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Responses of Rumen Microflora to High-Concentrate Low-Roughage Diets Containing Whey Products1
Responses of R u m e n M i c r o f l o r a t o H i g h - C o n c e n t r a t e L o w - R o u g h a g e Diets C o n t a i n i n g W h e y Products 1 V. L. METZGER, R. J. BAKER, and D. J. SCHINGOETHE Dairy Science Department South Dakota State University Brookings 57006
proteolytic organisms were not different among experimental rations but were generally lower during the post-experimental period.
ABSTRACT
After 3 wk on a standardization ration of alfalfa hay and corn silage ad libitum and concentrate at 1 kg/3 kg milk, 15 lactating Holstein cows were fed 2.3 kg hay/day and one of five concentrate rations ad libitum for a 6-wk experiment. Cows were returned to the standardization ration after the experimental period. Concentrate rations during experimental period were: 1) control, 2) 14% dried whole whey, 3) 5.9% high mineral whey product, 4) 11.8% demineralized whey product, and 5) 9.8% lactose. Ration 3 contained the same amount of minerals from whey as ration 2 while rations 4 and 5 contained the same amounts of lactose as ration 2. Whey products replaced portions of corn and soybean meal in the rations. Total protozoa numbers in rumen contents averaged 1.8 × lOS/ml and were not different among times although they tended to be less during the experimental period on ration 4. Dasytricba decreased on rations 1, 2, and 4, while trends in numbers of Iso'tricba, Entodinium, and Diplodinium were not consistent. Only a few Opbryscolex were in a couple of the rumen samples. Bacterial numbers increased from 4.0 × 109/mI during standardization to 5.8 × 109/ml during the high-grain period, then returned to 3.8 × 109/ml in the post-experimental period. The number of lactose fermenters increased on all diets containing whey or whey products but not on the control diet. No differences in numbers of starch digesters were detected between times or among experimental rations because of large variations in numbers. Numbers of
INTRODUCTION
Feeding high-concentrate, restricted-roughage rations often depresses milk fat yields and alters the proportions of volatile fatty acids (VFA) in the rumen (3, 6, 7, 8). With added dried whey or whey components to high-concentrate rations, the milk fat concentration usually returns toward that when forages are fed ad libitum (8, 9, 16, 17). Concentrations of rumen VFA also are returned usually toward normal except that rumen butyrate concentrations are elevated when rations contain whey or whey products (8, 9, 16, 17). Such responses indicate that switching rations of cows to high-concentrate, restricted-roughage may influence rumen microbial populations and that including whey or whey products in the ration may have an additional influence on rumen microorganisms. Large reductions in the proportion of forage in the ration influence the composition of the microbial population in the rumen, but detailed studies reporting these effects are limited (12). Latham et al. (12) observed small numbers of cellulolytic and fiber-digesting bacteria and large numbers of lactic and propionic acid-producing bacteria in the rumen of five cows given rations that depressed fat percentage. The purpose of our research was to evaluate further the effects of high concentrate rations on rumen microbes and evaluate the effects of dried whey and whey products on rumen microbial populations. Effects of these rations on milk yield, composition, a n d r u m e n V F A were in (17). The rumen microbial data were collected from one-half of the cows in the experiment of (17).
Received May 19, 1976. 1Published with the approval of the director of the South Dakota Agricultural Experiment Station on publication 1416 of the Journal Series.
EXPERIMENTAL PROCEDURE
After 3 wk of standardization, 15 lactating Holstein cows were assigned randomly to one
1769
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METZGER ET AL.
of five treatment groups of three cows each for a 6-wk experimental period. During standardization and post-experimental periods, the ration consisted of alfalfa hay and corn silage ad libitum and a concentrate ration fed at 1 kg/3 kg milk produced. Rations during the experimental period were 1 of 5 concentrate rations fed ad libitum and 2.3 kg hay as the only forage. Ingredients of concentrate rations are in Table 1. The high mineral whey ration was formulated to provide the same amount of ash as supplied by 14% dried whole whey whereas demineralized whey and lactose rations were designed to provide the same amount of lactose as supplied by 14% dried whole whey. Rations were changed over 3 to 5 days between periods. Rumen fluid samples for protozoal and bacterial observations were taken every 2 wk, starting the 3rd wk of the standardization period and ending 2 wk post-experimental. The rumen samples were collected via stomach tube
and syringe 2 to 3 h after morning feeding. After the first 20 to 40 ml of rumen fluid drawn were discarded, 80 to 100 ml of rumen fluid were collected in a 100 ml sample jar and taken immediately to the laboratory for observations. A protozoal counting procedure of Luther (13) used slides for rumen protozoal counts designed by Boyne et al. (4). Thirty fields from each slide were counted. Total protozoa were counted and were classified under five predominant genera according to the scheme of Becker and Talbott (2): Isotricba, Dasytricba, Entodinium, Diplodinium, and Opbryoscolex. To maintain conditions as anaerbic as possible, the Hungate (10) technique for anaerobiosis was used to enumerate the viable rumen bacteria and to determine the composition of several bacterial groups. The medium used for the total rumen bacterial count was described by Hungate (10). The rumen fluid-gtucose-cellobiose agar (RGCA medium) as prepaxed by
TABLE 1. Ingredients and proximate analysis of concentrates fed.
Ingredient
Ground shelled corn Rolled oats Soybean meal Urea Molasses Dried whole wheyc High mineral whey productd Demineralized whey productC,e
L ctoseC
Standardizationb
51.3 34.0 11.2 1.0 .
.
.
Control
Rationa Dried High whole mineral whey whey
79.5
66.5
1210 1.0 5.0
1110 1.0 5.0 14.0
. . . . . . . . . [. . . . . . . . . . . . .
(%) 76.0 . 9[6 1.0 5.0 . . 5[9
"'" :ii
.
