In Vitro Effects of the Thiopeptide A10255 on Ruminal Fermentation and Microbial Populatlons 1 R. S. TUNG2 and L. KUNG, JR.3 Delaware Agricultural Experiment Station Department of Animal Science and Agricultural Biochemistry College of Agricultural Sciences University of Delaware Newark 19717-1303
L. L. SLYTER USDA-ARS Beltsville Agricultural Research Center Ruminant Nutrition Laboratory Beltsville, MD 20705
ABSTRACT
total anaerobes and lactate-producing, lactate-utilizing, cellulolytic and amylolytic bacteria were unaffected by treatment. However, molar proportions of acetate, butyrate, and isovalerate were decreased, and propionate was increased, by addition of Al0255. (Key words: thiopeptide, ruminal fermentation)
Experiments used unadapted mixed cultures of ruminal microorganisms in batch or continuous culture fermentation to investigate the effect of a thiopeptide, Al0255, on ruminal fermentation and microbial populations. After 24 h of fermentation in batch culture, addition of Al0255 (.5 to 20 ppm of the culture fluid) to 0, 45, 60, and 75% concentrate diets had no effect on total VFA but increased molar proportion of propionate and decreased butyrate. The molar proportion of acetate was decreased by treatment only in the 0 and 75% concentrate diets. The increase in molar proportion of propionate by 20 ppm of Al0255 was less than the increase caused by a similar concentration of monensin. The same concentration of A10255 (20 ppm) decreased ADF digestion less than 20 ppm of monensin. In continuous culture, A10255 (33 mglkg of dietary DM) did not affect total VFA concentration, culture pH, OM digestion, or ADF digestion. Ruminal bacterial populations of
Abbreviation key: MPN number.
probable
INTRODUCTION
Received February 14, 1992. Accepted April 27, 1992. .Published as Miscellaneous Paper Number 1416 of the Delaware Agricultural Experiment Station. Funding for this research was supported by Lilly Research Laboratories, Inc. and a University of Delaware Unidel Grant. 2Present address: Land O'Lakes, Inc., PO Box 64089, St. Paul, MN 55164. 3To whom reprint requests should be addressed. 1992 J Dairy Sci 75:2494-2503
= most
Much emphasis has been placed on methods to enhance the production of ruminants by manipulating ruminal fermentation. In particular, efforts have centered on ways to increase ruminal propionic acid and to depress methanogenesis and ruminal proteolysis and deamination of dietary protein. Ionophores, such as monensin and lasalocid, increase feed efficiency in finishing beef cattle (22). Others have used antibiotics that contain sulfur to alter ruminal fermentation. For example, thiopeptin (a sulfur antibiotic) is active against Gram-positive bacteria (16), resulted in an increase in the molar proportion of ruminal propionate (17), and prevented lactic acidosis (17, 18). A new thiopeptide, Al0255, produced by Streptomyces gardneri has been proposed as a potent ruminal fermentation modifier (24), but information that characterizes its effects on ruminal fermentation and microbial populations is limited. The objectives of this study were to determine the effects of varying doses of A10255
2494
2495
THIOPEPTIDE FEED ADDITIVE TABLE 1. Composition of experimental diets. Diets 1 Items
0:100
45:55
60:40
75:25
(%, DM basis) Feed composition, % Alfalfa hay Orchardgrass hay Corn silage Corn Soybean meal, 48% CP Limestone Trace-mineralized salt 2 Chemical composition, % of DM CP ADF TDN
49.5 50.0
.2 .3
15.2 0 39.8 29.8 14.5 .2 .4
10.0 0 30.0 46.1 13.2 .3 .4
15.0 61.8 12.5 .2 .6
17.0 36.7 70.0
15.0 19.2 70.4
14.8 15.3 12.8
14.3 11.8 75.1
10.0
o
lDiets varied in concentrate:forage ratio. 210% P, 19% Ca, 3% Mg, .6% K, .75% S, .15% I, .004% Co, .025% Cu, .035% Fe, .2% Mn, .75% Zn, .006% Se, 660,000 IU of vitamin A, 165,000 IU of vitamin D and 2475 IU of vitamin FJIcg.
on short-term ruminal fermentation in batch cultures and long-term effects of Al0255 on ruminal fermentation and microbial populations in continuous cultures. MATERIALS AND METHODS
Dose Titration in Batch Cultures
Batch cultures of mixed ruminal microorganisms were established from fistulated steers fed maintenance diets (20) containing com silage, alfalfa hay, cracked com grain, and soybean meal. Varying concentrations of thiopeptide A10255 (Lilly Research Laboratories, Inc., Greenfield, IN) were added to cultures differing in concentrate:forage ratios of 0:100, 45: 55, 60:40, or 75:25 (Table 1). In separate concentrate:forage ratio studies, three fistulated Holstein steers were adapted to the same diet used as substrate in the in vitro cultures. Steers served to replicate fermentations (n = 3). The in vitro diets were ground through a l-mm mesh screen and used at the rate of .5 g in 40 ml of culture medium, which consisted of 20 m1 of ruminal fluid and 20 ml of a phosphatebicarbonate buffer (11). Ruminal fluid was collected 3 h after feeding, strained through four layers of cheesecloth, and processed to recover the particulate-bound microorganisms under anaerobic conditions (7). Thiopeptide A10255 was solubilized in 30% ethanol, and
appropriate dilutions were prepared such that .5 ml of solution yielded final concentrations of 0, .5, I, 2.5, 5, 10, and 20 ppm of the culture fluid. Monensin (20 ppm) was used as a positive control. Four l00-ml incubation tubes were prepared for each steer at each dose. After 24 h of fermentation, pH was determined in two incubation tubes for each steer, followed by addition of 1 mt of 25% mphosphoric acid to 5 ml of fermentation fluid. The acidified fluid was analyzed for ammonia N (21) and VFA by gas chromatography (model 5890A, Hewlett-Packard Co., Avondale, PA) using a lOom, 530-llm macrobore Carbowax M column Supelco, Inc., Bellefonte, PA). Helium was used as the carrier gas with a flow rate of 10 mVmin. One microliter of sample was injected with a split ratio of 8:1. Injection port temperature was 200·C, and the detector temperature was 250·C. The oven was temperature programed as follows: 70·C for I min, 5·C increase/min to lOO·C, 45·C increase/min to l70·C, and a final holding time of 5 min. The remaining incubation tubes were analyzed for residual ADF (1). Digestion of ADF was calculated by subtracting the residual ADF from the initial ADF in the diet. Data were analyzed by the general linear models procedure of SAS (28); main effects of steer and concentration of A10255 were tested against the residual error term. Comparisons among 20 ppm of monensin and 0 and 20 ppm Journal of Dairy Science Vol. 75, No.9, 1992
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TUNG ET AL.
