% without PEG and 78.1% with PEG, this difference was not significant. 3.2. Metabolism study Results of the metabolism study are shown in Table 3. There was no significant difference in animal weights between the treatments at the beginning of the experiment. Although DMI was numerically higher for the animals on the live-bacteria treatment Table 3 Apparent digestibility and crude protein balance of growing sheep fed diets containing peanut skins, inoculated with dead and live tannin-tolerant bacteria, during the N balance (collection) period Item

Treatment

SEM

p

dead

alive

Number of animals Average initial weight (kg) Dry-matter intake (g/kg0.75/day)

7 23.9 55.4

6 23.2 64.9

0.58 4.10

0.43 0.13

Apparent digestibility (%) Dry matter Crude protein (CP) NDF

58.8 48.8 56.8

59.6 51.1 57.7

1.47 2.18 2.13

0.70 0.48 0.77

CP balance(g/kg/day) CP intake Faecal CP Urinary CP CP retention CP retention/CP intake (%)

4.4 2.3 0.9 1.2 26.4

5.2 2.5 1.0 1.7 31.9

0.31 0.18 0.11 0.17 1.85

0.10 0.34 0.71 0.07 0.07

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(686.1 vs. 598.8 g/day for the dead-bacteria treatment), the difference was not significant (P = 0.13). Coefficients for apparent digestibility of DM, CP, and NDF were similar for animals in both the treatments. Crude protein intake for the animals on the live-bacteria treatment was higher than that for the sheep receiving dead bacteria (P = 0.10) (Table 3). On the other hand, faecal and urinary CP excretion were similar for animals on both the treatments. Therefore, CP retention was higher for animals on the live-bacteria treatment (P = 0.07). Finally, the higher CP intake and retention observed before resulted in a higher CP retention/CP intake coefficient (P = 0.07). Although faecal and urinary CP excretion were similar between treatments, when expressed as a percentage of CP intake, numerically higher excretion levels were observed for the dead-bacteria treatment. Faecal CP, as a percentage of CP intake, averaged 52.3% and 48.1% for the dead- and live-bacteria treatment, respectively, while urinary CP excretion represented 20.5% for the dead- and, 19.2% of the total-CP intake for the live-bacteria treatments, respectively. Urinary N was less than half the amount of faecal N, for both the treatments. 3.3. Post-inoculation periods Dry-matter and CP intake observed during the two different post-inoculation periods are shown in Table 4. No differences (P >0.10) were found between treatments in either Table 4 Dry matter and CP intake, average daily gain and feed intake and efficiency for sheep; animals consumed a highCTa diet during the first two-week period and a low-CT diet for the subsequent two-week period; no microbial treatments were administered during either period Item

Treatment dead

No. of animals Average initial weight (kg)

SEM

P

alive

7 24.5

5 23.8

1.06

0.62

High-CT diet period Dry-matter intake (g/kg0.75/day) Crude protein intake (g/kg/day) Average daily feed (g/day) Average daily gain, g/day Feed/gain Average final/initial weight (kg)

66.3 5.2 761.9 144.7 5.7 26.5

69.2 5.5 771.3 140.8 14.2 25.7

5.12 0.36 80.14 35.75 4.11 1.41

0.70 0.60 0.94 0.94 0.18 0.68

Low-CT diet period Dry-matter intake (g/kg0.75/day) Crude protein intake (g/kg/day) Average daily feed (g/day) Average daily gain (g/day) Feed/gain Average final weight (kg)

92.9 6.9 1149.3 346.2 3.4 31.2

88.6 6.7 1074.6 327.9 3.3 30.1

1.82 0.15 41.86 24.62 0.31 1.20

0.14 0.24 0.24 0.61 0.87 0.53

a

Condensed tannin.

