Characterization of the Bacterial Communities Associated with 180-day-old Calf Fetuses and Their Dams Fed High-concentrate or High-forage Diets to Alter Maternal Gut Microbiome

Abstract The objective of this study was to determine whether feeding high-concentrate (HC) or high-forage (HF) diets alters the gut microbiome of beef heifers and how these diet-induced microbial changes influence feto-maternal microbial crosstalk at mid-gestation. For this, 20 Angus crossbred heifers (approximately 14 months of age) fed either high forage (HF, 75% forage, 25% concentrate) or high concentrate (HC, 75% concentrate, 25% forage) (n = 10 per group) diets were euthanized for fetal harvesting. Both diets were formulated to meet nutrient requirements and to achieve a targeted daily gain of 0.45 kg per day. Heifers were individually fed using an Insentec feeding system and were fed on their respective diets for 90 days prior to artificial insemination with male-sexed semen from a single sire until euthanization at day 180 of gestation. A total of 263 samples representing 13 distinct sample types were analyzed using 16S rRNA (V4) amplicon sequencing with a host DNA blocking agent. Maternal samples included ruminal fluid, intestinal fluid, vaginal and uterine swabs, and whole blood collected from HF- and HC-fed dams. Fetal-associated samples included fetal ruminal fluid, intestinal tissue, allantoic and amniotic fluid, cord blood, caruncle, cotyledon tissue, and meconium. Multiple environmental (e.g., room air, tap water, and outer uterine swabs) and DNA extraction control samples were included to assess potential contamination. Sequencing of maternal samples revealed significant differences (P 0.05) in microbial community structure, diversity, and composition in the ruminal and intestinal microbiota of HF- and HC-fed dams, confirming diet-induced modulation of the gut microbiome. However, no significant differences (P 0.05) were observed in the vaginal, uterine, or blood microbiota between dietary groups. Fetal and environmental control samples were clearly distinct from each other and from maternal samples (PERMANOVA, R² = 0.503, P 0.00001), confirming minimal environmental contamination. No significant differences (P 0.05) in community structure, richness, or alpha diversity were observed between fetuses from HF- and HC-fed dams; however, site-specific bacterial communities were evident across fetal sample types. SourceTracker2 analysis is underway to identify potential maternal and environmental contributors to these fetal bacterial profiles. Overall, our findings indicate that microbial exposure of the calf intestine may occur during mid-gestation, but maternal gut microbiome alterations induced by HF or HC diets did not significantly impact the bacterial communities associated with 180-day-old bovine fetuses.