of A10255 also were processed by the general linear models procedure. The effect of dose was analyzed by orthogonal polynomial comparisons for linear, quadratic, and residual effects. Significance was declared at P < .05 unless otherwise noted. Continuous Culture Fermentation
Fermentors (29) consisted of a closed system with 500-ml reaction vessels with a single overflow port and were continuously stirred at 75 rpm and held at 39"C in a water bath. Effluent was collected in plastic bottles containing 15 ml of saturated mercuric chloride to inhibit microbial growth. Eight fermentors were filled to capacity with ruminal fluid from a steer fed a 60% concentrate diet (Table 1). Fermentors were purged with C02 prior to filling, after filling, and during feeding. A modified McDougall's artificial saliva was used as the buffer and was fed continuously by a peristaltic pump to obtain a turnover rate of approximately 1.5 volumesld (15). For the first 5 d of culturing, fermentors were fed daily 18 g (DM basis) of the diet in two equal offerings. The diet was the same composition and formulation fed to the ruminal fluid donor steer (Table I), except that com silage was air dried, and each ingredient was ground to pass a I-mm mesh screen. Steady state conditions were achieved after 5 d of culturing. After d 6, fermentors were divided randomly into control (no antibiotic, n = 4) and treated (with AI0255, n = 4) groups. The A10255 was dissolved in a 30% ethanol solution, and 1 ml of this solution containing a concentration of 33 mglkg of dietary DM was added to each of the treated fermentors at each feeding. Control fermentors were given 1 ml of 30% ethanol solution with no Al 0255 at each feeding. Culture effluents were collected every 24 h, and pH was recorded. Five milliliters of effluent were acidified with 1 ml of 25% mphosphoric acid, and the supernatant was obtained by centrifuging at 12,000 x g for 15 min and analyzed for ammonia N (21) and VFA by gas chromatography (model 5890A, HewlettPackard Co.) using a lO-m, 530-Ilm macrobore Carbowax M column (Supelco, Inc.). After 4 d of treatment, the daily effluents were composited by fermentor for 3 d. Composite effluents were stirred constantly, sampled by Journal of Dairy Science Vol. 75. No.9. 1992
using a wide-bore pipet, lyophilized, ground to pass a 1-mm mesh screen, and analyzed for ADF and ash (1). Organic matter and ADF digestibilities were calculated by subtracting the residual OM and ADF from the initial amount of OM and ADF in the diet, respectively. After 7 d of treatment, a representative sample of culture fluid from each fermentor was analyzed for total anaerobic bacteria using Bryant's 98-5 media (3), lactate-utilizing bacteria (30), and lactate-producing bacteria (25) in anaerobic roll tubes (14). Roll tubes with appropriate dilutions in duplicate were incubated at 39"C for 4 d before colony counting. All colonies were counted in tubes for enumeration of total anaerobic bacteria. Only medium and larger colonies were counted in lactate-utilizing and lactate-producing agar tubes. Total cellulolytic and amylolytic bacteria were cultured anaerobically using a modified cellulose-starch broth medium of Hungate (13). The anaerobic techniques for preparing and tubing the media and for inoculating the bacteria into the media were that of Hungate (12, 14), using an 02-free C02 gas phase. Tubes with appropriate dilutions were made in triplicate and incubated at 39"C for 7 d. Disappearance of cellulose (observed visually) and starch (iodine test) was measured, and the most probable number (MPN) technique was used for quantification (9). To determine proportions of ruminal bacteria sensitive or resistant to AI0255, culture samples from each fermentor also were inoculated into the various agar or broth media containing 4 ppm of A10255. The pH of all media was approximately 6.5. Data were analyzed by ANOVA using the general linear models procedure of SAS (28). The experimental design was a split plot with repeated measures (10). Effect of treatment was tested in the main plot, whereas day and day x treatment interaction were subplot treatments. Microbial data were log transformed (base 10) before analyses. Means were tested using an F protected (P < .05) pairwise comparison of the least squares means. RESULTS
Dose Titration In Batch Cultures
Table 2 shows the effects of A I 0255 and monensin on in vitro ruminal fermentation and
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THIOPEPTIDE FEED ADDITIVE
TABLE 2. Effect of thiopeptide A10255 and monensin on ruminal VFA production, culture pH, ADF digestion, and ammonia N concentration after 24 h in batch culture with a 100% hay diet. VFA Total VFA
Acetate
IsoPropionateButyrate butyrate
Isovalerate
Valerate
pH
(molJloo mol) - - - - - - -
(roM)
ADF Digestion Ammonia (%)
(mgldl)
35.9 38.9 36.9 36.4 34.9 34.8 33.1 7.1 8 1.2
33.3 31.9 31.0 31.5 31.8 28.8 29.0 22.7 a 1.8
Dose
o .5 1.0 2.5 5.0 10.0 20.0 M20 1 SE (n = 3) Significance2
95.5 86.4 87.7 83.1 88.0 96.2 87.0 73.1 8 4.4
73.0 73.2 72.8 71.9 71.4 72.5 71.3 69.0b .5 L*
16.3 17.0 17.7 18.7 19.1 18.6 18.9 19.6b .3 Q**
5.63 5.03 4.77 4.53 4.61 4.39 4.81 5.73c .14 Q**
1.73 1.68 1.67 1.71 1.73 1.62 1.75 1.86 .04
1.89 1.75 1.76 1.79 1.87 1.72 1.96 2.18 b .08 Q*
1.43 1.39 1.33 1.34 1.30 1.20 1.33 1.638 .06
6.53 6.65 6.57 6.77 6.67 6.61 6.67 6.71 b .05 L*
aM20 differs from control and 20 ppm of AI0255 (P < .05). bM20 differs from control (P < .05). cM20 differs from 20 ppm of AI0255 (P < .05). 1Monensin at 20 ppm. 2L = Linear effect of AI0255; Q = quadratic effect of AI0255. *p < .05. **p < .01.
ADF digestion in a 100% hay diet. Total VFA concentration was not affected by any dose of A10255 but was decreased by 20 ppm of monensin. Both A10255 and monensin increased the molar proportions of propionate. Addition of A10255 decreased the molar proportions of acetate, butyrate, and valerate but had no effect on the molar percentages of isobutyrate and isovalerate. In contrast with control cultures, 20 ppm of monensin had no effect on the molar proportions of butyrate but increased isovalerate and valerate. Culture pH increased linearly with increasing doses of AI0255. The pH of monensin-treated cultures was greater than in untreated cultures but similar to that of cultures treated with medium to high doses of A10255. Addition of A10255 had no effect on ADF digestion, but monensin caused an 80% reduction in fiber digestion relative to untreated cultures. Monensin, but not A10255, decreased the ammonia N concentration compared with that of untreated cultures. In 45% concentrate diets, total VFA concentration and molar proportions of acetate, isobutyrate, isovalerate, and valerate were not
affected by A10255 (Table 3). Increasing doses of AI0255 increased the molar proportion of propionate but decreased the proportion of butyrate. Monensin caused decreases in acetate, butyrate, and isobutyrate but increased propionate and valerate compared with untreated cultures. Compared with diets containing 20 ppm of monensin, cultures with 20 ppm of AI0255 had similar proportions of propionate, butyrate, and isovalerate but more total VFA. Proportions of acetate and isobutyrate also were higher with A10255, whereas the proportion of valerate was lower. The pH was greater in monensin-treated cultures but was unaffected by treatment with AI0255. Digestion of ADF was decreased by about 50% when concentrations of A10255 were greater than 5 ppm, but 20 ppm of monensin inhibited fiber digestion by more than 85%. Neither A10255 nor monensin had any effect on ammonia N concentrations in this diet. Compared with untreated cultures, total VFA concentration and the molar proportions of acetate, isobutyrate, isovalerate, and valerate were not affected by A10255 in 60% concentrate diets (Table 4). Increasing doses of Journal of Dairy Science Vol. 75, No.9, 1992
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TUNG ET AL.
TABLE 3. Effect of thiopeptide A10255 and monensin on ruminal VFA production, culture pH, ADF digestion, and ammonia concentration after 24 h in batch culture with a 45% concentrate diet. VFA Total VFA
Acetate
Propionate
122.6 114.1 113.1 108.0 119.6 117.5 122.2 l00.8 a 4.8
Isovalerate
Valerate
pH
(mol/l00 mol)
(mM)
Dose 0 .5 1.0 2.5 5.0 10.0 20.0 M20 1 SE (n = 3) Significance 2
IsoButyrate butyrate
52.3 53.1 51.9 52.7 51.7 51.7 51.6 50.2a .4
25.7 25.5 26.7 28.1 29.3 30.2 30.3 31.1 b .3 Q*
13.9 13.3 13.3 11.3 11.2 10.4 10.2 9.9.4 Q*
1.21 1.19 1.22 1.17 1.24 1.23 1.24 1.08a .04
3.13 3.23 3.25 3.22 3.00 3.14 3.28 2.80 .22
3.70 3.67 3.72 3.59 3.55 3.23 3.40 4.82a .13
5.55 5.51 5.50 5.54 5.52 5.53 5.55 5.73a .02
ADF Digestion Ammonia (%)
(mgldl)
31.8 23.5 21.5 28.2 15.8 14.4 15.6 3.8a 2.9 L*
25.7 28.2 28.2 29.3 28.7 30.5 29.8 28.0a .8
aM20 differs from control and 20 ppm of AI0255 (P < .05). bM20 differs from control (P < .05). IMonensin at 20 ppm. 2Q = Quadratic effect of A10255. *p < .01.
A10255 and 20 ppm of monensin increased the molar proportion of propionate, but the effect was greater with monensin. Total VFA and molar proportions of acetate and isobutyrate were greater in cultures with 20 ppm of A10255 than in cultures with 20 ppm of monensin. Both antibiotics similarly reduced the molar proportion of butyrate. Treatment with monensin resulted in higher pH than with the same concentration of AI0255 and untreated· cultures. Digestion of ADF was decreased by AI0255 and almost completely inhibited by monensin. Ammonia N concentration increased with greater doses of AI0255 compared with untreated cultures but was not affected by 20 ppm of monensin. Table 5 shows the effects of varying concentrations of A10255 and 20 ppm of monensin on ruminal fermentation characteristics in batch culture with a 75% concentrate diet. Addition of AI0255 had no effect on total VFA concentration, whereas monensin decreased total VFA compared with the control culture and 20 ppm of A10255. Addition of AI0255 increased the molar percentage of propionate and decreased butyrate relative to untreated cultures. As in the 60% concentrate diet, monensin decreased acetate and increased Journal of Dairy Science Vol. 75, No.9, 1992
propionate more than did 20 ppm of A10255. Culture pH was highest in monensin cultures. Although ADF digestion was decreased by more than 50% with 20 ppm of A10255, digestion was not significantly different from that of control cultures. As in all diets, monensin markedly decreased ADF digestion. Ammonia N concentration was not affected by A10255 or monensin. Continuous Culture Fermentation
Total VFA concentration, culture pH, OM digestion, and ADF digestion were not affected by A10255 (Table 6). Molar percentages of acetate, butyrate, and isovalerate were decreased by the antibiotic. However, addition of A10255 increased the molar proportion of propionate but did not affect molar percentages of isobutyrate and valerate. Addition of A10255 slightly increased ammonia N concentration in the culture. The colony counts of total anaerobes and lactate-producing and lactate-utilizing bacteria and the MPN of cellulolytic and amylolytic bacteria were not different between control and antibiotic-treated cultures (Table 7). Regardless of adaptation to A10255 in continuous culture, numbers of ma-
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THIOPEPTIDE FEED ADDITIVE
TABLE 4. Effect of thiopeptide A10255 and monensin on ruminal VFA production, culture pH, ADF digestion, and ammonia concentration after 24 h in batch culture with a 60% concentrate diet. VFA Total VFA
Acetate
Propionate
IsoButyrate butyrate
(mM)
Dose 0 .5 1.0 2.5 5.0 10.0 20.0 M201 SE (n 3) Significance2
=
118.9 123.8 123.6 128.9 129.8 127.4 124.9 105.3a 3.1
Isovalerate
Valerate
pH
(mol/100 mol) 50.7 50.7 51.3 50.9 50.9 51.5 50.6 42.0" .5
26.2 26.8 26.9 28.6 29.9 29.7 30.6 36.7a .3 Q*
15.8 15.5 14.7 13.4 12.3 11.9 11.8 11.7b
1.36 1.41 1.43 1.40 1.22 1.32 1.35 1.08a
.4
.06
2.95 2.78 2.75 2.84 2.69 2.70 2.76 3.17c .12
Q*
3.07 2.94 2.93 3.04 3.03 3.00 2.94 4.29a .15
5.61 5.59 5.61 5.63 5.64 5.70 5.66 5.90" .09 Q*
ADF Digestion Ammonia (%)
(mg/dl)
30.3 28.7 32.4 26.2 26.1 15.0 22.8 3.7a 2.8
20.4 20.3 20.6 23.5 23.5 23.7 25.0 19.3c .7 Q*
aM20 differs from control and 20 ppm of AI0255 (P < .05). bM20 differs from control (P < .05). cM20 differs from 20 ppm of AI0255 (P < .05). IMonensin at 20 ppm. 2Q
= Quadratic
effect of AI0255.