Detaineralized whey
Lactose
69.2
6 7.5
1015 1.0 5.0 . .
14[2 1.0 5.0 . ...
i 1[8 :i:
.
..
918
(% of dry matter) Proximate analyses Crude protein Ether extract Crude fiber Ash
16.8 3.9 6.8 3.7
17.0 3.0 2.7 3.9
17.0 2.5 2.8 5.3
17.2 2.5 2.7 4.9
16.7 2.4 3.0 4.4
17.5 2.6 3.0 4.4
aAll rations also contained 1,25% dicalcium phosphate, 1.25% trace mineralized salt, 4400 IU added vitamin A/kg, and 660 IU added vitamin D/kg. bAlso fed during post-experimental period. CFurnished through the courtesy of Foremost Foods Company, San Francisco, CA. dspray dried whey product purchased from Valley Queen Cheese factory, Milbank, SD. See (17) for detailed analysis. eNutritek 900, a 90% demineralized whey. Journal of Dairy Science Vol. 59, No. 10
RUMEN MICROBES AND WHEY Bryant and Burkey (5) was used as the basal medium for the lactose fermenters, starch fermenters, and proteolytic bacteria. Media for the three groups of bacteria were prepared the same as the RGCA medium except that the glucose and cellobiose were omitted and the following ingredients added: (a) .5% (wt/vol) lactose for lactose fermenters; (b) .2% (wt/vol) soluble starch for starch fermenters; and (c) 1 ml of sterile skim milk was added per 100 ml of medium for proteolytic bacteria. Culture tubes 25 × 200 mm that had been sterilized previously without stoppers were used for preparing the roll tubes. Upon removal from the autoclave the culture tubes were stoppered immediately with sterile rubber stoppers. Nine milliliters of a culture medium were pipetted into each tube and flushed with carbon dioxide as described previously (10). The tubes were stoppered and held in a 45 to 50 C water bath until inoculated. The desired dilution of the inoculum was added with a sterile pipette, and the tubes were flushed again with carbon dioxide before the stoppers were inserted firmly. A roll tube was prepared (10) and was rolled by hand under the cold tap water so an even film of agar covered the inside surface of the tube. The cultures were incubated at 39 C for 72 to 96 h. RESULTS A N D DISCUSSION Protozoa
Protozoa data are in Table 2. Total protozoal numbers averaged 1.8 × 10S/rnl throughout experiment. Numbers for the average of the three samples taken during the high-concentrate (experimental) period were not lower than for standardization or post-experimental periods as has been observed by others (11, 12, 13). However, part of this possible discrepancy is explained by the high numbers of protozoa with several rations in the first sample taken during the high-concentrate period. The large standard error reflects the large variation in protozoal numbers probably due to instability of protozoal populations especially during the high-concentrate period. Numbers were considerably lower in cows fed demineralized whey ration than in those fed the other diets, but this difference was significant (P<.05) only during the high-concentrate period.
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Changes in numbers of the various protozoal genera followed trends similar to those for numbers of total protozoa. Endodinium were the most predominant genera accounting for approximately 61% of all protozoa in these cows. Diplodinium accounted for about 30% of the protozoa while Isotricba and Dasytricba each accounted for less than 3% of the protozoa in these cows. Opbryscolex were absent from most of the cows so were not summarized in Table 2. These organisms were in low numbers in a few samples from six different cows. No ration effect on Opbryscolex numbers was apparent. The proportion of Entodinium in the total population tended to increase during the high-concentrate period on all but the demineralized whey ration while the proportion of Isotricba tended to decrease. Others (1, 15) have also observed increased numbers of Entodinium with high energy rations. Numbers o f all the protozoal genera observed tended to be highest in cows fed the high mineral whey ration and lowest in those fed the demineralized whey ration. Bacteria
Bacterial numbers are in Table 3. Direct microscopic counts during the high-concentrate period were higher (P<.05) than during standardization or post-experimental periods. Bacterial numbers increased more (P<.05) in cows fed rations containing dried whole whey or whey products than in cows fed the control ration. The higher bacterial numbers during the high-concentrate period agreed with results of Maki and Foster (14). Total viable counts were lower (6.6 x 107/ml) than direct microscopic counts (5.0 x 109/ml) and were more variable, which is usually expected (10). There were no significant differences (P>.05) in total viable bacteria between diets or time intervals although numbers were lowest during the post-experimental period possibly indicating a loss in bacterial population when the high-concentrate ration was discontinued. Numbers of lactose fermenters usually tended to be higher in cows fed all rations which contained lactose than in cows fed the control ration although the differences were not statistically significant (P>.05). On the average, numbers of lactose fermenters in cows Journal of Dairy Science Vol. 59, No. 10
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METZGER ET AL.
fed lactose-containing rations d o u b l e d during t h e h i g h - c o n c e n t r a t e p e r i o d as c o m p a r e d t o t h e
standardization and post-experimental periods w h i l e t h e r e w a s n o i n c r e a s e in l a c t o s e fer-
TABLE 2. Numbers of protozoa in tureen contents of cows fed high-concentrate rations containing whey or whey products. Period Parameter Ration
Std.
2 wk
Experimental 4 wk 6 wk
Mean
Postexptl.
