This study investigated the effect of diet and host on the rumen bacterial microbiome and the impact of an acidotic challenge on its composition. Using parallel pyrosequencing of the V3 hypervariable region of 16S rRNA gene, solid and liquid associated bacterial communities of 8 heifers were profiled. Heifers were exclusively fed forage, before being transitioned to a concentrate diet, subjected to an acidotic challenge and allowed to recover. Samples of rumen digesta were collected when heifers were fed forage, mixed forage, high grain, during challenge (4 h and 12 h) and recovery. A total of 560,994 high-quality bacterial sequences were obtained from the solid and liquid digesta. Using cluster analysis, prominent bacterial populations differed (P≤0.10) in solid and liquid fractions between forage and grain diets. Differences among hosts and diets were not revealed by DGGE, but real time qPCR showed that several bacteria taxon were impacted by changes in diet, with the exception of Streptococcus bovis. Analysis of the core rumen microbiome identified 32 OTU's representing 10 distinct bacterial taxa including Bacteroidetes (32.8%), Firmicutes (43.2%) and Proteobacteria (14.3%). Diversity of OTUs was highest with forage with 38 unique OTUs identified as compared to only 11 with the high grain diet. Comparison of the microbial profiles of clincial vs. subclinical acidotic heifers found a increases in the relative abundances of Acetitomaculum, Lactobacillus, Prevotella, and Streptococcus. Increases in Streptococcus and Lactobacillus likely reflect the tolerance of these species to low pH and their ability to proliferate on surplus fermentable carbohydrate. The acetogen, Acetitomaculum may thereforeplay a role in the conversion of lactate to acetate in acidotic animals. Further profiling of the bacterial populations associated with subclinical and clinical acidosis could establish a microbial fingerprint for these disorders and provide insight into whether there are causative microbial populations that could potentially be therapeutically manipulated.

The gastrointestinal epithelium of the dairy cow and calf faces the challenge of protecting the host from the contents of the luminal milieu while controlling the absorption and metabolism of nutrients. Adaptations of the gastrointestinal tract play an important role in animal energetics as the portal-drained viscera accounts for 20% of the total oxygen consumption of the ruminant. The mechanisms that govern growth and barrier function of the gastrointestinal epithelium have received particular attention over the past decade, especially with advancements in molecular-based techniques, such as microarrays and next-generation DNA sequencing. The rumen has been the focal point of dairy cow and calf nutritional physiology research, whereas the lower gut has received less attention. Three key areas that require discovery-based and applied research include (1) early-life intestinal gut barrier function and growth; (2) how the weaning transition affects function of the rumen and intestine; and (3) gastrointestinal adaptations during the transition to high-energy diets in early lactation. In dairy nutrition, nutrients are seen not only as metabolic substrates, but also as signals that can alter gastrointestinal growth and barrier function. Nutrients have been shown to affect epithelial cell gene expression directly and, in concert with insulin-like growth factor, growth hormone, and glucagon-like peptide 2, play a pivotal role in gut tissue growth. The latest research suggests that ruminal and intestinal barrier function is compromised during the preweaning phase, at weaning, and in early lactation. Gastrointestinal barrier function is influenced by the presence of metabolites, such as butyrate, the resident microbiota, and the microbes provided in feed. In the first studies that investigated barrier function in cows and calves, it was determined that the expression of genes encoding tight junction proteins, such as claudins, occludins, and desmosomal cadherins, are affected by age and diet. Recent evidence suggests that the upper and lower gut can communicate, but the exact mechanisms of gastrointestinal cross-talk in ruminants have not been studied in detail. A deeper understanding of how diet and microbiota can affect growth and barrier function of the intestinal tract may facilitate the development of specific management regimens that could effectively influence gut function.

Little is known about the nature of the rumen epithelial adherent (epimural) microbiome in cattle fed different diets. Using denaturing gradient gel electrophoresis (DGGE), quantitative real-time PCR (qPCR), and pyrosequencing of the V3 hypervariable coding region of 16S rRNA, epimural bacterial communities of 8 cattle were profiled during the transition from a forage to a high-concentrate diet, during acidosis, and after recovery. A total of 153,621 high-quality gene sequences were obtained, with populations exhibiting less taxonomic variability among individuals than across diets. The bacterial community composition exhibited clustering (P < 0.03) by diet, with only 14 genera, representing >1% of the rumen epimural population, differing (P < 0.05) among diets. During acidosis, levels of Atopobium, Desulfocurvus, Fervidicola, Lactobacillus, and Olsenella increased, while during the recovery, Desulfocurvus, Lactobacillus, and Olsenella reverted to levels similar to those with the high-grain diet and Sharpea and Succinivibrio reverted to levels similar to those with the forage diet. The relative abundances of bacterial populations changed during diet transition for all qPCR targets except Streptococcus spp. Less than 5% of total operational taxonomic units (OTUs) identified exhibited significant variability across diets. Based on DGGE, the community structures of epithelial populations differed (P < 0.10); segregation was most prominent for the mixed forage diet versus the grain, acidotic challenge, and recovery diets. Atopobium, cc142, Lactobacillus, Olsenella, RC39, Sharpea, Solobacterium, Succiniclasticum, and Syntrophococcus were particularly prevalent during acidosis. Determining the metabolic roles of these key genera in the rumens of cattle fed high-grain diets could define a clinical microbial profile associated with ruminal acidosis.