*p < .01.
jor ruminal bacteria were not affected when grown in media containing 4 ppm of A10255. DISCUSSION
Regardless of concentrate:forage ratio, increasing doses of A10255 increased the molar proportions of propionate and decreased butyrate but did not alter total VFA concentrations in batch cultures, which agrees with results of Richardson et al. (24), who found that A10255 had no effect on total VFA concentration but increased the molar proportion of propionate in an in vitro, batch fermentation with a 60% concentrate diet with ruminal microflora adapted to the same diet. However, in that study (24), AI0255 tended to increase VFA concentration in an alfalfa hay diet, which is in contrast to our results with an all forage diet. Perhaps the difference in diets and in the fermentation time could explain the differences in those findings. For example, our 100% forage diet consisted of 50% alfalfa and 50% orchardgrass hay instead of 100% alfalfa hay, and the fermentation time in our system was 24 h rather than 12 h in the study of Richardson et al. (24). The molar percentage of
acetate was not affected consistently and decreased only with high concentrations of A10255 in the 0 and 75% concentrate diets. Monensin (20 ppm) depressed total VFA concentrations in all diets compared with untreated cultures and those treated with 20 ppm of AI0255. Richardson et al, (23) noted that addition of monensin (at 1 ppm or less) to an in vitro system increased total VFA concentration in a concentrate but not in a forage diet. In the same study (23), 25 ppm of monensin did not decrease total VFA concentration compared with the controls, which differs from findings in our study with 20 ppm of monensin. In diets containing concentrate, cultures with monensin had lower molar percentage of acetate and higher pH than cultures with a similar dose of AI0255. In the 0 and 45% concentrate diets, 20 ppm of monensin or A10255 similarly increased the molar percentage of propionate, but propionate was increased more by monensin in the higher concentrate diets. Increasing doses of A10255 reduced the molar proportion of butyrate in all diets. The reduction in butyrate caused by 20 ppm of A10255 was similar to that observed Journal of Dairy Science Vol. 75, No.9, 1992
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TABLE 5. Effect of thiopeptide A10255 and monensin on ruminal VFA production. culture pH. ADF digestion and ammonia N concentration after 24 h in batch culture with a 75% concentrate diet. VFA Total VFA
Acetate
Propionate
Isovalerate
Valerate
pH
(moUl00 mol)
(roM)
Dose 0 .5 1.0 2.5 5.0 10.0 20.0 M20 1 SE (n = 3) Significance 2
IsoButyrate butyrate
125.9 127.7 129.4 127.8 127.2 126.7 125.0 102.5" 1.2
54.1 53.8 53.9 53.2 52.9 52.3 52.5 49.Ql .4 Q*
23.1 27.7 28.5 29.6 30.0 30.1 29.9 34.3" .3 Q**
17.7 13.6 12.7 12.2 12.2 12.5 12.6 l1.Ql .2 Q**
2.12 2.06 2.10 2.15 2.17 2.24 2.24 2.18 .02 Q**
1.20 1.15 1.17 1.18 1.19 1.21 1.24 1.15 .01 Q**
1.78 1.72 1.69 1.60 1.55 1.56 1.57 2.35"
5.79 5.78 5.82 5.88 5.83 5.91 5.94 5.99" .02
.07
ADF Digestion Ammonia (%)
(mg/d!)
22.5 22.7 19.4 17.0 15.5 10.1 9.4 3.5b 4.9
31.3 33.4 34.6 34.2 33.3 33.9 35.3 30.9 2.3
"M20 differs from control and 20 ppm of A10255 (P < .05). bM20 differs from control (P < .05). IMonensin at 20 ppm.
2L = Linear effect of AI025; Q = quadratic effect of A10255. *p < .05. "P < .01.
with 20 ppm of monensin in all diets except the l()()q'o forage diet. A dose-dependent decrease in butyrate by thiopeptin was reported by Nagaraja et al. (19); in our diet, addition of monensin decreased butyrate more than did a similar concentration of AI0255. Increases in ruminal propionate production and decreases in acetate and butyrate by monensin (23),
lasalocid (2), and lysocellin (15) have been documented. Reduction of butyrate in the rumen by ionophores may be caused by the selectivity of antibiotics against butyrateproducing microorganisms, such as Butyrivibrio fibrisolvens (8). Monensin is stimulatory for bacteria producing succinate and propionate (4). The effect of A10255 on ruminal
TABLE 6. Effect of thiopeptide AI0255 on ruminal fermentation and digestion in continuous culture. VFA PropioIsoAcetate nate Butyrate butyrate
Treatment Total VFA (rnM)
Control 131.7 AI025S3 133.6 SE (n = 4) 2.7 Significance
Isovalerate
Valerate
- - - - - - (moVl00 mol) - - - - - 2.4 2.2 28.3 10.4 .6 56.1 2.7 1.7 50.9 36.2 8.0 .6 .2 1.5 1.7 .4 .02 .2 t t ** *
10M Digestion. 2ADF Digestion. 3AI025S was used at the rate of 33 mglkg of dietary DM.
tp < .1. *p < .05.
"P < .01. Journal of Dairy Science Vol. 75. No.9. 1992
pH
OMDI ADFD2
Ammonia
-(%)-
(mg/dl)
5.71 5.77
50.2 51.8
.04
.8
13.3 16.1 .9
38.1 37.3 3.6
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TIiIOPEPTIDE FEED ADDITIVE
TABLE 7. Interaction between thiopeptide AI0255 in continuous culture and in enumerating media on numbers of ruminal bacteria (log to) per milliliter.
Bacterial populations Total anaerobes Lactate producers Lactate utilizers Cellulolytics Amylolytics
4 lLg/mIl
10.1 8.0 9.1 8.0 9.0
AI0255 in media 0 lLg/ml2
10.2 7.9 9.2
9.7 8.3 9.0
8.2
8.6
9.0
9.0
4 lLg/ml2
SE3
9.8
.6 .5 .7 .8 .9
8.2 8.9 8.8 9.0
ISamples from fennentors without AI0255. 2Samples from fermentors with A10255, 33 ppm of the dietary DM. 3D = 4.
