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Holstein steers were fed corn silage supplemented with either wet or dried brewers' grains to determine effects of heat drying commercial brewers' grains. Four rumen-fistulated steers were fed a 12.5% crude protein diet in a single reversal design experiment. Brewers' grains supplied 45% of the protein of the diet. Bacterial numbers, concentration of ciliated protozoa, and ammonia concentration in the rumen were higher, and rumen pH was lower, for steers fed wet brewers' grains. Concentrations of rumen volatile fatty acids were similar for both diets. Ruminal digestibility of dry matter decreased when wet versus dried brewers' grains were fed (56.9 versus 39.3%). The rate of dry matter passage from the rumen was faster with wet brewers' grains. In Experiment 2, 12 steers were in a 2 X 2 factorial design. Diets contained wet or dried brewers' grains supplemented at 22 or 40% of the diet dry matter (12.5 and 14.5% crude protein). Nitrogen retention was increased in steers fed the higher crude protein diet. Apparent digestible nitrogen, acid detergent fiber nitrogen, and nitrogen retention were higher with wet versus dried brewers' grains. Plasma essential and nonessential amino acids were also higher in steers fed wet brewers' grains. Alteration in microbial numbers, fermentation measurements, and nitrogen utilization were associated with more soluble nitrogen with wet (13.4%) versus dried (3.3%) brewers' grains.

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By anaerobic procedures, the total number of adherent bacteria was determined on tissue samples obtained from the roof of the dorsal rumen of three sheep. After four washings, 1.91 x 10, 0.34 x 10, and 1.23 x 10 bacteria per cm were still attached to the rumen epithelium in sheep 1, 2, and 3, respectively. A total of 95 strains of bacteria were isolated from these three samples. Based on morphology, Gram stain, anaerobiosis, motility, and fermentation end products, they were presumptively identified as follows: Butyrivibrio fibrisolvens, 30 strains; atypical Butyrivibrio, 5 strains; Bacteroides ruminicola, 22 strains; Lactobacillus, 1 strain; and unknown Bacteroides species, 37 strains. For sheep 3, washing the rumen epithelium a total of 10 times reduced the adherent bacterial population by 93% (8.4 x 10 bacteria per cm). Of 30 strains isolated from this sample, 22 were presumptively identified as Butyrivibrio and Bacteroides types. These results suggest that the epithelium on the roof of the dorsal rumen is primarily colonized by two genera of bacteria, Butyrivibrio and Bacteroides. Most Butyrivibrio and Bacteroides ruminicola strains appeared to be similar to previously isolated rumen strains. However, the unknown Bacteroides species differed considerably from the three species of this genus which are commonly isolated from rumen contents.

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Anaerobic storage of whole rumen contents at 0 degrees C for 8 and 24 h resulted in viable colony counts which were 113 and 92%, respectively, of the colony count obtained with an unstored sample. No significant differences in the percentages of the total population capable of utilizing glucose, cellobiose, starch, or xylose occurred with storage. Numerous factors were investigated as possible explanations for the increase in bacterial numbers observed after storage for 8 h in ice. Growth and multiplication of bacteria, subsampling of rumen contents, susceptibility to oxygen, lysis of protozoa with the release of viable bacteria, and rumen sampling time did not appear to be involved. Compilation of the data from all 29 of the above experiments gave a mean value for samples stored for 8 h in ice which was 134.8% of the control (P < 0.005). The effect of storage time at 0 degrees C indicated that a significant increase in colony count occurred after 4 h, and, based on these data, 6 h was subsequently used as the standard cold-storage period. Circumstantial evidence supported the hypothesis that storage of rumen contents for 6 h at 0 degrees C appears to alter or to break down the material responsible for cell-to-cell or cell-to-particulate matter attachment. Addition of a surfactant to the anaerobic dilution solution significantly increased total colony count of rumen contents to an extent similar to chilling in ice for 6 h. However, an additive effect was observed when surfactant-containing anaerobic dilution solution was used with samples stored for 6 h at 0 degrees C.

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A total of 44 strains of bacteria were isolated from rumen contents of the goat. Based on morphology, Gram stain, anaerobiosis, motility, and fermentation end products, they were grouped into 11 different types. For each type, all or representative strains were characterized in detail. The type, number of strains characterized over total number of strains, and identification were as follows: type 1, 6/21, atypical Butyrivibrio fibriosolvens; type 2, 6/9, atypical Butyrivibrio fibrisolvens; type 3, 3/3, genus uncertain; type 4, 2/2, genus uncertain; type 5, 3/3, Streptoccous bovis; type 6, 1/1, Butyrivibrio fibrisolvens; type 7, 1/1, Bacteroides ruminicola subsp. ruminicola; type 8, 1/1, Bacteroides ruminicola subsp. brevis; type 9, 1/1, family Peptococcaceae, genus uncertain; type 10, 1/1, genus Bacteroides; type 11, 1/1, genus Bacteroides. About 70% of the isolated strains were classified as Butyrivibrio, which is quite high compared with previous studies in cattle on similar rations. Of the 30 strains listed as type 1 and 2, the 12 studied further were characterized as atypical Butyrivibrio fibrisolvens, which differed from the species description primarily by their inability to hydrolyze starch and lack of gas production.

