An Improved Nonselective Culture Medium for Ruminal Bacteria and Its Use in Determining Diurnal Variation in Numbers of Bacteria in the Rumen

A basal (BC) medium devoid of added carbohydrates, a complete (CC) medium containing nine carbohydrates were developed for enumerating rumen bacteria The colony counts on the BC medium were 85 to 100% of those obtained on the CC medium These colonies were pinpoint size (less than or equal to mm in diameter) but increased in size (2 to 5 mm in diameter) when carbohydrates were subsequently added With the CC medium or other media tested, the colony counts were 20 to 50% higher on plates than on roll tubes and were about 35% of the direct cell counts The lower colony counts on roll tubes were shown to result primarily from the loss of viability due to heat stress The DC media were found by plating techniques to be suitable for differentiating mixed rumen bacterial populations into subgroups based upon carbohydrate utilization as shown by differences in subgroup profiles found within solid and liquid fractions of rumen contents, within rumen contents from animals fed high-forage and high-grain diets, and by correct colony formations by pure cultures of rumen bacteria on appropriate DC media With simple modifications and use of an anaerobic glove box, replica plating methods and the CC and DC media were found to be a suitable means of rapidly determining the range of utilizable carbohydrate energy sources of rumen bacteria

Differential carbohydrate media and anaerobic replica plating techniques in delineating carbohydrate-utilizing subgroups in rumen bacterial populations.

Establishment of microbes in the young ruminant is primarily dependent upon its exposure to an adult animal and having a rumen environment compatible with growth of the microorganisms. The major portion of the rumen microbial population consists of strictly anaerobic bacteria and ciliate protozoa, which appear to account for most of the fermentative activity in this organ. Anaerobic fungi have recently been found also to be normal inhabitants of the rumen; however, they occur in lower concentrations and their ecological niche in the rumen fermentation is uncertain (Chapter 4). Microbial numbers and the composition of the population are affected by a number of factors, of which diet is probably one of the most important. Smaller numbers of facultatively anaerobic bacteria (which may be of importance in very young ruminants), aerobic bacteria, flagellate protozoa and mycoplasmas are also present; however, their contribution to the overall fermentation is considered minimal.

Development of, and natural fluctuations in, rumen microbial populations

Differential carbohydrate media and anaerobic replica plating techniques were used to assess the degrees of diurnal variations in the direct and viable cell counts as well as the carbohydrate-specific subgroups within the mixed rumen bacterial populations in cattle fed maintenance (metabolizable energy) levels of either a high-forage or a high-concentrate diet once daily. The rumen was sampled at 1 h before feeding and 2, 4, 8, 12, and 16 h after feeding, and selected microbiological parameters of the isolated bacterial populations were assessed. Corresponding samples of ruminal fluid were assayed for fermentation acids, carbohydrate, ammonia, and pH changes. The data showed that regardless of diet, total bacterial numbers remained fairly constant throughout the day. The number of viable bacteria declined 40 to 60% after feeding and then increased to a maximum at 16 h postfeeding. Changes occurred in the carbohydrate-specific subgroups within the bacterial populations, and some of the changes were consistent with a predicted scheme of ruminal feedstuff carbohydrate fermentation. Regardless of diet, however, soluble-carbohydrate-utilizing bacteria predominated at all times. Xylan-xylose and pectin subgroups respectively comprised about one-half and one-third of the population when the high-forage diet was given. These subgroups, along with the cellulolytics, constituted lesser proportions of the population when the high-concentrate diet was given. The cellulolytic subgroup was the least numerous of all subgroups regardless of diet but followed a diurnal pattern similar to that predicted for cellulose fermentation. There were few diurnal variations or differences in bacterial cell compositions and ruminal fluid parameters between diets. The observed similarities and dissimilarities of the rumen bacterial populations obtained when the two diets were given are discussed. The data are consistent with the versatility and constancy of the rumen as a stable, mature microbial system under the specific low-level feeding regimens used.

Diurnal variations in bacterial numbers and fluid parameters in ruminal contents of animals fed low- or high-forage diets.

SUMMARY: In sheep fed once daily, the concentrations of micro-organisms in the rumen changed with the time after feeding, some organisms fluctuating in numbers more than others; peak concentrations were reached at different times for different organisms. These changes in concentration were reflected in changes in the proportion of dividing cells, for example, in Dasytricha ruminantium this proportion varied from less than 0.2% throughout most of the day to a maximum of 23 % a few hours before feeding; for Entodinium spp. the variation was less: it was possible to calculate the minimum doubling time for these latter organisms as about 5 hr.
One animal at different times, or different animals, on the same ration and dietary regime had very different rumen microbial populations, these differences being particularly marked in the case of some organisms. Reasons why these marked differences in microbial population are not reflected in similarly marked differences in over-all rumen metabolism are discussed.
Feeding different quantities of the same ration had little effect on the concentration of rumen microbes, provided the ration was above a minimal level; it is, however, suggested that output of microbial cells from the rumen may have varied with the amount of feed given. Starvation for a few days or prolonged under-nutrition had a marked effect, some organisms being drastically reduced in numbers or dying out completely. When the qualitative nature of the diet was changed, about 10 days were needed to complete the major adjustments in the rumen microbial population.

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Some factors influencing the rumen microbial population.

A single ration made up of commercial sheep cubes (20 per cent. protein) together with oaten and lucerne chaff was fed to five sheep in five different ways. A Latin square design was used. Measurements were made on the ruminal population, the metabolism within the rumen, and the overall nitrogen balance and dry matter digestibility. Each of the different treatments resulted in a distinctive pattern in the ruminal population, pH, ammonia nitrogen, and volatile fatty acid levels. There were significant differences in dry matter digestibility and nitrogen retention. The daily ration given as a single feed gave the lowest dry matter digestibility and nitrogen retention. It also caused the greatest fall in pH values and bacterial counts, and the lowest protozoal counts. The highest ruminal ammonia nitrogen levels were also associated with this treatment. The best performance was obtained by feeding the concentrate in the morning and roughage in the afternoon, or feeding the whole ration in four equal portions at 2-hr intervals. The other two treatments – half ration morning and afternoon, or chaff in the morning and concentrates in the afternoon – gave intermediate results. Some implications of the various data are discussed, and the strong influence of feeding pattern is stressed.

Ruminal flora studies. VIII. The influence of rate and method of feeding a ration upon its digestibility, upon ruminal function, and upon the ruminal population

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Ruminal flora studies. VIII. The influence of rate and method of feeding a ration upon its digestibility, upon ruminal function, and upon the ruminal population