fermentation may provide more energy; if so, A10255 could be used in the diets for lactating cows early in lactation. However, in vivo studies with lactating cows are needed to verify our assumptions and to ensure that ruminal bacteria do not adapt to the compound. Continuous cultures differ from batch cultures because of turnover rate, supplies of substrate and buffer, and removal of fermentation end products. These difference can have profound effects on microbial populations and fermentation patterns. In our continuous culture study, addition of Al0255 to a 60% concentrate diet increased the molar proportion of propionate and decreased butyrate with no effect on total VFA concentration and culture pH. Those findings were similar to results from our batch culture studies. However, the increase in propionate was greater in continuous than in batch cultures, and A10255 decreased the molar proportions of acetate and isovalerate only in continuous culture. As in batch culture, A10255 had little effect on the percentages of isobutyrate and valerate in continuous culture. Although we did not measure methane production, initial studies by Richardson et al. (24) reported that, unlike monensin, A10255 has no effect on methane production. The addition of monensin to unadapted ruminal cultures markedly depressed (greater than 80%) fiber digestion (26). Chen and Wolin (4) reported that 2.5 ppm of monensin inhibited cellulolytic bacteria, including Ruminococcus albus, Ruminococcus flavefaciens, and B. fibrisolvens, and delayed the growth of Bacteroides succinogenes and Bacteroides ruminicola. In contrast, high concentrations of A10255 moderately decreased ADF digestion
in diets contammg concentrates and had no effect on fiber digestion in the 100% forage diet. The reason for that finding is unknown, but it suggests an interaction between diet and treatment. Muir and Barreto (17) suggested that, in high forage diets, thiopeptin would have little effect on "normal" ruminal fermentation because of its narrow effectiveness against Gram-positive organisms. We could not find reports documenting the direct effect of thiopeptin on fiber digestion in ruminants. Perhaps depressions in ADF digestion in our concentrate diets, but not in our 100% forage diet, occurred for similar reasons; that is, A10255 had little effect on fiber digestion in the all forage diet because more bacteria likely were Gram-negative in the forage cultures, and actual fiber concentrations were high. In contrast, more Gram-positive organisms might have prevailed in the concentrate diets, which presumably were more susceptible to the effect of A10255, and the effect was magnified because of less fiber in the diets. Russell and Strobel (27) reported an inhibition of casein degradation by thiopeptin, but in our study A10255 had no effect on ammonia N. Similarly, Richardson et al. (24) reported that Al0255 had no effect on protein or AA degradation. In past studies (26), ammonia N concentrations generally have been reduced by monensin, suggesting a decrease in proteolysis. However, the effect of monensin on ruminal ammonia N was not consistent in our studies. Monensin decreased ammonia N in the 100% forage diet, increased ammonia N in the 45% concentrate diet, and had no effect in the 60 and 75% concentrate diets. We are unable to explain these findings. Journal of Dairy Science Vol. 75, No.9, 1992
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The ruminal bacteria populations were not affected by 33 ppm (dietary DM basis) of A10255 in continuous culture or by 4 ppm of AlO255 (of the fluid) in the enumerating media; this suggests that bacterial populations in fermentors were resistant to AI0255 or that A10255 had only moderate effects on these organisms in our culturing system. Although Counter et al. (6) reported that AI0255 inhibited several Bacteroides sp. and Clostridium sp. with no effect on Gram-negative bacteria, those bacteria were not ruminal isolates. Furthermore, when the sensitivity of bacteria to antibiotics is tested, the pH of media is critical (5). Unfortunately, the pH of media was not reported by Counter et al. (6). Our media pH was approximately 6.5 and may not have allowed us to detect the sensitivity of ruminal bacteria to AI0255 (R. S. Tung et al., 1992, unpublished data). Further studies to investigate the effect of AI0255 on the growth of pure cultures of ruminal bacteria with an emphasis on the culture pH are suggested. CONCLUSIONS
Decreases in fiber digestion, total VFA concentration, and the molar percentage of acetate are contraindicated for use of ionophores in dairy cattle diets. Our finding suggest that AlO255 caused a marked shift in ruminal fermentation patterns toward more propionate and less acetate and butyrate, but with no adverse effect on OM or ADF digestion. ACKNOWLEDGMENTS
The authors thank Leo Richardson for his support and encouragement. We also thank John Pesek for statistical consultation and Caroline Golt and Aideen Hession for analytical assistance. REFERENCES I Association Official Analytical Chemists. 1984. Official Methods of Analysis. 14th ed. AOAC, Washington, DC. 2 Bogaert, C., L. Gomez, I. P. Iouany, and G. Ieminet. 1989. Effect of the ionophore antibiotics 1asalocid and cationomycin on ruminal fennentation in vitro (RUSITEC). Anim. Feed Sci. Technol. 27:1. 3 Bryant, M. P., and I. M. Robinson. 1961. An improved nonselective culture medium for ruminal bacteria and its use in determining diurnal variation in Iournal of Dairy Science Vol. 75, No.9, 1992
numbers of bacteria in the rumen. I. Dairy Sci. 44:
1446. 4 Chen, M., and M. I. Wolin. 1979. Effect of monensin and lasalocid-sodium on the growth of methanogenic and rumen saccharolytic bacteria. Appl. Environ. Microbiol. 38:72. 5 Chow, I. M., and I. B. Russen. 1990. Effect of ionophores and pH on growth of Streptococcus bovis in batch and continuous culture. Appl. Environ. Microbiol. 56:1588. 6 Counter, F. T., P. W. Ensminger, and C.Y.E. Wu. 1989. A10255, a new thiopeptide antibiotic complex, produced by Streptomyces gard1leri. 3. Microbiological evaluation. Page 169 in Proc 29th Mtg. Intersci. Conf. Antimicrob. Agents Chemother. Am. Soc. MicrobioL, Houston, TX.(Abstr.) 7 Craig, W. M., B. I. Hong, G. A. Broderick, and R. I. BUla. 1984. In vitro inoculum enriched with particle associated microorganisms for determining rates of fiber digestion and protein degradation. 1. Dairy Sci. 67:2902. 8 Dennis, S. M., T. G. Nagaraja, and E. E. Bartley. 1981. Effect of lasalocid or monensin on lactateproducing or-using rumen bacteria. I. Anim. Sci. 52: 418. 9 Galton, M. M., G. K. Morris, and W. T. Martin. 1968. Salmonellae in Food and Feeds. Nail. Communicable Dis. Ctr., Ctr. Dis. Ctrl., Atlanta, GA. 10 Gill, J. L., and H. D. Hafs. 1971. Analysis of repeated measurements of animals. I. Anim. Sci. 33:331. 11 Goering, H. K., and P. I. Van Soest. 1970. Forage fiber analyses (apparatus, reagents, procedures. and some applications). Agric. Handbook No. 379. ARSUSDA, Washington, DC. 12 Hungate, R. E. 1950. The anaerobic mesophilus cellulolytic bacteria. Bacteriol. Rev. 14:1. 13 Hungate, R. E. 1957. Microorganisms in the rumen of cattle fed a constant ration. Can. I. Microbiol. 3:289. 14 Hungate, R. E. 1969. A ron tube method for cultivation of strict anaerobes. Page 117 in Methods in Microbiology. Vol. 3. I. R. Norris and E. W. Ribbons, ed. Academic Press, Inc., New York, NY. 1.5 Kung, L., R. S. Tung, and L. L. Slyter. 1992. In vitro effects of the ionophore Iysocellin on ruminal fermentation and microbial populations. J. Anim. Sci. 70: 281. 16 Miyairi, N., T. Miyoshi, H. Aoki, M. Kohsaka, H. Ikushima, K. Kunugita, H. Sakai, and H. Imanaka. 1972. Thiopeptin, a new feed additive antibiotic: microbiological and chemical studies. Antirnicrob. Agents Chemother. 1:192. 17 Muir, L. A., and A. Barreto, Ir. 1979. Sensitivity of Streptococcus bovis to various antibiotics. 1. Anim. Sci. 48:468. 18 Muir, L. A., E. L. Rickes, P. F. Duquette, and G. E. Smith. 1980. Control of wheat-induced lactic acidosis in sheep by thiopeptin and related antibiotics. I. Anim. Sci. 51:547. 19 Nagaraja, T. G., S. M. Dennis, S. 1. Galitzer, and D. L. Harmon. 1986. Effect of lasalocid, monensin and thiopeptin on lactate production from in vitro rumen fennentation of starch. Can. I. Anim. Sci. 66:129. 20 National Research Council. 1984. Nutrient Requirements of Beef Cattle. 5th rev. ed. Natl. Acad. Sci., Washington, DC.
lHIOPEPTIDE FEED ADDITIVE 21 Okuda, H., S. Fuji, and Y. Kawashima. 1965. A direct colorimetric method for blood ammonia Tokushima J. Exp. Med. 12:11. 22 Raun, A. P., C. O. Cooley, E. L. Potter, R. P. Rathmacher, and L. F. Richardson. 1976. Effect of monensin on feed efficiency of feedlot cattle. J. Anim. Sci. 43:670. 23 Richardson, L. F., A. P. Rann, E. L. Potter, C. O. Cooley, and R. P. Rathmacher. 1976. Effect of monensin on rumen fermentation in vitro and in vivo. J. Anim. Sci. 43:657. 24 Richardson, L. F., C. C. Scheifmger, and D. A. Becker. 1989. A10255, a new thiopeptide antibiotic complex produced by Streptomyces gardneri. 4. Rumen fermentation efficiency enhancement. Page 169 in Prot. 29th Mtg. Intersci. Conf. Antimicrob. Agents Chemother. Am. Soc. Microbiol., Houston, TX.(Abstr.)
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25 Rogosa, M., J. A. Mitchell, and R. R. Wiseman. 1951. A selective medium for the isolation and enumeration of oral and fecal lactobacilli. J. Bacteriol. 62:132. 26 Russell, J. B., W. G. Bottje, and M. A. Cotta. 1981. Degradation of protein by mixed cultures of rumen bacteria: identification of Streptococcus bovis as an actively proteolytic rumen bacterium. 1. Anim. Sci. 53:242. 27 Russell, 1. B., and H. J. Strobel. 1988. Effects of additives on in vitro ruminal fermentation: a comparison of monensin and bacitracin, another Grampositive antibiotic. J. Anim. Sci. 66:552. 28 SASe User's Guide: Statistics, Version 5 Edition. 1985. SAS Inst., Inc., Cary, NC. 29 Slyter, L. L. 1975. Automatic pH control and soluble and insoluble substrate input for continuous culture of rumen microorganisms. Appl. Microbiol. 30:330. 30 Slyter, L. L., and P. A. Putnam. 1967. In vivo vs. in vitro continuous culture of ruminal microbial populations. J. Anim. Sci. 26:1421.