Random amplified polymorphic DNA (RAPD) analysis appears to offer a cost-and time-effective alternative to restriction fragment-length polymorphlsm (RFLP) analysis. However, concerns about the ability to compare RAPD results from one laboratory to another have not been addressed effectively. DNA fragments that were amplified by five primers and shown to be reproducibly polymorphic between two oat cultivars (within the Ottawa laboratory) were tested in six other laboratories in North America. Four of the six participants amplified very few or no fragments using the Ottawa protocol. These same participants were able to generate a considerable number of amplified fragments by using their own protocols. The reproducibility of results among laboratories was affected by two factors. First, different laboratories amplified different size ranges of DNA fragments, and, consequently, small and large polymorphic fragments were not always reproduced. Second, although reproducible results were obtained with four of the primers, reproducible resuits were not obtained with the fifth primer, using the same reaction conditions. It is suggested that if the overall temperature profiles (especially the annealing temperature) in-MATERIALS AND METHODS RAPD primers were obtained from Dr.

The objectives of this study were 1) to develop and evaluate the accuracy and precision of a new stand-alone submersible continuous ruminal pH measurement system called the Lethbridge Research Centre ruminal pH measurement system (LRCpH; Experiment 1); 2) to establish the accuracy and precision of a well-documented, previously used continuous indwelling ruminal pH system (CIpH) to ensure that the new system (LRCpH) was as accurate and precise as the previous system (CIpH; Experiment 2); and 3) to determine the required frequency for pH electrode standardization by comparing baseline millivolt readings of pH electrodes in pH buffers 4 and 7 after 0, 24, 48, and 72 h of ruminal incubation (Experiment 3). In Experiment 1, 6 pregnant Holstein heifers, 3 lactating, primiparous Holstein cows, and 2 Black Angus heifers were used. All experimental animals were fitted with permanent ruminal cannulas. In Experiment 2, the 3 cannulated, lactating, primiparous Holstein cows were used. In both experiments, ruminal pH was determined continuously using indwelling pH electrodes. Subsequently, mean pH values were then compared with ruminal pH values obtained using spot samples of ruminal fluid (MANpH) obtained at the same time. A correlation coefficient accounting for repeated measures was calculated and results were used to calculate the concordance correlation to examine the relationships between the LRCpH-derived values and MANpH, and the CIpH-derived values and MANpH. In Experiment 3, the 6 pregnant Holstein heifers were used along with 6 new submersible pH electrodes. In Experiments 1 and 2, the comparison of the LRCpH output (1- and 5-min averages) to MANpH had higher correlation coefficients after accounting for repeated measures (0.98 and 0.97 for 1- and 5-min averages, respectively) and concordance correlation coefficients (0.96 and 0.97 for 1- and 5-min averages, respectively) than the comparison of CIpH to MANpH (0.88 and 0.87, correlation coefficient and concordance correlation coefficient, respectively). The concordance correlation analysis indicated that the ruminal pH data for LRCpH (1- and 5-min averages) vs. MANpH had location shifts that were smaller than those of the CIpH vs. MANpH. However, the scale shift was similar between the LRCpH and the CIpH. The plotted data from both systems closely resembled the line y = x, indicating that both systems were accurate and precise. In Experiment 3, changes in baseline millivolt readings for pH readings after 24, 48, or 72 h of ruminal incubation were not significantly different than zero, indicating that daily standardization of new electrodes was not essential. Results from this study indicate that the LRCpH system can accurately and precisely measure ruminal pH; thus, it provides increased opportunity for researchers to measure ruminal pH and the occurrence of ruminal acidosis in unrestrained cattle.