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When glucose-1-phosphate was used as the only added energy source in a selective roll tube medium, colony counts for rumen contents ranged from 17.8 to 84.8% of the total culturable count. Percentages were highest in rumen contents from sheep fed high-concentrate rations. From a total of 73 cultures isolated from glucose-1-phosphate roll rubes, only 15.1% were presumptively identified as Bacteroides species. Strains presumptively identified as Butyrivibrio, Selenomonas, Treponema, Streptococcus bovis, and Lachnospira also fermented glucose-1-phosphate. Thus, glucose-1-phosphate would not be useful as a selective substrate for isolation or enumeration of Bacteroides species from the rumen.

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A 40% rumen fluid basal medium has been developed that without added substrate will support growth of about 10% or less of the total colony count obtained with 40% rumen fluid-glucose-cellobiose-starch-agar medium (RGCSA). The basal medium is prepared by anaerobic incubation of all ingredients in RGCSA medium except the carbohydrates, Na2CO3, and cysteine for 7 days at 38 degrees C. After incubation, substrate(s), Na2CO3 and cysteine are added and the medium is tubed and sterilized as in normal medium preparation. When xylose was included with glucose, cellobiose, and starch as added carbohydrates in the incubated medium, colony counts were comparable to those obtained with RGCSA medium. The addition of specific carbohydrates or other substrates as energy sources to the basal medium suggested that the percentage of the bacterial population capable of utilizing these energy sources was influenced by the ration of the animal; however, considerable animal variation and day-to-day variation in a given animal was observed. Comparison of the population in animals fed either orchardgrass hay or 60% corn-40% orchardgrass (60-40) indicated little or no difference for the percentage of bacteria utilizing glucose, pectin, xylan, or mannitol. Increases in the percentages of xylose-, cellobiose-, Glycerol-, and lactate-utilizing bacteria occurred with the orchardgrass hay ration, whereas the percentage of starch-digesting bacteria was increased significantly (P less than 0.01) in the animals fed the 60-40 ration. A limited number of bacterial strains were isolated from the basal medium without added substrate, most of which were atypical with respect to the predominant rumen bacteria. Growth of these strains, even in complex media, was very slow and limited. Based on these data with isolated strains and colony counts obtained in roll tube medium containing only minerals, resazurin, agar, Na2CO3, and cysteine, the selective medium overestimated the percentage of bacteria able to use a specific energy source. This overestimate was 6 to 7% of the total culturable count.

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Volume and type of medium, carbohydrate concentration, carbohydrate ratios, and inoculum level were investigated as possible factors influencing total colony counts of anaerobic rumen bacteria obtained in roll tubes (18 by 150 mm). Colony counts were lower when the rumen fluid was clarified by centrifugation before inclusion in the medium; however, decreasing the volume of 40% rumen fluid glucose-cellobiose-starch-agar medium (RGCSA medium with 0.025% each of glucose and cellobiose and 0.05% starch, 4 ml per tube) was compared to the clarified rumen fluid medium and non-rumen fluid medium (medium 10) of Caldwell and Bryant (1966), 9 ml of each per tube. Total counts of rumen contents from sheep consuming four different types of rations were higher with the 4 ml of RGCSA medium than with the other two media. Dilution of the basal medium as a result of inoculum volume, as much as 1.5 ml per 4 ml of medium, did not appear to affect total counts. Colony counts and the simplicity of medium preparation and inoculation would favor the present method for routine use in estimating numbers of total viable anaerobic rumen bacteria, especially when large numbers of samples are involved.

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When three sheep were abruptly changed from a ration of 100% orchardgrass hay to 60% cracked corn-40% orchardgrass hay, fed at equal dry-matter intakes, significant increases in concentration were observed in the rumen microbial population. Bacterial numbers (colony counts) per gram of rumen contents did not appear to have stabilized within 21 days after the ration change; however, protozoan numbers per milliliter plateaued after 5 days. The concentration of cellulose-digesting bacteria varied considerably between animals and decreased in all animals with the change. Changes were observed in total and molar percentages of volatile fatty acids, which were typical for the two types of rations. Although the concentration of protozoa increased after the ration change, only minor differences were observed in their percent generic distribution. A significant decrease in rumen volume was measured in two of the three sheep with the change in ration; however, fluid turnover rates were not significantly affected. Rates of rumen dry-matter turnover were slower with the concentrate ration, although rumen dry-matter digestion was increased. Calculation of total bacterial numbers based on total rumen volume completely negated the effect of ration change in one animal, whereas total numbers in the other two animals were still significantly different between rations and very similar between animals. Adjustment of total protozoa numbers did not alter the trends seen previously with concentration values.

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