Journal of Dairy Science Vol. 75, No.9, 1992
L. L. SLYTER USDA-ARS Beltsville Agricultural Research Center Ruminant Nutrition Laboratory Beltsville, MD 20705
ABSTRACT
total anaerobes and lactate-producing, lactate-utilizing, cellulolytic and amylolytic bacteria were unaffected by treatment. However, molar proportions of acetate, butyrate, and isovalerate were decreased, and propionate was increased, by addition of Al0255. (Key words: thiopeptide, ruminal fermentation)
Experiments used unadapted mixed cultures of ruminal microorganisms in batch or continuous culture fermentation to investigate the effect of a thiopeptide, Al0255, on ruminal fermentation and microbial populations. After 24 h of fermentation in batch culture, addition of Al0255 (.5 to 20 ppm of the culture fluid) to 0, 45, 60, and 75% concentrate diets had no effect on total VFA but increased molar proportion of propionate and decreased butyrate. The molar proportion of acetate was decreased by treatment only in the 0 and 75% concentrate diets. The increase in molar proportion of propionate by 20 ppm of Al0255 was less than the increase caused by a similar concentration of monensin. The same concentration of A10255 (20 ppm) decreased ADF digestion less than 20 ppm of monensin. In continuous culture, A10255 (33 mglkg of dietary DM) did not affect total VFA concentration, culture pH, OM digestion, or ADF digestion. Ruminal bacterial populations of
Abbreviation key: MPN number.
probable
INTRODUCTION
Received February 14, 1992. Accepted April 27, 1992. .Published as Miscellaneous Paper Number 1416 of the Delaware Agricultural Experiment Station. Funding for this research was supported by Lilly Research Laboratories, Inc. and a University of Delaware Unidel Grant. 2Present address: Land O'Lakes, Inc., PO Box 64089, St. Paul, MN 55164. 3To whom reprint requests should be addressed. 1992 J Dairy Sci 75:2494-2503
= most
Much emphasis has been placed on methods to enhance the production of ruminants by manipulating ruminal fermentation. In particular, efforts have centered on ways to increase ruminal propionic acid and to depress methanogenesis and ruminal proteolysis and deamination of dietary protein. Ionophores, such as monensin and lasalocid, increase feed efficiency in finishing beef cattle (22). Others have used antibiotics that contain sulfur to alter ruminal fermentation. For example, thiopeptin (a sulfur antibiotic) is active against Gram-positive bacteria (16), resulted in an increase in the molar proportion of ruminal propionate (17), and prevented lactic acidosis (17, 18). A new thiopeptide, Al0255, produced by Streptomyces gardneri has been proposed as a potent ruminal fermentation modifier (24), but information that characterizes its effects on ruminal fermentation and microbial populations is limited. The objectives of this study were to determine the effects of varying doses of A10255
2494
2495
THIOPEPTIDE FEED ADDITIVE TABLE 1. Composition of experimental diets. Diets 1 Items
0:100
45:55
60:40
75:25
(%, DM basis) Feed composition, % Alfalfa hay Orchardgrass hay Corn silage Corn Soybean meal, 48% CP Limestone Trace-mineralized salt 2 Chemical composition, % of DM CP ADF TDN
49.5 50.0
.2 .3
15.2 0 39.8 29.8 14.5 .2 .4
10.0 0 30.0 46.1 13.2 .3 .4
15.0 61.8 12.5 .2 .6
17.0 36.7 70.0
15.0 19.2 70.4
14.8 15.3 12.8
14.3 11.8 75.1
10.0
o
lDiets varied in concentrate:forage ratio. 210% P, 19% Ca, 3% Mg, .6% K, .75% S, .15% I, .004% Co, .025% Cu, .035% Fe, .2% Mn, .75% Zn, .006% Se, 660,000 IU of vitamin A, 165,000 IU of vitamin D and 2475 IU of vitamin FJIcg.
on short-term ruminal fermentation in batch cultures and long-term effects of Al0255 on ruminal fermentation and microbial populations in continuous cultures. MATERIALS AND METHODS
Dose Titration in Batch Cultures
Batch cultures of mixed ruminal microorganisms were established from fistulated steers fed maintenance diets (20) containing com silage, alfalfa hay, cracked com grain, and soybean meal. Varying concentrations of thiopeptide A10255 (Lilly Research Laboratories, Inc., Greenfield, IN) were added to cultures differing in concentrate:forage ratios of 0:100, 45: 55, 60:40, or 75:25 (Table 1). In separate concentrate:forage ratio studies, three fistulated Holstein steers were adapted to the same diet used as substrate in the in vitro cultures. Steers served to replicate fermentations (n = 3). The in vitro diets were ground through a l-mm mesh screen and used at the rate of .5 g in 40 ml of culture medium, which consisted of 20 m1 of ruminal fluid and 20 ml of a phosphatebicarbonate buffer (11). Ruminal fluid was collected 3 h after feeding, strained through four layers of cheesecloth, and processed to recover the particulate-bound microorganisms under anaerobic conditions (7). Thiopeptide A10255 was solubilized in 30% ethanol, and
appropriate dilutions were prepared such that .5 ml of solution yielded final concentrations of 0, .5, I, 2.5, 5, 10, and 20 ppm of the culture fluid. Monensin (20 ppm) was used as a positive control. Four l00-ml incubation tubes were prepared for each steer at each dose. After 24 h of fermentation, pH was determined in two incubation tubes for each steer, followed by addition of 1 mt of 25% mphosphoric acid to 5 ml of fermentation fluid. The acidified fluid was analyzed for ammonia N (21) and VFA by gas chromatography (model 5890A, Hewlett-Packard Co., Avondale, PA) using a lOom, 530-llm macrobore Carbowax M column Supelco, Inc., Bellefonte, PA). Helium was used as the carrier gas with a flow rate of 10 mVmin. One microliter of sample was injected with a split ratio of 8:1. Injection port temperature was 200·C, and the detector temperature was 250·C. The oven was temperature programed as follows: 70·C for I min, 5·C increase/min to lOO·C, 45·C increase/min to l70·C, and a final holding time of 5 min. The remaining incubation tubes were analyzed for residual ADF (1). Digestion of ADF was calculated by subtracting the residual ADF from the initial ADF in the diet. Data were analyzed by the general linear models procedure of SAS (28); main effects of steer and concentration of A10255 were tested against the residual error term. Comparisons among 20 ppm of monensin and 0 and 20 ppm Journal of Dairy Science Vol. 75, No.9, 1992
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TUNG ET AL.