SE
(104/mL) Total protozoa Control Dwwa HMwb DMW c Lactose
14.0 19.0 27.0 7.8 20.0
28.0 18.0 83.0 3.4 42.0
14.0 19.0 21.0 2.8 19.0
15.0 4.6 13.0 1.4 18.0
19.0 def 13.9 ef 39.0 d 2.5 f 26.3 de
17.0 15.5 11.5 10.0 16.5
Mean
17.6
34.9
15.2
10.4
20.1
14.1
6.66
Dasytricba Control DWW HMW DMW Lactose Mean
.48 de .30 e .10 e .13 e 1.00 d
.39 .30 1.70 .12 .99
.27 .31 .55 .08 .49
.27 .04 .18 .00 .49
.31 def .22 ef .81 d .07£ .66 de
.40
.70
.34
.20
.41
.17 e .32 e .38 e .30 e 1.20 d .47
.16
Diplodinium Control DWW HMW DMW Lactose Mean
3.5 8.6 6.6 2.4 4.3
11.0 4.7 32.0 .8 12.5
4.3 4.7 5.3 .8 4.1
3.9 1.4 3.3 .3 3.5
6.4 de 3.6 e 13.5 d .6 e 6.7 de
4.8 5.3 3.8 3.5 4.2
5.1
12.2
3.8
2.5
6.1
4.3
8.6 7.0 18.0xy 5.3 11.5
16.0 11.4 43.0 2.3 26.0
10.9 13.7 17.0 2.0 13.2
10.5 3.2 8.8 .9 14.1
10.1
19.7
11.4
7.5
2.57
Entodinium Control DWW HMW DMW Lactose Mean
12.5 de 9.4 de 23.0 dx 1.7 e 17.8 d 12.9
10.0 7.6 8.3Y 5.0 8.8 7.9
4.43
lsotricba Control DWW HMW DMW Lactose Mean
.50 f .65 ef 1.40 de .17 f 1.70 d
.30 .02 1.00 .09 .62
.30 .25 1.10 .09 .39
.09 .06 .12 .11 .22
.23 .11 .74 .10 .41
.43 .32 .53 .70 1.50
.88
.41
.43
.12
.32
.70
.24
aDWW = dried whole whey ration. bHMW = high mineral whey product ration. CDMW = demineralized whey product ration. d'e'fMearts in the same column sharing a c o m m o n letter or no letters are not significantly different (P<.05). x'YMeans for standardization, experimental, and post-experimental periods sharing a c o m m o n letter or no letter are not significantly different (P<.05). Journal of Dairy Science Vol. 59, No. 10
RUMEN MICROBES AND WHEY
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TABLE 3. Numbers of bacteria in rumen contents o f coves fed high-concentrate rations containing whey or whey products. Period Parameter Radon
Std.
2 wk
Experimental 4 wk 6 wk
Mean
Postexptl.
SE
(10 9/ml) Direct microscopic count Control DWWa HMwb DMW c Lactose
4.1dy 4.0dy 4.2dy 3.6 fz 3.9ey
5.2 6.2 6.0 5.9 6.4
5.4 6.3 7.8 7.0 5.3
4.7 5.8 4.4 5.5 5.3
5.1 fx 6.1dx 6.1 dx 6. ldX 5.7 ex
3.8 efz 3.7 fz 4.1 dy 4.0dy 3.6dy
Mean
4.0Y
5.9
6.4
5.1
5.8 x
3.8 z
.06
(107/ml) Total viable count Control DWW HMW DMW Lactose Mean
5.3 e 8.2 e 5.7 e 9.0 e 20.5 d
5.6 17.5 1.8 10.5 21.0
4.0 .9 6.7 5.7 4.5
5.3 4.4 10.2 3.7 5.7
5.0 7. 6 6.2 6.6 10.4
1.8 1.4 2.6 2.1 1.8
9.7
11.3
4.4
5.9
7.2
2.0
2.60
(106/ml) Lactose fermenters Control DWW HMW DMW Lactose
4.3 e 2.5exy 3.5 e 9.5 dx 5.2 x
3.8 2.6 6.2 3.5 5.5
6.0 7.6 4.7 13.0 8.5
2.2 3.3 4.1 3.7 2.7
4.0 4.5 x 5.0 6.7 x 5.6 x
3.8 .6Y 1.3 2.0Y 1.2Y
Mean
5.0
4.3
8.0
3.2
5.2
1.8
1.13
( 10 6/ml) Starch~rmente~ Control DWW HMW DMW Lactic Mean
2.5 e 2.1 e 1.8 e 13.0 d 4.2 e
3.3 2.8 5.8 4.7 9.2
21.5 2.8 5.6 7.3 3.7
4.4 4.6 1.6 8.4 3.6
9.7 3.4 4.3 6.8 5.5
3.0 1.4 1.2 1.4 1.7
4.7
5.2
8.2
4.5
6.0
1.8
.5 de .2 e .5dey .4dey 1.6 d
2.30
(106/ml) Proteolytic bacteria Control DWW HMW DMW Lactose
.9 1.0 .8Y 1.2xy 1.0
2.4 2.0 4.2 3.2 3.3
1.2 .6 2.0 1.4 .8
1.2 1.1 1.7 1.4 2.3
1.6 de 1.2 e 2.6 dx 2.0 dex 2.1de
Mean
1.0xy
3.0
1.2
1.5
1.9 x
.6Y
.38
aDWW = dried whole whey ration. bHMW = high mineral whey product ration. CDMW = demineralized whey product ration. d'e'fMeans in the same column sharing a c o m m o n letter or no letters are not significantly different (P<.05). x'Y'ZMeans for standardization, experimental, and post-experimental periods sharing a c o m m o n letter or no letter are not significantly different (P<.05). Journal of Dairy Science Vol. 59, No. 10
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METZGER ET AL.
