Biodiversity and predicted metabolic functions of the rumen microbiota depending on feeding habits at different stages of the physiological cycle of dairy cows

Under intensified cattle breeding, combined stress factors, in particular, extremely high milk productivity, inconsistency of neuro-humoral and hormonal regulation of feed intake and milk production, negative energy balance, feeds excessive in starch negatively impact the rumen microbiota and, consequently, a cow’s physiology. This paper for the first time shows the phases of dairy cow lactation cycle as an important factor that determines the relative abundance of non-attributable bacteria from the candidate families vadinBE97 and WCHB1-41 which functions are practically not studied. The most pronounced changes in the metabolic potential of the microbiota, namely the inhibition of various metabolic pathways in the rumen chyme, e.g., energy (tricarboxylic acid cycle), protein, carbohydrate, lipid, including volatile fatty acid (VFA) synthesis, occurred in cows during stable and declining milk production phases as compared to dry, fresh and milked cows. The aim of this work is to study the composition and metabolic potential of the rumen microbiome in dairy cows during different physiological phases. The experiment (the JSC Agrofirma Dmitrova Gora, Tver Province, the summer 2020) was performed on 15 black-and-white Holsteinized dairy cows ( Bos taurus ) of the second and third lactations. The cows were assigned to five groups (5 cows each), including the dry cows (on average 30 days before calving, group I), the cows of 20 milking days (group II), of 90 milking days (group III), at day 208 of lactation (group IV), and in late lactation phase when the milk production is declining (day 310, group V). Dairy cows’ diets were calculated using AMTS.Cattle.Professional software in accordance with the accepted requirements. Total DNA was extracted from rumen chyme samples (a Genomic DNA Purification Kit, Fermentas, Inc., Lithuania). The NGS procedure (a MiSeq platform, Illumina, Inc., USA) was performed using primers to the 16S rRNA V3-V4 region and reagents for NGS library preparation (Nextera® XT IndexKit, Illumina Inc., USA), PCR product purification (Agencourt AMPure XP, Beckman Coulter Inc., USA), and sequencing (MiSeq® ReagentKit v2, 500 cycle, Illumina Inc., USA). Bioinformatic analysis was performed with Qiime2 ver. 2020.8 software. Noise sequences were filtered by the Deblur method. The de novo phylogeny was constructed using the MAFFT software package. To analyze the taxonomy, the reference database Silva 138 (https://www.arb-silva.de/documentation/release-138/) was used. Reconstruction and prediction of the functional content of the metagenome was performed using PICRUSt2 software package v.2.3.0 with MetaCyc database for metabolic pathways and enzymes. Total RNA was isolated from the chyme samples (Aurum Total RNA kit, Bio-Rad, United States) followed by cDNA synthesis (iScript RT Supermix kit, BioRad, USA). The relative expression of the bacterial L-lactate dehydrogenase gene Ldh-L and the Ldb 0813 gene associated with D-lactate dehydrogenase synthesis was assessed using quantitative PCR (SsoAdvanced Universal SYBR Green Supermix kit, Bio-Rad, USA). The16S metagenomic sequencing revealed a decrease (p ≤ 0.05) in the rumen bacteria a-diversity in group IV and group V. We have found twelve superphila and phyla of microorganisms. The superphylum Bacteroidota and the phylum Firmicutes we refer to the dominant rumen bacteria (up to 59.94±1.86 and 46.82±14.40 % of the population, respectively). The superphylum Actinobacteriota bacteria not found in lactating cows appeared only in dry cows. The bacteria of the superphylum Armatimonadota disappeared from the rumen of fresh cows and during stable lactation phase, and of the phylum Chloroflexi - during early and stable lactation phases. The cows differed significantly in eight bacterial families, the Muribaculaceae , Prevotellaceae , Erysipelatoclostridiaceae , Oscillospiraceae , Ruminococcaceae , Saccharimonadaceae , and candidate families WCHB1-41 and vadinBE97. The rumen genera Asteroleplasma , Sharpea , Moryella , Oribacterium , Shuttleworthia appeared after calving and persisted in the next phases of lactation. These bacteria were absent in dry cows. The predicted functional capability of 17 metabolic pathways of the microbiome varied (p ≤ 0.01) in cows of different groups. The most pronounced changes, namely the suppression of various metabolic pathways in the rumen chyme, occurred in groups IV and V compared to group I, group II, and group III (p ≤ 0.01). An increase in the expression of the Ldh-L (p ≤ 0.01) and Ldb 0813 (p ≤ 0.05) genes associated with the synthesis of lactate dehydrogenases was characteristic of fresh cows compared to dry cows. There was a significant increase in the expression of the rumen bacteria genes Ldh-L (10.6-fold, p ≤ 0.001) and Ldb 0813 (2.8-fold, p ≤ 0.05) when lactation declined as compared to group IV.