of A10255 also were processed by the general linear models procedure. The effect of dose was analyzed by orthogonal polynomial comparisons for linear, quadratic, and residual effects. Significance was declared at P < .05 unless otherwise noted. Continuous Culture Fermentation
Fermentors (29) consisted of a closed system with 500-ml reaction vessels with a single overflow port and were continuously stirred at 75 rpm and held at 39"C in a water bath. Effluent was collected in plastic bottles containing 15 ml of saturated mercuric chloride to inhibit microbial growth. Eight fermentors were filled to capacity with ruminal fluid from a steer fed a 60% concentrate diet (Table 1). Fermentors were purged with C02 prior to filling, after filling, and during feeding. A modified McDougall's artificial saliva was used as the buffer and was fed continuously by a peristaltic pump to obtain a turnover rate of approximately 1.5 volumesld (15). For the first 5 d of culturing, fermentors were fed daily 18 g (DM basis) of the diet in two equal offerings. The diet was the same composition and formulation fed to the ruminal fluid donor steer (Table I), except that com silage was air dried, and each ingredient was ground to pass a I-mm mesh screen. Steady state conditions were achieved after 5 d of culturing. After d 6, fermentors were divided randomly into control (no antibiotic, n = 4) and treated (with AI0255, n = 4) groups. The A10255 was dissolved in a 30% ethanol solution, and 1 ml of this solution containing a concentration of 33 mglkg of dietary DM was added to each of the treated fermentors at each feeding. Control fermentors were given 1 ml of 30% ethanol solution with no Al 0255 at each feeding. Culture effluents were collected every 24 h, and pH was recorded. Five milliliters of effluent were acidified with 1 ml of 25% mphosphoric acid, and the supernatant was obtained by centrifuging at 12,000 x g for 15 min and analyzed for ammonia N (21) and VFA by gas chromatography (model 5890A, HewlettPackard Co.) using a lO-m, 530-Ilm macrobore Carbowax M column (Supelco, Inc.). After 4 d of treatment, the daily effluents were composited by fermentor for 3 d. Composite effluents were stirred constantly, sampled by Journal of Dairy Science Vol. 75. No.9. 1992
using a wide-bore pipet, lyophilized, ground to pass a 1-mm mesh screen, and analyzed for ADF and ash (1). Organic matter and ADF digestibilities were calculated by subtracting the residual OM and ADF from the initial amount of OM and ADF in the diet, respectively. After 7 d of treatment, a representative sample of culture fluid from each fermentor was analyzed for total anaerobic bacteria using Bryant's 98-5 media (3), lactate-utilizing bacteria (30), and lactate-producing bacteria (25) in anaerobic roll tubes (14). Roll tubes with appropriate dilutions in duplicate were incubated at 39"C for 4 d before colony counting. All colonies were counted in tubes for enumeration of total anaerobic bacteria. Only medium and larger colonies were counted in lactate-utilizing and lactate-producing agar tubes. Total cellulolytic and amylolytic bacteria were cultured anaerobically using a modified cellulose-starch broth medium of Hungate (13). The anaerobic techniques for preparing and tubing the media and for inoculating the bacteria into the media were that of Hungate (12, 14), using an 02-free C02 gas phase. Tubes with appropriate dilutions were made in triplicate and incubated at 39"C for 7 d. Disappearance of cellulose (observed visually) and starch (iodine test) was measured, and the most probable number (MPN) technique was used for quantification (9). To determine proportions of ruminal bacteria sensitive or resistant to AI0255, culture samples from each fermentor also were inoculated into the various agar or broth media containing 4 ppm of A10255. The pH of all media was approximately 6.5. Data were analyzed by ANOVA using the general linear models procedure of SAS (28). The experimental design was a split plot with repeated measures (10). Effect of treatment was tested in the main plot, whereas day and day x treatment interaction were subplot treatments. Microbial data were log transformed (base 10) before analyses. Means were tested using an F protected (P < .05) pairwise comparison of the least squares means. RESULTS
Dose Titration In Batch Cultures
Table 2 shows the effects of A I 0255 and monensin on in vitro ruminal fermentation and
2497
THIOPEPTIDE FEED ADDITIVE
TABLE 2. Effect of thiopeptide A10255 and monensin on ruminal VFA production, culture pH, ADF digestion, and ammonia N concentration after 24 h in batch culture with a 100% hay diet. VFA Total VFA
Acetate
IsoPropionateButyrate butyrate
Isovalerate
Valerate
pH
(molJloo mol) - - - - - - -
(roM)
ADF Digestion Ammonia (%)
(mgldl)
35.9 38.9 36.9 36.4 34.9 34.8 33.1 7.1 8 1.2
33.3 31.9 31.0 31.5 31.8 28.8 29.0 22.7 a 1.8
Dose
o .5 1.0 2.5 5.0 10.0 20.0 M20 1 SE (n = 3) Significance2
95.5 86.4 87.7 83.1 88.0 96.2 87.0 73.1 8 4.4
73.0 73.2 72.8 71.9 71.4 72.5 71.3 69.0b .5 L*
16.3 17.0 17.7 18.7 19.1 18.6 18.9 19.6b .3 Q**
5.63 5.03 4.77 4.53 4.61 4.39 4.81 5.73c .14 Q**
1.73 1.68 1.67 1.71 1.73 1.62 1.75 1.86 .04
1.89 1.75 1.76 1.79 1.87 1.72 1.96 2.18 b .08 Q*
1.43 1.39 1.33 1.34 1.30 1.20 1.33 1.638 .06
6.53 6.65 6.57 6.77 6.67 6.61 6.67 6.71 b .05 L*
aM20 differs from control and 20 ppm of AI0255 (P < .05). bM20 differs from control (P < .05). cM20 differs from 20 ppm of AI0255 (P < .05). 1Monensin at 20 ppm. 2L = Linear effect of AI0255; Q = quadratic effect of AI0255. *p < .05. **p < .01.
ADF digestion in a 100% hay diet. Total VFA concentration was not affected by any dose of A10255 but was decreased by 20 ppm of monensin. Both A10255 and monensin increased the molar proportions of propionate. Addition of A10255 decreased the molar proportions of acetate, butyrate, and valerate but had no effect on the molar percentages of isobutyrate and isovalerate. In contrast with control cultures, 20 ppm of monensin had no effect on the molar proportions of butyrate but increased isovalerate and valerate. Culture pH increased linearly with increasing doses of AI0255. The pH of monensin-treated cultures was greater than in untreated cultures but similar to that of cultures treated with medium to high doses of A10255. Addition of A10255 had no effect on ADF digestion, but monensin caused an 80% reduction in fiber digestion relative to untreated cultures. Monensin, but not A10255, decreased the ammonia N concentration compared with that of untreated cultures. In 45% concentrate diets, total VFA concentration and molar proportions of acetate, isobutyrate, isovalerate, and valerate were not
affected by A10255 (Table 3). Increasing doses of AI0255 increased the molar proportion of propionate but decreased the proportion of butyrate. Monensin caused decreases in acetate, butyrate, and isobutyrate but increased propionate and valerate compared with untreated cultures. Compared with diets containing 20 ppm of monensin, cultures with 20 ppm of AI0255 had similar proportions of propionate, butyrate, and isovalerate but more total VFA. Proportions of acetate and isobutyrate also were higher with A10255, whereas the proportion of valerate was lower. The pH was greater in monensin-treated cultures but was unaffected by treatment with AI0255. Digestion of ADF was decreased by about 50% when concentrations of A10255 were greater than 5 ppm, but 20 ppm of monensin inhibited fiber digestion by more than 85%. Neither A10255 nor monensin had any effect on ammonia N concentrations in this diet. Compared with untreated cultures, total VFA concentration and the molar proportions of acetate, isobutyrate, isovalerate, and valerate were not affected by A10255 in 60% concentrate diets (Table 4). Increasing doses of Journal of Dairy Science Vol. 75, No.9, 1992
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TUNG ET AL.
TABLE 3. Effect of thiopeptide A10255 and monensin on ruminal VFA production, culture pH, ADF digestion, and ammonia concentration after 24 h in batch culture with a 45% concentrate diet. VFA Total VFA
Acetate
Propionate
122.6 114.1 113.1 108.0 119.6 117.5 122.2 l00.8 a 4.8
Isovalerate
Valerate
pH
(mol/l00 mol)
(mM)
Dose 0 .5 1.0 2.5 5.0 10.0 20.0 M20 1 SE (n = 3) Significance 2
IsoButyrate butyrate
52.3 53.1 51.9 52.7 51.7 51.7 51.6 50.2a .4
25.7 25.5 26.7 28.1 29.3 30.2 30.3 31.1 b .3 Q*
13.9 13.3 13.3 11.3 11.2 10.4 10.2 9.9.4 Q*
1.21 1.19 1.22 1.17 1.24 1.23 1.24 1.08a .04
3.13 3.23 3.25 3.22 3.00 3.14 3.28 2.80 .22
3.70 3.67 3.72 3.59 3.55 3.23 3.40 4.82a .13
5.55 5.51 5.50 5.54 5.52 5.53 5.55 5.73a .02
ADF Digestion Ammonia (%)
(mgldl)
31.8 23.5 21.5 28.2 15.8 14.4 15.6 3.8a 2.9 L*
25.7 28.2 28.2 29.3 28.7 30.5 29.8 28.0a .8
aM20 differs from control and 20 ppm of AI0255 (P < .05). bM20 differs from control (P < .05). IMonensin at 20 ppm. 2Q = Quadratic effect of A10255. *p < .01.
A10255 and 20 ppm of monensin increased the molar proportion of propionate, but the effect was greater with monensin. Total VFA and molar proportions of acetate and isobutyrate were greater in cultures with 20 ppm of A10255 than in cultures with 20 ppm of monensin. Both antibiotics similarly reduced the molar proportion of butyrate. Treatment with monensin resulted in higher pH than with the same concentration of AI0255 and untreated· cultures. Digestion of ADF was decreased by AI0255 and almost completely inhibited by monensin. Ammonia N concentration increased with greater doses of AI0255 compared with untreated cultures but was not affected by 20 ppm of monensin. Table 5 shows the effects of varying concentrations of A10255 and 20 ppm of monensin on ruminal fermentation characteristics in batch culture with a 75% concentrate diet. Addition of AI0255 had no effect on total VFA concentration, whereas monensin decreased total VFA compared with the control culture and 20 ppm of A10255. Addition of AI0255 increased the molar percentage of propionate and decreased butyrate relative to untreated cultures. As in the 60% concentrate diet, monensin decreased acetate and increased Journal of Dairy Science Vol. 75, No.9, 1992
propionate more than did 20 ppm of A10255. Culture pH was highest in monensin cultures. Although ADF digestion was decreased by more than 50% with 20 ppm of A10255, digestion was not significantly different from that of control cultures. As in all diets, monensin markedly decreased ADF digestion. Ammonia N concentration was not affected by A10255 or monensin. Continuous Culture Fermentation
Total VFA concentration, culture pH, OM digestion, and ADF digestion were not affected by A10255 (Table 6). Molar percentages of acetate, butyrate, and isovalerate were decreased by the antibiotic. However, addition of A10255 increased the molar proportion of propionate but did not affect molar percentages of isobutyrate and valerate. Addition of A10255 slightly increased ammonia N concentration in the culture. The colony counts of total anaerobes and lactate-producing and lactate-utilizing bacteria and the MPN of cellulolytic and amylolytic bacteria were not different between control and antibiotic-treated cultures (Table 7). Regardless of adaptation to A10255 in continuous culture, numbers of ma-
2499
THIOPEPTIDE FEED ADDITIVE
TABLE 4. Effect of thiopeptide A10255 and monensin on ruminal VFA production, culture pH, ADF digestion, and ammonia concentration after 24 h in batch culture with a 60% concentrate diet. VFA Total VFA
Acetate
Propionate
IsoButyrate butyrate
(mM)
Dose 0 .5 1.0 2.5 5.0 10.0 20.0 M201 SE (n 3) Significance2
=
118.9 123.8 123.6 128.9 129.8 127.4 124.9 105.3a 3.1
Isovalerate
Valerate
pH
(mol/100 mol) 50.7 50.7 51.3 50.9 50.9 51.5 50.6 42.0" .5
26.2 26.8 26.9 28.6 29.9 29.7 30.6 36.7a .3 Q*
15.8 15.5 14.7 13.4 12.3 11.9 11.8 11.7b
1.36 1.41 1.43 1.40 1.22 1.32 1.35 1.08a
.4
.06
2.95 2.78 2.75 2.84 2.69 2.70 2.76 3.17c .12
Q*
3.07 2.94 2.93 3.04 3.03 3.00 2.94 4.29a .15
5.61 5.59 5.61 5.63 5.64 5.70 5.66 5.90" .09 Q*
ADF Digestion Ammonia (%)
(mg/dl)
30.3 28.7 32.4 26.2 26.1 15.0 22.8 3.7a 2.8
20.4 20.3 20.6 23.5 23.5 23.7 25.0 19.3c .7 Q*
aM20 differs from control and 20 ppm of AI0255 (P < .05). bM20 differs from control (P < .05). cM20 differs from 20 ppm of AI0255 (P < .05). IMonensin at 20 ppm. 2Q
= Quadratic
effect of AI0255.
