Application of molecular techniques to assess changes in ruminal microbial populations and protozoal generation time in cows and continuous culture

Apart from formulating rations to ensure economic production of high-quality food products, the latest challenge for ruminant nutritionists is to minimize excretion of environmentally hazardous wastes by cattle overfed protein. Manipulation of the fermentation of the feeds by the microorganisms in the fore stomach of cattle (rumen) offers a potential approach to minimize waste excretion. However, the conventional evaluation of digestibility of feeds with in vitro feed digestion and animal studies do not describe the microbial mechanisms involved in ruminal digestion. Examining microbial diversity using culture-independent techniques such as small subunit rDNA analysis allows greater understanding of the function of complex microbial ecosystems such as the rumen. For the first set of experiments, ruminal and omasal samples were obtained from cows supplemented with different sources of methionine (Met). Prior research had shown that supplementing Met in diets with low crude protein maintained or even increased milk protein production, and this response was mediated by changes in ruminal bacterial and protozoal populations. The objectives in this set of experiments were to study how Met supplementation affected microbial populations in the rumen and if omasal sampling provided a representative sample of ruminal bacteria. The changes in bacterial populations were studied using denaturing gradient gel electrophoresis (DGGE) and ribosomal intergenic spacer length polymorphism (RIS-LP). Protozoal populations in the same samples were compared using microscopic counts and DGGE. The flow of protozoal cells from the rumen was quantified by multiplying protozoal cell count in omasal fluid by the omasal fluid flow (using CoEDTA as a liquid flow marker) or was estimated by rumen pool size of cells multiplied by either the ruminal dilution rate of CoEDTA (after termination of CoEDTA dosing) or the passage rate of Yb-marked particles. Neither the protozoal counts nor the DGGE banding patterns derived from protozoa were different among the dietary treatments or for ruminal vs. omasal samples. As revealed by both DGGE and RIS-LP, bacterial populations clustered by treatments in ruminal and especially in omasal samples. Compared with the omasal fluid flow measurement (16.4 h), protozoal generation time was estimated much more closely using the particulate rather than the fluid passage rate from the rumen (generation times of 15.7 and 7.5 h, respectively).; The next experiments were performed in dual flow continuous culture fermenters which had been modified to retain protozoa. The objectives were to use microbial inhibitors to selectively suppress different functional groups of microbes to quantitatively evaluate their specific roles in methane production and ruminal fermentation. The 4 continuous culture vessels were incubated in 4 periods in a 4 x 4 Latin square design with each period divided into faunated and defaunated sub-periods. The fermenters were fed either no additive, 5% animal-vegetable fat (toxic to ciliate protozoa and methanogens), monensin (2.5 muM, toxic to Gram-positive bacteria but not to Gram-negatives, methanogens or ciliate protozoa) or bromoethanesulfonate (BES, 250 muM, inhibits only methanogens). Protozoal counts were used to calculate their generation times. For the defaunated sub-period, total N flow and digestibilities of NDF and OM were significantly higher and ammonia concentration was lower, but treatment had no effect on these measurements. Protozoal counts were not different between treatments, but BES increased the generation time from 43.2 to 55.6 h. Defaunation did not affect total VFA production but decreased the acetate: propionate ratio; monensin increased isovalerate production in both sub-periods but more in faunated. Biohydrogenation of unsaturated fatty acids was impaired in the defaunated fermenters because flow (mg/d) of oleic, linoleic and linolenic acids were significantly higher in the effluent.