menters in cows fed the control ration. While numbers of lactose fermenters tended to be higher throughout the experiment in cows fed the demineralized whey ration, as compared to the average for standardization and post-experimental periods, their numbers increased less than in cows fed the other three whey product rations and actually decreased when only compared to the standardization period. The response in lactose fermenters was just as great with the high mineral whey ration as with the other whey product rations even though the high mineral ration contained only 20% as much lactose as was contained in each of the other whey product rations. This may indicate that only a limited increase in ruminal lactose fermentation can be achieved despite the amount of lactose fed, but more likely indicates that the precision of these studies was not sensitive enough to detect a "dose-response" to the amounts of lactose fed. The numbers of starch fermenters were higher during the high-concentrate period although there were no significant differences (P>.05) between diets or time intervals. Such a response would be expected when comparing bacterial numbers obtained with high-concentrate, restricted-roughage rations to numbers obtained when forages were fed ad libitum (10). Numbers of proteolytic bacteria increased during the high-concentrate period although the increase was significant (P<.O5) only in cows fed the high mineral whey ration. That ration also contained more of the readily solubilized whey proteins than the other whey product rations. All rations were essentially isonitrogenous. The higher numbers of proteolytic bacteria during the high-concentrate period correspond to the higher total bacterial numbers occurring with those rations. I nterrelationships
The milk yield, composition, and rumen VFA data from the 15 cows were essentially identical to the data from all 30 cows previously reported (17) so will not be repeated here but will be summarized briefly. The dried whole whey and high mineral whey rations were most effective in maintaining milk fat percentages during the high-concentrate period while the lactose ration was slightly less effecJournal of Dairy Science Vol. 59, No. 10
tive, and the control and demineralized whey rations were ineffective. Rumen propionate concentration was highest in cows fed the control and demineralized whey rations while butyrate concentration was highest in cows fed rations containing the most lactose (i.e., dried whole whey, demineralized whey, and lactose). Relationships between rumen microbial populations and rumen VFA and/or milk composition are not readily apparent primarily because of large variations in microbial data although some probably exist. Protozoal numbers were consistently lower in cows fed the demineralized whey ration, but were unaffected by the high-concentrate control ration. Of the bacterial groups evaluated, starch fermenters were in highest numbers with the two rations (control and demineralized whey) which depressed milk fat percentages the most and caused the highest rumen propionate (17). Numbers of lactose fermenters were related to rumen butyrate, but high butyrate fermentation did not maintain necessarily milk fat percentages as suggested by Latham et al. (12) as butyrate concentrations were among the highest in cows fed the demineralized whey ration. ACKNOWLEDGMENT
The authors are grateful to the staff of the Dairy Cattle Research Unit at South Dakota State University for care of the cows; to W. L. Tucker, Experiment Station Statistician, for assistance with statistical analysis of the data; and to Foremost Foods Company, San Francisco, for supplying some of the dried whey products in this research. This research was supported partially by funds from the Foremost-McKesson Foundation. REFERENCES
1 Abe, M., H. Shibui, T. Iriki, and F. Kumeno. 1973. Relation between diet and protozoal population in the rumen. Br. J. Nutr. 29:197. 2 Becker, E. R., and M. Talbott. 1927. The protozoa fauna of the rumen and reticulum of American Cattle. Iowa State Coll. J. Sci. 1:345. 3 Beitz, D. C., and C. L. Davis. 1964. Relationship of certain milk fat depressing diets to changes in the proportions of the volatile fatty acids produced in the rumen. J. Dairy Sci. 47:1213. 4 Boyne, A. W., J. M. Eadie, and K. Raitt. 1957. The development and testing of a method of counting rumen ciliate protozoa. J. Gen. Microbiol. 17:414. 5 Bryant, M. P., and L . . ~ Burkey. 1953. Cultural
RUMEN MICROBES AND WHEY
6
7
8
9
10 11
m e t h o d s a n d some characteristics o f s o m e of the more n u m e r o u s groups of bacteria in the bovine tureen. J. Dairy Sci. 36:205. Davis, C. L., R. E. Brown, and D. C. Beitz. 1964. Effect of feeding high-grain restricted-roughage rations with and w i t h o u t bicarbonates on the fat c o n t e n t of milk produced and proportions of volatile fatty acids in the rumen. J. Dairy Sci. 47:1217. Emery, R. S., L. D. Brown, and J. W. Thomas. 1964. Effect of sodium and calcium carbonates on milk production and composition o f milk, blood, and r u m e n contents o f cows fed grain ad libitum with restricted roughage. J. Dairy Sci. 47:1325. Huber, J. T., R. S. Emery, J. W. T h o m a s , and I. M. Yousef. 1969. Milk fat synthesis on restrictedroughage rations containing whey, sodium bicarbonate, and m a g n e s i u m oxide. J. Dairy Sci. 52:54. Huber, J. T., C. E. Polan, and R. A. Rosser. 1967. Effect o f whey on milk composition and r u m e n volatile fatty acids in restricted-roughage rations. J. Dairy Sci. 50:687. Hungate, R. E. 1966. The r u m e n and its microbes. Academic Press, New York. Latham, M. J., M. E. Sharpe, and J. D. Sutton.
12
13
14
15
16
17
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1971. The microbial flora of the r u m e n o f cows fed hay and high cereal rations and its relationship to the r u m e n fermentation. J. Appl. Bacteriol. 34:425. Latham, M. J., J. D. Sutton, and M. E. Sharpe. 1974. Fermentation and microorganisms in the r u m e n and t h e c o n t e n t of fat in the milk o f cows given low roughage rations. J. Dairy Sci. 57:803. Luther, R. M. 1967. Effect of diet on ruminal ciliate protozoa populations in cattle and sheep. Proc. S. D. Acad. Sci. 46:107. Maki, L. R., and E. M. Foster. 1957. Effect of roughage in the bovine ration on types of bacteria in the rumen. J. Dairy Sci. 40:905. Nakamura, K., and S. Kanegasaki. 1969. Densities of ruminal protozoa of sheep established u n d e r different dietary conditions. J. Dairy Sci. 52:250. Rosser, R. A., C. E. Polan, P. T. Chandler, and T. L. Bibb. 1971. Effects of whey c o m p o n e n t s and methionine analog on bovine milk fat production. J. Dairy Sci. 54:1807. Schingoethe, D. J., P. E. Stake, and M. J. Owens. 1973. Whey c o m p o n e n t s in restricted-roughage rations, milk composition, and r u m e n volatile fatty acids. J. Dairy Sci. 56:909.