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of these two periods. However, when fed the normal diet, animals on the dead-bacteria treatment had numerically higher intakes (P = 0.14). It is clear that the high level of tannins present on the high-CT diet reduced DMI. During the first two weeks, when fed the high-CT diet, DMI for all the animals averaged 68 g/kg0.75/day. On the other hand, DMI increased significantly (P <0.001) to 90.5 g/kg0.75/day when the animals were offered the normal diet, during the final two weeks. Table 4 shows data on animal performance during the two post-inoculation periods. No significant differences were found between treatments. During the period when animals were fed the high-CT diet, the feed : gain ratio was very high for animals on the live-bacteria treatment, largely due to the low weight gain of one animal. However, during the period on the normal diet, the feed : gain ratio was similar for both groups of animals. 4. Discussion 4.1. Metabolism study When cattle were fed with a 24% peanut skin (6.2% tannin) diet, West et al. (1993) found an apparent depression in intake, which they attributed to the decreased palatability. Since tannins can depress intake, and thereby confound interpretation of changes in digestive efficiency due to the experimental treatments, animals were fed at a maintenance level (90% of the average consumption from the previous two weeks) in this study. This insured that almost all the feed was consumed. On average, <10 g of orts were removed prior to the next feeding. The lower intake of tannin-rich feeds may be attributed to the astringent taste of tannins (Mueller-Harvey and McAllan, 1992). However, besides unpleasant taste, the adverse effects of tannins on the rate of digestion of feeds could be another mechanism by which feed intake is decreased (Makkar et al., 1995b). When digestion rate is reduced (causing higher rumen fill), feed intake is also decreased (Van Soest, 1994). Decreased digestibility of organic matter and fibre has been partially attributed to the irreversible binding of tannins to fibre fractions or to fibrolytic enzymes (Barry and Manley, 1986; Reed et al., 1990). Reduced protein digestibility may be due to formation of tannin±protein complexes with dietary and endogenous (e.g. microbial or mammalian enzymes or epithelium cells) proteins (Butler, 1989). When considering the small effect of the inoculations with the tannin-tolerant bacteria on diet digestibility, it is important to consider that two explanations are possible: (a) dietary tannin levels did not affect digestibility, or (b) addition of the tannin-resistant bacteria was ineffective in improving digestibility. The first explanation, however, is unlikely because we showed that digestibility of the high-CT diet was increased when PEG was added to the culture medium. Nitrogen is often the first limiting nutrient for animals and, when animals are fed high-tannin forages, they may suffer a net N loss. Crude protein intake and retention in this study were increased due to the inoculations with the tannin-resistant bacteria,

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even with the small number of animals. Therefore, CP retention as a function of intake (CP retention/CP intake) was also greater (P = 0.07) due to inoculations with live bacteria. This higher CP retention/CP intake ratio could be attributed solely to the ability of the microbes inoculated into the rumen to withstand high levels of tannins, since both groups of animals received the same diet and the same amount of supplemental bacterial protein. The main purpose of inoculating animals with dead bacteria was to eliminate the differences in the amount of supplemental extra (bacterial) protein and to ensure that the inoculation process did not affect intake or animal performance. In view of the fact that both treatment groups received inoculations, the effects of stress should have been similar across groups. Ruminal microorganisms from animals adapted to high-tannin diets are potentially transferable to non-adapted ruminants to improve CP digestion of high-tannin diets (Miller et al., 1995). The potential of this approach is supported by these results. However, a longer and more detailed study involving a larger number of animals would be necessary to confirm these results. Although the results of this study obtained by inoculating a single species of bacteria seem promising, it is important not to forget results of the research of Miller et al. (1996); Miller et al. (1997). They suggested that a consortium of bacteria, rather than a single species, may be required to improve the digestibility of forages or diets that contain high levels of tannins. 4.2. Post-inoculation periods No post-inoculation effects were observed in animal performance in this study, but it is important to stress the short duration of the experiment. Dry matter and CP intake were similar between groups when the high-CT diet was offered, suggesting either absence or ineffectiveness of the tannin-tolerant bacteria in the rumen. During the final period, when the animals were fed the low-tannin diet, DM intake was numerically higher for animals that had received the inoculations of dead bacteria. When fed the low-CT diet, a compensatory effect was observed in all the animals during the experiment expressed as higher daily gain. The feed : gain ratio was also lower than when the high-CT diet was fed and there were no differences between treatments. The increase in DMI for all animals during this phase of the study is evidence that DMI was reduced due to the presence of tannins. 5. Conclusion In this study, the inoculation of the tannin-tolerant isolate did not improve DM or CP digestibility of sheep consuming a diet containing 29.5% peanut skins. However, even with the small number of animals in our study, we did see a positive effect in the crude protein balance in animals receiving the lab-cultured live inoculum. No post-inoculation effects were detected in animal performance, but the presence (persistence) or absence of the inoculated bacteria in the rumen of the animals remains to be studied.

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