Dairy cattle are globally important agricultural animals. Central to their biology is the rumen, which houses an essential microbial community, or microbiome, important for providing nutrition from otherwise host-inaccessible dietary components. The rumen environment is noted for its substantial spatial heterogeneity, as illustrated by the stratification into ruminal solid and liquid phases. A third microbiota found directly attached to the ruminal epithelium (the epimural microbiota) also exists but is less well understood because of challenges in sampling the ruminal epithelium. As a result, our understanding of the epimural microbiota is based on analyses of cannulated animals sampled at a single location-the ventral sac-and does not account for other ruminal locations, which may have importance for overall rumen function. To address this knowledge gap, we hypothesize that the epimural microbiota at different ruminal locations differs due to known morphological, physiological, and functional differences across the geographic spread of the rumen epithelium. Here, we characterized bacterial epimural communities at different sites within 8 lactating Holstein dairy cows using 16S rRNA gene sequencing. Four different sites were sampled via rumen tissue biopsy: cranial sac (CS), ventral sac (VS), caudodorsal blind sac (CDBS), and caudoventral blind sac (CVBS). We found that locations differed in both epimural bacterial community structure and composition, with the CDBS community displaying the greatest diversity. Across all sampling sites, epimural bacterial communities were dominated by members of the phyla Bacteroidetes, Firmicutes, and Proteobacteria. Bacteria within Prevotellaceae, Butyrivibrio, Campylobacter, Mogibacterium, and Desulfobulbus all showed high relative sequence abundance and differential distributions according to sample location. There appears to be a core epimural microbiota present across all locations in all cows, although relative abundance was highly variable. The difference in relative abundance in epimural microbial communities, perhaps influenced by host physiology and the diversity within rumen contents, likely has important consequences for nutrition acquisition and general health. To the best of our knowledge, this work represents the first characterization of the ruminal epimural microbiota across different epithelial locations for any bovine ruminant.

Maternal consumption of a high-fat diet (HFD) during pregnancy has established adverse effects on the developing neonate. In this study, we aimed to investigate the effect of an HFD on the murine mammary gland during midlactation. Female C57BL/6J mice were placed on either a low-fat diet (LFD/10% fat) or HFD (60% fat) from 3 wk of age through peak lactation (lactation day 11/L11). After 4 wk of consuming either the LFD or HFD, female mice were bred. There were no significant differences in milk yield between treatment groups, which was measured from L1 to L9. On L10, mice were subjected to an overnight fast and then euthanized on the morning of L11. Total RNA was isolated from inguinal mammary glands for whole transcriptome sequencing. We found 628 genes that were differentially expressed between the treatment groups. Notably, HFD feeding resulted in expression alterations of genes involved in collagen and cytoplasmic components. Additionally, genes related to inflammatory and immune responses were also impacted. Differential expression in gene transcript isoforms between the treatment groups was detected in three genes related to mammary duct development. This study sheds light as to how an HFD may affect the mammary gland transcriptome during midlactation.

In lactating dairy cattle, the corpus luteum (CL) is a dynamic endocrine tissue vital for pregnancy maintenance, fertility, and cyclicity. Understanding processes underlying luteal physiology is therefore necessary to increase reproductive efficiency in cattle. A common technique for investigating luteal physiology is reversetranscription quantitative PCR (RT-qPCR), a valuable tool for quantifying gene expression. However, reference-gene-based RT-qPCR quantification methods require utilization of stably expressed genes to accurately assess mRNA expression. Historically, selection of reference genes in cattle has relied on subjective selection of a small pool of reference genes, many of which may have significant expression variation among different tissues or physiologic states. This is particularly concerning in dynamic tissues such as the CL, with its capacity for rapid physiologic changes during luteolysis, and likely in the less characterized period of CL maintenance during pregnancy. Thus, there is a clear need to identify reference genes well suited for the bovine CL over a wide range of physiological states. Whole-transcriptome RNA sequencing stands as an effective method to identify new reference genes by enabling the assessment of the expression profile of the entire pool of mRNA transcripts. We report the identification of 13 novel putative reference genes using RNA sequencing in the bovine CL throughout early pregnancy and luteolysis: RPL4,