Due to their high energy requirements, high-yielding dairy cows receive high-grain diets. This commonly jeopardises their gastrointestinal health by causing subacute ruminal acidosis (SARA) and hindgut acidosis. These disorders can disrupt nutrient utilisations, impair the functionalities of gastrointestinal microbiota, and reduce the absorptive and barrier capacities of gastrointestinal epithelia. They can also trigger inflammatory responses. The symptoms of SARA are not only due to a depressed rumen pH. Hence, the diagnosis of this disorder based solely on reticulo-rumen pH values is inaccurate. An accurate diagnosis requires a combination of clinical examinations of cows, including blood, milk, urine and faeces parameters, as well as analyses of herd management and feed quality, including the dietary contents of NDF, starch and physical effective NDF. Grain-induced SARA increases acidity and shifts availabilities of substrates for microorganisms in the reticulo-rumen and hindgut and can result in a dysbiotic microbiota that are characterised by low richness, diversity and functionality. Also, amylolytic microorganisms become more dominant at the expense of proteolytic and fibrolytic ones. Opportunistic microorganisms can take advantage of newly available niches, which, combined with reduced functionalities of epithelia, can contribute to an overall reduction in nutrient utilisation and increasing endotoxins and pathogens in digesta and faeces. The reduced barrier function of epithelia increases translocation of these endotoxins and other immunogenic compounds out of the digestive tract, which may be the cause of inflammations. This needs to be confirmed by determining the toxicity of these compounds. Cows differ in their susceptibility to poor gastrointestinal health, due to variations in genetics, feeding history, diet adaptation, gastrointestinal microbiota, metabolic adaptation, stress and infections. These differences may also offer opportunities for the management of gastrointestinal health. Strategies to prevent SARA include balancing the diet for physical effective fibre, non-fibre carbohydrates and starch, managing the different fractions of non-fibre carbohydrates, and consideration of the type and processing of grain and forage digestibility. Gastrointestinal health disorders due to high grain feeding may be attenuated by a variety of feed supplements and additives, including buffers, antibiotics, probiotics/direct fed microbials and yeast products. However, the efficacy of strategies to prevent these disorders must be improved. This requires a better understanding of the mechanisms through which these strategies affect the functionality of gastrointestinal microbiota and epithelia, and the immunity, inflammation and 'gastrointestinal-health robustness' of cows. More representative models to induce SARA are also needed.

Rumen health is of vital importance in ensuring healthy and efficient dairy cattle production. Current feeding programs for cattle recommend concentrate-rich diets to meet the high nutritional needs of cows during lactation and enhance cost-efficiency. These diets, however, can impair rumen health. The term "subacute ruminal acidosis" (SARA) is often used as a synonym for poor rumen health. In this review, we first describe the physiological demands of cattle for dietary physically effective fiber. We also provide background information on the importance of enhancing salivary secretions and short-chain fatty acid absorption across the stratified squamous epithelium of the rumen; thus, preventing the disruption of the ruminal acid-base balance, a process that paves the way for acidification of the rumen. On-farm evaluation of dietary fiber adequacy is challenging for both nutritionists and veterinarians; therefore, this review provides practical recommendations on how to evaluate the physical effectiveness of the diet based on differences in particle size distribution, fiber content, and the type of concentrate fed, both when the latter is part of total mixed ration and when it is supplemented in partial mixed rations. Besides considering the absolute amount of physically effective fiber and starch types in the diet, we highlight the role of several feeding management factors that affect rumen health and should be considered to control and mitigate SARA. Most importantly, transitional feeding to ensure gradual adaptation of the ruminal epithelium and microbiota; monitoring and careful management of particle size distribution; controlling feed sorting, meal size, and meal frequency; and paying special attention to primiparous cows are some of the feeding management tools that can help in sustaining rumen health in high-producing dairy herds. Supplementation of feed additives including yeast products, phytogenic compounds, and buffers may help attenuate SARA, especially during stress periods when the risk of a deficiency of physically effective fiber in the diet is high, such as during early lactation. However, the usage of feed additives cannot fully compensate for suboptimal feeding management.

With the development of genetic maps and the identification of the most-likely positions of quantitative trait loci (QTLs) on these maps, molecular markers for lodging resistance can be identified. Consequently, marker-assisted selection (MAS) has the potential to improve the efficiency of selection for lodging resistance in a breeding program. This study was conducted to identify genetic loci associated with lodging resistance, plant height and reaction to mycosphaerella blight in pea. A population consisting of 88 recombinant inbred lines (RILs) was developed from a cross between Carneval and MP1401. The RILs were evaluated in 11 environments across the provinces of Manitoba, Saskatchewan and Alberta, Canada in 1998, 1999 and 2000. One hundred and ninety two amplified fragment length polymorphism (AFLP) markers, 13 random amplified polymorphic DNA (RAPD) markers and one sequence tagged site (STS) marker were assigned to ten linkage groups (LGs) that covered 1,274 centi Morgans (cM) of the pea genome. Six of these LGs were aligned with the previous pea map. Two QTLs were identified for lodging resistance that collectively explained 58% of the total phenotypic variation in the mean environment. Three QTLs were identified each for plant height and resistance to mycosphaerella blight, which accounted for 65% and 36% of the total phenotypic variation, respectively, in the mean environment. These QTLs were relatively consistent across environments. The AFLP marker that was associated with the major locus for lodging resistance was converted into the sequence-characterized amplified-region (SCAR) marker. The presence or absence of the SCAR marker corresponded well with the lodging reaction of 50 commercial pea varieties.