Journal of Dairy Science Vol. 59, No. 10
proteolytic organisms were not different among experimental rations but were generally lower during the post-experimental period.
ABSTRACT
After 3 wk on a standardization ration of alfalfa hay and corn silage ad libitum and concentrate at 1 kg/3 kg milk, 15 lactating Holstein cows were fed 2.3 kg hay/day and one of five concentrate rations ad libitum for a 6-wk experiment. Cows were returned to the standardization ration after the experimental period. Concentrate rations during experimental period were: 1) control, 2) 14% dried whole whey, 3) 5.9% high mineral whey product, 4) 11.8% demineralized whey product, and 5) 9.8% lactose. Ration 3 contained the same amount of minerals from whey as ration 2 while rations 4 and 5 contained the same amounts of lactose as ration 2. Whey products replaced portions of corn and soybean meal in the rations. Total protozoa numbers in rumen contents averaged 1.8 × lOS/ml and were not different among times although they tended to be less during the experimental period on ration 4. Dasytricba decreased on rations 1, 2, and 4, while trends in numbers of Iso'tricba, Entodinium, and Diplodinium were not consistent. Only a few Opbryscolex were in a couple of the rumen samples. Bacterial numbers increased from 4.0 × 109/mI during standardization to 5.8 × 109/ml during the high-grain period, then returned to 3.8 × 109/ml in the post-experimental period. The number of lactose fermenters increased on all diets containing whey or whey products but not on the control diet. No differences in numbers of starch digesters were detected between times or among experimental rations because of large variations in numbers. Numbers of
INTRODUCTION
Feeding high-concentrate, restricted-roughage rations often depresses milk fat yields and alters the proportions of volatile fatty acids (VFA) in the rumen (3, 6, 7, 8). With added dried whey or whey components to high-concentrate rations, the milk fat concentration usually returns toward that when forages are fed ad libitum (8, 9, 16, 17). Concentrations of rumen VFA also are returned usually toward normal except that rumen butyrate concentrations are elevated when rations contain whey or whey products (8, 9, 16, 17). Such responses indicate that switching rations of cows to high-concentrate, restricted-roughage may influence rumen microbial populations and that including whey or whey products in the ration may have an additional influence on rumen microorganisms. Large reductions in the proportion of forage in the ration influence the composition of the microbial population in the rumen, but detailed studies reporting these effects are limited (12). Latham et al. (12) observed small numbers of cellulolytic and fiber-digesting bacteria and large numbers of lactic and propionic acid-producing bacteria in the rumen of five cows given rations that depressed fat percentage. The purpose of our research was to evaluate further the effects of high concentrate rations on rumen microbes and evaluate the effects of dried whey and whey products on rumen microbial populations. Effects of these rations on milk yield, composition, a n d r u m e n V F A were in (17). The rumen microbial data were collected from one-half of the cows in the experiment of (17).
Received May 19, 1976. 1Published with the approval of the director of the South Dakota Agricultural Experiment Station on publication 1416 of the Journal Series.
EXPERIMENTAL PROCEDURE
After 3 wk of standardization, 15 lactating Holstein cows were assigned randomly to one
1769
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METZGER ET AL.
of five treatment groups of three cows each for a 6-wk experimental period. During standardization and post-experimental periods, the ration consisted of alfalfa hay and corn silage ad libitum and a concentrate ration fed at 1 kg/3 kg milk produced. Rations during the experimental period were 1 of 5 concentrate rations fed ad libitum and 2.3 kg hay as the only forage. Ingredients of concentrate rations are in Table 1. The high mineral whey ration was formulated to provide the same amount of ash as supplied by 14% dried whole whey whereas demineralized whey and lactose rations were designed to provide the same amount of lactose as supplied by 14% dried whole whey. Rations were changed over 3 to 5 days between periods. Rumen fluid samples for protozoal and bacterial observations were taken every 2 wk, starting the 3rd wk of the standardization period and ending 2 wk post-experimental. The rumen samples were collected via stomach tube
and syringe 2 to 3 h after morning feeding. After the first 20 to 40 ml of rumen fluid drawn were discarded, 80 to 100 ml of rumen fluid were collected in a 100 ml sample jar and taken immediately to the laboratory for observations. A protozoal counting procedure of Luther (13) used slides for rumen protozoal counts designed by Boyne et al. (4). Thirty fields from each slide were counted. Total protozoa were counted and were classified under five predominant genera according to the scheme of Becker and Talbott (2): Isotricba, Dasytricba, Entodinium, Diplodinium, and Opbryoscolex. To maintain conditions as anaerbic as possible, the Hungate (10) technique for anaerobiosis was used to enumerate the viable rumen bacteria and to determine the composition of several bacterial groups. The medium used for the total rumen bacterial count was described by Hungate (10). The rumen fluid-gtucose-cellobiose agar (RGCA medium) as prepaxed by
TABLE 1. Ingredients and proximate analysis of concentrates fed.
Ingredient
Ground shelled corn Rolled oats Soybean meal Urea Molasses Dried whole wheyc High mineral whey productd Demineralized whey productC,e
L ctoseC
Standardizationb
51.3 34.0 11.2 1.0 .
.
.
Control
Rationa Dried High whole mineral whey whey
79.5
66.5
1210 1.0 5.0
1110 1.0 5.0 14.0
. . . . . . . . . [. . . . . . . . . . . . .
(%) 76.0 . 9[6 1.0 5.0 . . 5[9
"'" :ii
.
Detaineralized whey
Lactose
69.2
6 7.5
1015 1.0 5.0 . .
14[2 1.0 5.0 . ...
i 1[8 :i:
.
..