*p < .01.
jor ruminal bacteria were not affected when grown in media containing 4 ppm of A10255. DISCUSSION
Regardless of concentrate:forage ratio, increasing doses of A10255 increased the molar proportions of propionate and decreased butyrate but did not alter total VFA concentrations in batch cultures, which agrees with results of Richardson et al. (24), who found that A10255 had no effect on total VFA concentration but increased the molar proportion of propionate in an in vitro, batch fermentation with a 60% concentrate diet with ruminal microflora adapted to the same diet. However, in that study (24), AI0255 tended to increase VFA concentration in an alfalfa hay diet, which is in contrast to our results with an all forage diet. Perhaps the difference in diets and in the fermentation time could explain the differences in those findings. For example, our 100% forage diet consisted of 50% alfalfa and 50% orchardgrass hay instead of 100% alfalfa hay, and the fermentation time in our system was 24 h rather than 12 h in the study of Richardson et al. (24). The molar percentage of
acetate was not affected consistently and decreased only with high concentrations of A10255 in the 0 and 75% concentrate diets. Monensin (20 ppm) depressed total VFA concentrations in all diets compared with untreated cultures and those treated with 20 ppm of AI0255. Richardson et al, (23) noted that addition of monensin (at 1 ppm or less) to an in vitro system increased total VFA concentration in a concentrate but not in a forage diet. In the same study (23), 25 ppm of monensin did not decrease total VFA concentration compared with the controls, which differs from findings in our study with 20 ppm of monensin. In diets containing concentrate, cultures with monensin had lower molar percentage of acetate and higher pH than cultures with a similar dose of AI0255. In the 0 and 45% concentrate diets, 20 ppm of monensin or A10255 similarly increased the molar percentage of propionate, but propionate was increased more by monensin in the higher concentrate diets. Increasing doses of A10255 reduced the molar proportion of butyrate in all diets. The reduction in butyrate caused by 20 ppm of A10255 was similar to that observed Journal of Dairy Science Vol. 75, No.9, 1992
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TUNG ET AL.
TABLE 5. Effect of thiopeptide A10255 and monensin on ruminal VFA production. culture pH. ADF digestion and ammonia N concentration after 24 h in batch culture with a 75% concentrate diet. VFA Total VFA
Acetate
Propionate
Isovalerate
Valerate
pH
(moUl00 mol)
(roM)
Dose 0 .5 1.0 2.5 5.0 10.0 20.0 M20 1 SE (n = 3) Significance 2
IsoButyrate butyrate
125.9 127.7 129.4 127.8 127.2 126.7 125.0 102.5" 1.2
54.1 53.8 53.9 53.2 52.9 52.3 52.5 49.Ql .4 Q*
23.1 27.7 28.5 29.6 30.0 30.1 29.9 34.3" .3 Q**
17.7 13.6 12.7 12.2 12.2 12.5 12.6 l1.Ql .2 Q**
2.12 2.06 2.10 2.15 2.17 2.24 2.24 2.18 .02 Q**
1.20 1.15 1.17 1.18 1.19 1.21 1.24 1.15 .01 Q**
1.78 1.72 1.69 1.60 1.55 1.56 1.57 2.35"
5.79 5.78 5.82 5.88 5.83 5.91 5.94 5.99" .02
.07
ADF Digestion Ammonia (%)
(mg/d!)
22.5 22.7 19.4 17.0 15.5 10.1 9.4 3.5b 4.9
31.3 33.4 34.6 34.2 33.3 33.9 35.3 30.9 2.3
"M20 differs from control and 20 ppm of A10255 (P < .05). bM20 differs from control (P < .05). IMonensin at 20 ppm.
2L = Linear effect of AI025; Q = quadratic effect of A10255. *p < .05. "P < .01.
with 20 ppm of monensin in all diets except the l()()q'o forage diet. A dose-dependent decrease in butyrate by thiopeptin was reported by Nagaraja et al. (19); in our diet, addition of monensin decreased butyrate more than did a similar concentration of AI0255. Increases in ruminal propionate production and decreases in acetate and butyrate by monensin (23),
lasalocid (2), and lysocellin (15) have been documented. Reduction of butyrate in the rumen by ionophores may be caused by the selectivity of antibiotics against butyrateproducing microorganisms, such as Butyrivibrio fibrisolvens (8). Monensin is stimulatory for bacteria producing succinate and propionate (4). The effect of A10255 on ruminal
TABLE 6. Effect of thiopeptide AI0255 on ruminal fermentation and digestion in continuous culture. VFA PropioIsoAcetate nate Butyrate butyrate
Treatment Total VFA (rnM)
Control 131.7 AI025S3 133.6 SE (n = 4) 2.7 Significance
Isovalerate
Valerate
- - - - - - (moVl00 mol) - - - - - 2.4 2.2 28.3 10.4 .6 56.1 2.7 1.7 50.9 36.2 8.0 .6 .2 1.5 1.7 .4 .02 .2 t t ** *
10M Digestion. 2ADF Digestion. 3AI025S was used at the rate of 33 mglkg of dietary DM.
tp < .1. *p < .05.
"P < .01. Journal of Dairy Science Vol. 75. No.9. 1992
pH
OMDI ADFD2
Ammonia
-(%)-
(mg/dl)
5.71 5.77
50.2 51.8
.04
.8
13.3 16.1 .9
38.1 37.3 3.6
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TIiIOPEPTIDE FEED ADDITIVE
TABLE 7. Interaction between thiopeptide AI0255 in continuous culture and in enumerating media on numbers of ruminal bacteria (log to) per milliliter.
Bacterial populations Total anaerobes Lactate producers Lactate utilizers Cellulolytics Amylolytics
4 lLg/mIl
10.1 8.0 9.1 8.0 9.0
AI0255 in media 0 lLg/ml2
10.2 7.9 9.2
9.7 8.3 9.0
8.2
8.6
9.0
9.0
4 lLg/ml2
SE3
9.8
.6 .5 .7 .8 .9
8.2 8.9 8.8 9.0
ISamples from fennentors without AI0255. 2Samples from fermentors with A10255, 33 ppm of the dietary DM. 3D = 4.
fermentation may provide more energy; if so, A10255 could be used in the diets for lactating cows early in lactation. However, in vivo studies with lactating cows are needed to verify our assumptions and to ensure that ruminal bacteria do not adapt to the compound. Continuous cultures differ from batch cultures because of turnover rate, supplies of substrate and buffer, and removal of fermentation end products. These difference can have profound effects on microbial populations and fermentation patterns. In our continuous culture study, addition of Al0255 to a 60% concentrate diet increased the molar proportion of propionate and decreased butyrate with no effect on total VFA concentration and culture pH. Those findings were similar to results from our batch culture studies. However, the increase in propionate was greater in continuous than in batch cultures, and A10255 decreased the molar proportions of acetate and isovalerate only in continuous culture. As in batch culture, A10255 had little effect on the percentages of isobutyrate and valerate in continuous culture. Although we did not measure methane production, initial studies by Richardson et al. (24) reported that, unlike monensin, A10255 has no effect on methane production. The addition of monensin to unadapted ruminal cultures markedly depressed (greater than 80%) fiber digestion (26). Chen and Wolin (4) reported that 2.5 ppm of monensin inhibited cellulolytic bacteria, including Ruminococcus albus, Ruminococcus flavefaciens, and B. fibrisolvens, and delayed the growth of Bacteroides succinogenes and Bacteroides ruminicola. In contrast, high concentrations of A10255 moderately decreased ADF digestion
in diets contammg concentrates and had no effect on fiber digestion in the 100% forage diet. The reason for that finding is unknown, but it suggests an interaction between diet and treatment. Muir and Barreto (17) suggested that, in high forage diets, thiopeptin would have little effect on "normal" ruminal fermentation because of its narrow effectiveness against Gram-positive organisms. We could not find reports documenting the direct effect of thiopeptin on fiber digestion in ruminants. Perhaps depressions in ADF digestion in our concentrate diets, but not in our 100% forage diet, occurred for similar reasons; that is, A10255 had little effect on fiber digestion in the all forage diet because more bacteria likely were Gram-negative in the forage cultures, and actual fiber concentrations were high. In contrast, more Gram-positive organisms might have prevailed in the concentrate diets, which presumably were more susceptible to the effect of A10255, and the effect was magnified because of less fiber in the diets. Russell and Strobel (27) reported an inhibition of casein degradation by thiopeptin, but in our study A10255 had no effect on ammonia N. Similarly, Richardson et al. (24) reported that Al0255 had no effect on protein or AA degradation. In past studies (26), ammonia N concentrations generally have been reduced by monensin, suggesting a decrease in proteolysis. However, the effect of monensin on ruminal ammonia N was not consistent in our studies. Monensin decreased ammonia N in the 100% forage diet, increased ammonia N in the 45% concentrate diet, and had no effect in the 60 and 75% concentrate diets. We are unable to explain these findings. Journal of Dairy Science Vol. 75, No.9, 1992
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TUNG ET AL.
