Bacterial Communities during Heifer Development and their Impact on the Uterine Environment

Uterine and ruminal bacterial communities are major contributors to reproductive success and feed efficiency by their impact on their respective environments. The first objective is to evaluate the effect of protein supplementation on uterine and ruminal bacterial communities during heifer development. Pre-pubertal Angus heifers were assigned to a level of crude protein supplementation: 10% control, 20%, or 40%. Every 56 days, uterine flushes and rumen content were collected for bacterial DNA extraction and amplification of the 16S gene. Bacterial analyses were performed in R 4.1 and statistical analyses in SAS 9.4. Observed amplicon sequence variants increased overtime and by pubertal status (P < 0.05), each indicating significant clustering by principal coordinate analyses (P < 0.001) in the uterus. Numerous bacterial taxa abundances differed by protein treatment, pubertal status, and day in the uterus and rumen (P < 0.05). The second objective was to evaluate the impact of lipopolysaccharide (LPS) on the bovine uterine endometrium during the follicular and luteal phases. Explants were collected from the endometrium of the contralateral and ipsilateral horns of uteri in mid-luteal or follicular phases. Explants were cultured at standard conditions for 8 hours in 1 mL complete media with or without LPS (1 µg/mL). Extraction of RNA from explants was reverse transcribed to cDNA for targeted RNA sequencing. Normalized total reads counts for gene expression were analyzed in Metaboanalyst 5.0 and SAS 9.4. Seven genes increased in LPS treatment compared to other groups (P < 0.05). Multiple genes were affected by estrous cycle phase and uterine horn, independent of treatment (P < 0.05). Clustering analysis indicated overall gene expression differences with similar clustering between luteal and follicular phase explants of controls, but distinct separate clustering between phases with LPS treatment (P = 0.001). Mucins contributed the most to differences observed between phases by treatment. Therefore, estrous cycle phase resulted in differing overall endometrial gene expression profiles of immune response to LPS treatment. In conclusion, uterine bacterial communities develop through puberty and may be affected by nutrition. Differing immune responses of the endometrium to bacteria between estrous cycle phases may influence uterine environment and pregnancy success.