918
(% of dry matter) Proximate analyses Crude protein Ether extract Crude fiber Ash
16.8 3.9 6.8 3.7
17.0 3.0 2.7 3.9
17.0 2.5 2.8 5.3
17.2 2.5 2.7 4.9
16.7 2.4 3.0 4.4
17.5 2.6 3.0 4.4
aAll rations also contained 1,25% dicalcium phosphate, 1.25% trace mineralized salt, 4400 IU added vitamin A/kg, and 660 IU added vitamin D/kg. bAlso fed during post-experimental period. CFurnished through the courtesy of Foremost Foods Company, San Francisco, CA. dspray dried whey product purchased from Valley Queen Cheese factory, Milbank, SD. See (17) for detailed analysis. eNutritek 900, a 90% demineralized whey. Journal of Dairy Science Vol. 59, No. 10
RUMEN MICROBES AND WHEY Bryant and Burkey (5) was used as the basal medium for the lactose fermenters, starch fermenters, and proteolytic bacteria. Media for the three groups of bacteria were prepared the same as the RGCA medium except that the glucose and cellobiose were omitted and the following ingredients added: (a) .5% (wt/vol) lactose for lactose fermenters; (b) .2% (wt/vol) soluble starch for starch fermenters; and (c) 1 ml of sterile skim milk was added per 100 ml of medium for proteolytic bacteria. Culture tubes 25 × 200 mm that had been sterilized previously without stoppers were used for preparing the roll tubes. Upon removal from the autoclave the culture tubes were stoppered immediately with sterile rubber stoppers. Nine milliliters of a culture medium were pipetted into each tube and flushed with carbon dioxide as described previously (10). The tubes were stoppered and held in a 45 to 50 C water bath until inoculated. The desired dilution of the inoculum was added with a sterile pipette, and the tubes were flushed again with carbon dioxide before the stoppers were inserted firmly. A roll tube was prepared (10) and was rolled by hand under the cold tap water so an even film of agar covered the inside surface of the tube. The cultures were incubated at 39 C for 72 to 96 h. RESULTS A N D DISCUSSION Protozoa
Protozoa data are in Table 2. Total protozoal numbers averaged 1.8 × 10S/rnl throughout experiment. Numbers for the average of the three samples taken during the high-concentrate (experimental) period were not lower than for standardization or post-experimental periods as has been observed by others (11, 12, 13). However, part of this possible discrepancy is explained by the high numbers of protozoa with several rations in the first sample taken during the high-concentrate period. The large standard error reflects the large variation in protozoal numbers probably due to instability of protozoal populations especially during the high-concentrate period. Numbers were considerably lower in cows fed demineralized whey ration than in those fed the other diets, but this difference was significant (P<.05) only during the high-concentrate period.
1771
Changes in numbers of the various protozoal genera followed trends similar to those for numbers of total protozoa. Endodinium were the most predominant genera accounting for approximately 61% of all protozoa in these cows. Diplodinium accounted for about 30% of the protozoa while Isotricba and Dasytricba each accounted for less than 3% of the protozoa in these cows. Opbryscolex were absent from most of the cows so were not summarized in Table 2. These organisms were in low numbers in a few samples from six different cows. No ration effect on Opbryscolex numbers was apparent. The proportion of Entodinium in the total population tended to increase during the high-concentrate period on all but the demineralized whey ration while the proportion of Isotricba tended to decrease. Others (1, 15) have also observed increased numbers of Entodinium with high energy rations. Numbers o f all the protozoal genera observed tended to be highest in cows fed the high mineral whey ration and lowest in those fed the demineralized whey ration. Bacteria
Bacterial numbers are in Table 3. Direct microscopic counts during the high-concentrate period were higher (P<.05) than during standardization or post-experimental periods. Bacterial numbers increased more (P<.05) in cows fed rations containing dried whole whey or whey products than in cows fed the control ration. The higher bacterial numbers during the high-concentrate period agreed with results of Maki and Foster (14). Total viable counts were lower (6.6 x 107/ml) than direct microscopic counts (5.0 x 109/ml) and were more variable, which is usually expected (10). There were no significant differences (P>.05) in total viable bacteria between diets or time intervals although numbers were lowest during the post-experimental period possibly indicating a loss in bacterial population when the high-concentrate ration was discontinued. Numbers of lactose fermenters usually tended to be higher in cows fed all rations which contained lactose than in cows fed the control ration although the differences were not statistically significant (P>.05). On the average, numbers of lactose fermenters in cows Journal of Dairy Science Vol. 59, No. 10
1772
METZGER ET AL.
fed lactose-containing rations d o u b l e d during t h e h i g h - c o n c e n t r a t e p e r i o d as c o m p a r e d t o t h e
standardization and post-experimental periods w h i l e t h e r e w a s n o i n c r e a s e in l a c t o s e fer-
TABLE 2. Numbers of protozoa in tureen contents of cows fed high-concentrate rations containing whey or whey products. Period Parameter Ration
Std.
2 wk
Experimental 4 wk 6 wk
Mean
Postexptl.
