Gut microbiota, metabolome and hormone profiles associate with pregnancy-related biomarkers in sheep with frozen embryo transfer.

Recipients' gut microbiota and serum metabolites are linked to pregnancy outcomes in frozen embryo transfer (FET), but the underlying integrative mechanisms remain unclear. Here we compared the gut microbiome, serum metabolome, and reproductive hormones between pregnant (P group) and non-pregnant (N group) Small-Tailed Han ewes at three time points: prior to oestrus detection (1st), prior to embryo transfer (2nd), and prior to pregnancy diagnosis (3rd). Of 55 recipients, 24 became pregnant (43.6%). Beta-diversity of the gut microbiota partially separated P from N at 1st (R2 = 0.0597, P = 0.159), 2nd (R2 = 0.0330, P = 0.293), and 3rd (R2 = 0.0062, P = 0.405). On the day of oestrus detection, Prevotellaceae UCG-001 was more abundant in the P group than in the N group (P < 0.05). On the day of embryo transfer, the opportunistic pathogen Escherichia-Shigella was enriched in the N group (P < 0.05). On the day of pregnancy diagnosis, the probiotic Lactobacillus was higher in the P group. Untargeted metabolomics identified 24, 24, and 33 stage-specific differential metabolites. Glycerophospholipid metabolism was consistently enriched across all stages. Notably, oleoyltaurine was a differential metabolite at both 2nd and 3rd (FC = 2.11 and 3.60, respectively; both P < 0.05). At oestrus, serum luteinizing hormone (LH) and prostaglandin E2 (PGE2) were significantly higher in the P group than in the N group (P < 0.05 for both). On the day of embryo transfer, cortisol and prostaglandin F2α (PGF2α) were markedly elevated in the N group (P < 0.01 for both). Microbiota-metabolite integration revealed that oleoyltaurine correlated negatively with Escherichia-Shigella (r = -0.51, P < 0.05) and positively with Lactobacillus (r = 0.64, P < 0.05). Adequate LH/PGE2 priming at oestrus was associated with a receptive peri-transfer period in ovine FET. This endocrine profile aligns with a distinct gut microbial succession and a systemic metabolomic signature, with oleoyltaurine emerging as a candidate metabolite putatively linking gut microbial shifts with systemic metabolic changes. These findings offer a basis for developing multi-target candidate biomarkers to improve recipient selection in sheep FET.