The ruminal bacteria populations were not affected by 33 ppm (dietary DM basis) of A10255 in continuous culture or by 4 ppm of AlO255 (of the fluid) in the enumerating media; this suggests that bacterial populations in fermentors were resistant to AI0255 or that A10255 had only moderate effects on these organisms in our culturing system. Although Counter et al. (6) reported that AI0255 inhibited several Bacteroides sp. and Clostridium sp. with no effect on Gram-negative bacteria, those bacteria were not ruminal isolates. Furthermore, when the sensitivity of bacteria to antibiotics is tested, the pH of media is critical (5). Unfortunately, the pH of media was not reported by Counter et al. (6). Our media pH was approximately 6.5 and may not have allowed us to detect the sensitivity of ruminal bacteria to AI0255 (R. S. Tung et al., 1992, unpublished data). Further studies to investigate the effect of AI0255 on the growth of pure cultures of ruminal bacteria with an emphasis on the culture pH are suggested. CONCLUSIONS
Decreases in fiber digestion, total VFA concentration, and the molar percentage of acetate are contraindicated for use of ionophores in dairy cattle diets. Our finding suggest that AlO255 caused a marked shift in ruminal fermentation patterns toward more propionate and less acetate and butyrate, but with no adverse effect on OM or ADF digestion. ACKNOWLEDGMENTS
The authors thank Leo Richardson for his support and encouragement. We also thank John Pesek for statistical consultation and Caroline Golt and Aideen Hession for analytical assistance. REFERENCES I Association Official Analytical Chemists. 1984. Official Methods of Analysis. 14th ed. AOAC, Washington, DC. 2 Bogaert, C., L. Gomez, I. P. Iouany, and G. Ieminet. 1989. Effect of the ionophore antibiotics 1asalocid and cationomycin on ruminal fennentation in vitro (RUSITEC). Anim. Feed Sci. Technol. 27:1. 3 Bryant, M. P., and I. M. Robinson. 1961. An improved nonselective culture medium for ruminal bacteria and its use in determining diurnal variation in Iournal of Dairy Science Vol. 75, No.9, 1992
numbers of bacteria in the rumen. I. Dairy Sci. 44:
1446. 4 Chen, M., and M. I. Wolin. 1979. Effect of monensin and lasalocid-sodium on the growth of methanogenic and rumen saccharolytic bacteria. Appl. Environ. Microbiol. 38:72. 5 Chow, I. M., and I. B. Russen. 1990. Effect of ionophores and pH on growth of Streptococcus bovis in batch and continuous culture. Appl. Environ. Microbiol. 56:1588. 6 Counter, F. T., P. W. Ensminger, and C.Y.E. Wu. 1989. A10255, a new thiopeptide antibiotic complex, produced by Streptomyces gard1leri. 3. Microbiological evaluation. Page 169 in Proc 29th Mtg. Intersci. Conf. Antimicrob. Agents Chemother. Am. Soc. MicrobioL, Houston, TX.(Abstr.) 7 Craig, W. M., B. I. Hong, G. A. Broderick, and R. I. BUla. 1984. In vitro inoculum enriched with particle associated microorganisms for determining rates of fiber digestion and protein degradation. 1. Dairy Sci. 67:2902. 8 Dennis, S. M., T. G. Nagaraja, and E. E. Bartley. 1981. Effect of lasalocid or monensin on lactateproducing or-using rumen bacteria. I. Anim. Sci. 52: 418. 9 Galton, M. M., G. K. Morris, and W. T. Martin. 1968. Salmonellae in Food and Feeds. Nail. Communicable Dis. Ctr., Ctr. Dis. Ctrl., Atlanta, GA. 10 Gill, J. L., and H. D. Hafs. 1971. Analysis of repeated measurements of animals. I. Anim. Sci. 33:331. 11 Goering, H. K., and P. I. Van Soest. 1970. Forage fiber analyses (apparatus, reagents, procedures. and some applications). Agric. Handbook No. 379. ARSUSDA, Washington, DC. 12 Hungate, R. E. 1950. The anaerobic mesophilus cellulolytic bacteria. Bacteriol. Rev. 14:1. 13 Hungate, R. E. 1957. Microorganisms in the rumen of cattle fed a constant ration. Can. I. Microbiol. 3:289. 14 Hungate, R. E. 1969. A ron tube method for cultivation of strict anaerobes. Page 117 in Methods in Microbiology. Vol. 3. I. R. Norris and E. W. Ribbons, ed. Academic Press, Inc., New York, NY. 1.5 Kung, L., R. S. Tung, and L. L. Slyter. 1992. In vitro effects of the ionophore Iysocellin on ruminal fermentation and microbial populations. J. Anim. Sci. 70: 281. 16 Miyairi, N., T. Miyoshi, H. Aoki, M. Kohsaka, H. Ikushima, K. Kunugita, H. Sakai, and H. Imanaka. 1972. Thiopeptin, a new feed additive antibiotic: microbiological and chemical studies. Antirnicrob. Agents Chemother. 1:192. 17 Muir, L. A., and A. Barreto, Ir. 1979. Sensitivity of Streptococcus bovis to various antibiotics. 1. Anim. Sci. 48:468. 18 Muir, L. A., E. L. Rickes, P. F. Duquette, and G. E. Smith. 1980. Control of wheat-induced lactic acidosis in sheep by thiopeptin and related antibiotics. I. Anim. Sci. 51:547. 19 Nagaraja, T. G., S. M. Dennis, S. 1. Galitzer, and D. L. Harmon. 1986. Effect of lasalocid, monensin and thiopeptin on lactate production from in vitro rumen fennentation of starch. Can. I. Anim. Sci. 66:129. 20 National Research Council. 1984. Nutrient Requirements of Beef Cattle. 5th rev. ed. Natl. Acad. Sci., Washington, DC.
lHIOPEPTIDE FEED ADDITIVE 21 Okuda, H., S. Fuji, and Y. Kawashima. 1965. A direct colorimetric method for blood ammonia Tokushima J. Exp. Med. 12:11. 22 Raun, A. P., C. O. Cooley, E. L. Potter, R. P. Rathmacher, and L. F. Richardson. 1976. Effect of monensin on feed efficiency of feedlot cattle. J. Anim. Sci. 43:670. 23 Richardson, L. F., A. P. Rann, E. L. Potter, C. O. Cooley, and R. P. Rathmacher. 1976. Effect of monensin on rumen fermentation in vitro and in vivo. J. Anim. Sci. 43:657. 24 Richardson, L. F., C. C. Scheifmger, and D. A. Becker. 1989. A10255, a new thiopeptide antibiotic complex produced by Streptomyces gardneri. 4. Rumen fermentation efficiency enhancement. Page 169 in Prot. 29th Mtg. Intersci. Conf. Antimicrob. Agents Chemother. Am. Soc. Microbiol., Houston, TX.(Abstr.)
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25 Rogosa, M., J. A. Mitchell, and R. R. Wiseman. 1951. A selective medium for the isolation and enumeration of oral and fecal lactobacilli. J. Bacteriol. 62:132. 26 Russell, J. B., W. G. Bottje, and M. A. Cotta. 1981. Degradation of protein by mixed cultures of rumen bacteria: identification of Streptococcus bovis as an actively proteolytic rumen bacterium. 1. Anim. Sci. 53:242. 27 Russell, 1. B., and H. J. Strobel. 1988. Effects of additives on in vitro ruminal fermentation: a comparison of monensin and bacitracin, another Grampositive antibiotic. J. Anim. Sci. 66:552. 28 SASe User's Guide: Statistics, Version 5 Edition. 1985. SAS Inst., Inc., Cary, NC. 29 Slyter, L. L. 1975. Automatic pH control and soluble and insoluble substrate input for continuous culture of rumen microorganisms. Appl. Microbiol. 30:330. 30 Slyter, L. L., and P. A. Putnam. 1967. In vivo vs. in vitro continuous culture of ruminal microbial populations. J. Anim. Sci. 26:1421.
Journal of Dairy Science Vol. 75, No.9, 1992