SE
(104/mL) Total protozoa Control Dwwa HMwb DMW c Lactose
14.0 19.0 27.0 7.8 20.0
28.0 18.0 83.0 3.4 42.0
14.0 19.0 21.0 2.8 19.0
15.0 4.6 13.0 1.4 18.0
19.0 def 13.9 ef 39.0 d 2.5 f 26.3 de
17.0 15.5 11.5 10.0 16.5
Mean
17.6
34.9
15.2
10.4
20.1
14.1
6.66
Dasytricba Control DWW HMW DMW Lactose Mean
.48 de .30 e .10 e .13 e 1.00 d
.39 .30 1.70 .12 .99
.27 .31 .55 .08 .49
.27 .04 .18 .00 .49
.31 def .22 ef .81 d .07£ .66 de
.40
.70
.34
.20
.41
.17 e .32 e .38 e .30 e 1.20 d .47
.16
Diplodinium Control DWW HMW DMW Lactose Mean
3.5 8.6 6.6 2.4 4.3
11.0 4.7 32.0 .8 12.5
4.3 4.7 5.3 .8 4.1
3.9 1.4 3.3 .3 3.5
6.4 de 3.6 e 13.5 d .6 e 6.7 de
4.8 5.3 3.8 3.5 4.2
5.1
12.2
3.8
2.5
6.1
4.3
8.6 7.0 18.0xy 5.3 11.5
16.0 11.4 43.0 2.3 26.0
10.9 13.7 17.0 2.0 13.2
10.5 3.2 8.8 .9 14.1
10.1
19.7
11.4
7.5
2.57
Entodinium Control DWW HMW DMW Lactose Mean
12.5 de 9.4 de 23.0 dx 1.7 e 17.8 d 12.9
10.0 7.6 8.3Y 5.0 8.8 7.9
4.43
lsotricba Control DWW HMW DMW Lactose Mean
.50 f .65 ef 1.40 de .17 f 1.70 d
.30 .02 1.00 .09 .62
.30 .25 1.10 .09 .39
.09 .06 .12 .11 .22
.23 .11 .74 .10 .41
.43 .32 .53 .70 1.50
.88
.41
.43
.12
.32
.70
.24
aDWW = dried whole whey ration. bHMW = high mineral whey product ration. CDMW = demineralized whey product ration. d'e'fMearts in the same column sharing a c o m m o n letter or no letters are not significantly different (P<.05). x'YMeans for standardization, experimental, and post-experimental periods sharing a c o m m o n letter or no letter are not significantly different (P<.05). Journal of Dairy Science Vol. 59, No. 10
RUMEN MICROBES AND WHEY
1773
TABLE 3. Numbers of bacteria in rumen contents o f coves fed high-concentrate rations containing whey or whey products. Period Parameter Radon
Std.
2 wk
Experimental 4 wk 6 wk
Mean
Postexptl.
SE
(10 9/ml) Direct microscopic count Control DWWa HMwb DMW c Lactose
4.1dy 4.0dy 4.2dy 3.6 fz 3.9ey
5.2 6.2 6.0 5.9 6.4
5.4 6.3 7.8 7.0 5.3
4.7 5.8 4.4 5.5 5.3
5.1 fx 6.1dx 6.1 dx 6. ldX 5.7 ex
3.8 efz 3.7 fz 4.1 dy 4.0dy 3.6dy
Mean
4.0Y
5.9
6.4
5.1
5.8 x
3.8 z
.06
(107/ml) Total viable count Control DWW HMW DMW Lactose Mean
5.3 e 8.2 e 5.7 e 9.0 e 20.5 d
5.6 17.5 1.8 10.5 21.0
4.0 .9 6.7 5.7 4.5
5.3 4.4 10.2 3.7 5.7
5.0 7. 6 6.2 6.6 10.4
1.8 1.4 2.6 2.1 1.8
9.7
11.3
4.4
5.9
7.2
2.0
2.60
(106/ml) Lactose fermenters Control DWW HMW DMW Lactose
4.3 e 2.5exy 3.5 e 9.5 dx 5.2 x
3.8 2.6 6.2 3.5 5.5
6.0 7.6 4.7 13.0 8.5
2.2 3.3 4.1 3.7 2.7
4.0 4.5 x 5.0 6.7 x 5.6 x
3.8 .6Y 1.3 2.0Y 1.2Y
Mean
5.0
4.3
8.0
3.2
5.2
1.8
1.13
( 10 6/ml) Starch~rmente~ Control DWW HMW DMW Lactic Mean
2.5 e 2.1 e 1.8 e 13.0 d 4.2 e
3.3 2.8 5.8 4.7 9.2
21.5 2.8 5.6 7.3 3.7
4.4 4.6 1.6 8.4 3.6
9.7 3.4 4.3 6.8 5.5
3.0 1.4 1.2 1.4 1.7
4.7
5.2
8.2
4.5
6.0
1.8
.5 de .2 e .5dey .4dey 1.6 d
2.30
(106/ml) Proteolytic bacteria Control DWW HMW DMW Lactose
.9 1.0 .8Y 1.2xy 1.0
2.4 2.0 4.2 3.2 3.3
1.2 .6 2.0 1.4 .8
1.2 1.1 1.7 1.4 2.3
1.6 de 1.2 e 2.6 dx 2.0 dex 2.1de
Mean
1.0xy
3.0
1.2
1.5
1.9 x
.6Y
.38
aDWW = dried whole whey ration. bHMW = high mineral whey product ration. CDMW = demineralized whey product ration. d'e'fMeans in the same column sharing a c o m m o n letter or no letters are not significantly different (P<.05). x'Y'ZMeans for standardization, experimental, and post-experimental periods sharing a c o m m o n letter or no letter are not significantly different (P<.05). Journal of Dairy Science Vol. 59, No. 10
1774
METZGER ET AL.
