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
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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
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
