Directory UMM :Data Elmu:jurnal:A:Animal Feed Science and Technology:Vol81.Issue1-2.Sep1999:
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
70
D.O. Molina et al. / Animal Feed Science and Technology 81 (1999) 69±80
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.
D.O. Molina et al. / Animal Feed Science and Technology 81 (1999) 69±80
71
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.
72
D.O. Molina et al. / Animal Feed Science and Technology 81 (1999) 69±80
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
D.O. Molina et al. / Animal Feed Science and Technology 81 (1999) 69±80
73
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).
74
D.O. Molina et al. / Animal Feed Science and Technology 81 (1999) 69±80
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 Ti Pj TPij 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 0.10) 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.
D.O. Molina et al. / Animal Feed Science and Technology 81 (1999) 69±80
77
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
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
70
D.O. Molina et al. / Animal Feed Science and Technology 81 (1999) 69±80
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.
D.O. Molina et al. / Animal Feed Science and Technology 81 (1999) 69±80
71
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.
72
D.O. Molina et al. / Animal Feed Science and Technology 81 (1999) 69±80
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
D.O. Molina et al. / Animal Feed Science and Technology 81 (1999) 69±80
73
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).
74
D.O. Molina et al. / Animal Feed Science and Technology 81 (1999) 69±80
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 Ti Pj TPij 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 0.10) 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.
D.O. Molina et al. / Animal Feed Science and Technology 81 (1999) 69±80
77
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