menters in cows fed the control ration. While numbers of lactose fermenters tended to be higher throughout the experiment in cows fed the demineralized whey ration, as compared to the average for standardization and post-experimental periods, their numbers increased less than in cows fed the other three whey product rations and actually decreased when only compared to the standardization period. The response in lactose fermenters was just as great with the high mineral whey ration as with the other whey product rations even though the high mineral ration contained only 20% as much lactose as was contained in each of the other whey product rations. This may indicate that only a limited increase in ruminal lactose fermentation can be achieved despite the amount of lactose fed, but more likely indicates that the precision of these studies was not sensitive enough to detect a "dose-response" to the amounts of lactose fed. The numbers of starch fermenters were higher during the high-concentrate period although there were no significant differences (P>.05) between diets or time intervals. Such a response would be expected when comparing bacterial numbers obtained with high-concentrate, restricted-roughage rations to numbers obtained when forages were fed ad libitum (10). Numbers of proteolytic bacteria increased during the high-concentrate period although the increase was significant (P<.O5) only in cows fed the high mineral whey ration. That ration also contained more of the readily solubilized whey proteins than the other whey product rations. All rations were essentially isonitrogenous. The higher numbers of proteolytic bacteria during the high-concentrate period correspond to the higher total bacterial numbers occurring with those rations. I nterrelationships
The milk yield, composition, and rumen VFA data from the 15 cows were essentially identical to the data from all 30 cows previously reported (17) so will not be repeated here but will be summarized briefly. The dried whole whey and high mineral whey rations were most effective in maintaining milk fat percentages during the high-concentrate period while the lactose ration was slightly less effecJournal of Dairy Science Vol. 59, No. 10
tive, and the control and demineralized whey rations were ineffective. Rumen propionate concentration was highest in cows fed the control and demineralized whey rations while butyrate concentration was highest in cows fed rations containing the most lactose (i.e., dried whole whey, demineralized whey, and lactose). Relationships between rumen microbial populations and rumen VFA and/or milk composition are not readily apparent primarily because of large variations in microbial data although some probably exist. Protozoal numbers were consistently lower in cows fed the demineralized whey ration, but were unaffected by the high-concentrate control ration. Of the bacterial groups evaluated, starch fermenters were in highest numbers with the two rations (control and demineralized whey) which depressed milk fat percentages the most and caused the highest rumen propionate (17). Numbers of lactose fermenters were related to rumen butyrate, but high butyrate fermentation did not maintain necessarily milk fat percentages as suggested by Latham et al. (12) as butyrate concentrations were among the highest in cows fed the demineralized whey ration. ACKNOWLEDGMENT
The authors are grateful to the staff of the Dairy Cattle Research Unit at South Dakota State University for care of the cows; to W. L. Tucker, Experiment Station Statistician, for assistance with statistical analysis of the data; and to Foremost Foods Company, San Francisco, for supplying some of the dried whey products in this research. This research was supported partially by funds from the Foremost-McKesson Foundation. REFERENCES
1 Abe, M., H. Shibui, T. Iriki, and F. Kumeno. 1973. Relation between diet and protozoal population in the rumen. Br. J. Nutr. 29:197. 2 Becker, E. R., and M. Talbott. 1927. The protozoa fauna of the rumen and reticulum of American Cattle. Iowa State Coll. J. Sci. 1:345. 3 Beitz, D. C., and C. L. Davis. 1964. Relationship of certain milk fat depressing diets to changes in the proportions of the volatile fatty acids produced in the rumen. J. Dairy Sci. 47:1213. 4 Boyne, A. W., J. M. Eadie, and K. Raitt. 1957. The development and testing of a method of counting rumen ciliate protozoa. J. Gen. Microbiol. 17:414. 5 Bryant, M. P., and L . . ~ Burkey. 1953. Cultural
RUMEN MICROBES AND WHEY
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m e t h o d s a n d some characteristics o f s o m e of the more n u m e r o u s groups of bacteria in the bovine tureen. J. Dairy Sci. 36:205. Davis, C. L., R. E. Brown, and D. C. Beitz. 1964. Effect of feeding high-grain restricted-roughage rations with and w i t h o u t bicarbonates on the fat c o n t e n t of milk produced and proportions of volatile fatty acids in the rumen. J. Dairy Sci. 47:1217. Emery, R. S., L. D. Brown, and J. W. Thomas. 1964. Effect of sodium and calcium carbonates on milk production and composition o f milk, blood, and r u m e n contents o f cows fed grain ad libitum with restricted roughage. J. Dairy Sci. 47:1325. Huber, J. T., R. S. Emery, J. W. T h o m a s , and I. M. Yousef. 1969. Milk fat synthesis on restrictedroughage rations containing whey, sodium bicarbonate, and m a g n e s i u m oxide. J. Dairy Sci. 52:54. Huber, J. T., C. E. Polan, and R. A. Rosser. 1967. Effect o f whey on milk composition and r u m e n volatile fatty acids in restricted-roughage rations. J. Dairy Sci. 50:687. Hungate, R. E. 1966. The r u m e n and its microbes. Academic Press, New York. Latham, M. J., M. E. Sharpe, and J. D. Sutton.
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1971. The microbial flora of the r u m e n o f cows fed hay and high cereal rations and its relationship to the r u m e n fermentation. J. Appl. Bacteriol. 34:425. Latham, M. J., J. D. Sutton, and M. E. Sharpe. 1974. Fermentation and microorganisms in the r u m e n and t h e c o n t e n t of fat in the milk o f cows given low roughage rations. J. Dairy Sci. 57:803. Luther, R. M. 1967. Effect of diet on ruminal ciliate protozoa populations in cattle and sheep. Proc. S. D. Acad. Sci. 46:107. Maki, L. R., and E. M. Foster. 1957. Effect of roughage in the bovine ration on types of bacteria in the rumen. J. Dairy Sci. 40:905. Nakamura, K., and S. Kanegasaki. 1969. Densities of ruminal protozoa of sheep established u n d e r different dietary conditions. J. Dairy Sci. 52:250. Rosser, R. A., C. E. Polan, P. T. Chandler, and T. L. Bibb. 1971. Effects of whey c o m p o n e n t s and methionine analog on bovine milk fat production. J. Dairy Sci. 54:1807. Schingoethe, D. J., P. E. Stake, and M. J. Owens. 1973. Whey c o m p o n e n t s in restricted-roughage rations, milk composition, and r u m e n volatile fatty acids. J. Dairy Sci. 56:909.
Journal of Dairy Science Vol. 59, No. 10