Features of the rumen microbial gene expression in dry and lactating cows

The quality and quantity of feed consumed by lactating and dry cows varies greatly. Dry cows are usually fed a high in roughage and low in compound feed diet, which slows down the rate of fermentation in the rumen. Immediately after calving, cows are fed with low in fiber and high in compound feed diets, which usually have a high fermentation rate due to the high content of easily digestible polysaccharides such as starch. In the present work, for the first time it was established that a change in dairy cows diet, associated with an increase in the proportion of starch, leads to changes in the expression of numerous genes of rumen microorganisms, especially the L-lactate dehydrogenase gene. Our goal was to analyze the expression of genes involved in the key reactions of rumen metabolism depending on the physiological period of the animal and the crude fiber content in the diet. Samples were taken in 2020 at Agrofirma Dmitrova Gora (Tver region) from 15 dairy cows ( Bos taurus ) of the black-and-white Holsteinized breed of the 2nd-3rd lactation. Animals were kept in the same conditions on a tie-up housing. Six cows were selected for the experiment and two groups of animals ( n = 3) were formed: group I - dry cows (on average, 30 days before calving), group II - cows in lactation (day 208 of lactation). Chyme samples (30-50 g from each cow) were taken from the upper part of the ventral rumen sac manually with a sterile probe. Total DNA was isolated from the studied samples using the Genomic DNA Purification Kit (Fermentas, Inc., Lithuania). The rumen bacterial community was analysed by NGS sequencing on the MiSeq platform (Illiumina, Inc., USA) using primers for the V3-V4 region of 16S rRNA. Bioinformatic data analysis was performed using Qiime2 ver. 2020.8 (https://docs.qiime2.org/2020.8/). Taxonomy was analyzed using the Silva 138 reference database (https://www.arbsilva.de/documentation/release-138/). Total RNA was isolated from cicatricial samples using the Aurum Total RNA kit (Bio-Rad, USA). cDNA was obtained on an RNA template (iScript RT Supermix kit, Bio-Rad, USA). The relative expression of genes was analyzed by quantitative PCR, which was carried out on a detection amplifier DT Lite-4 624 (DNA-Technology, Russia). It was shown that a change in the diet of cows, associated with an increase in the proportion of starch, contributed to a decrease in the proportion of cellulolytic bacteria of the families Ruminococcaceae and Lachnospiraceae and an increase in the number of bacteria of the family Prevotellaceae associated with the decomposition of starch. Changes in the expression of bacterial genes depending on the diet have also been shown. Thus, the expression of the L-lactate dehydrogenase gene increased in the group of lactating cows (p ≤ 0.05) receiving a high-starch diet. This is probably due to the high content of lactate in the rumen of cows consuming high concentrations of easily digestible carbohydrates and to the formation of adaptive mechanisms in the microbial community of the rumen. Also, in lactating cows, the expression of the phosphofructokinase gene (p ≤ 0.05), one of the regulatory enzymes of glycolysis, increased. Improving the accessibility of monosaccharides from compound feed contributes to the intensification of the process of glycolysis by rumen microorganisms. In this regard, the Ldh-L gene can be considered as a candidate for biomarkers that can give an idea of the activity of lactic acid synthesis processes and, as a result, a decrease in pH in the rumen of cows.