Giving the GnRH drug triptorelin to sheep 48 hours after stopping a progesterone treatment lowered their pregnancy success and changed blood chemicals linked to uterine tissue and inflammation, suggesting it harms embryo implantation.

Gonadotropin-releasing hormone (GnRH) is widely used in the timed artificial insemination protocol for sheep. However, there remains a debate regarding its impact on pregnancy rates during artificial insemination. This study aims to evaluate the effect of GnRH on the pregnancy rates in Huyang ewes, analyze the pre-implantation metabolite changes caused by GnRH using metabolomics, and elucidate the mechanism effect on pregnancy rates. All ewes were administered a vaginal progesterone sponge containing 45&#x2009;mg of flurogestone acetate for 12&#x2009;days and received 330&#x2009;units of equine chorionic gonadotropin (eCG) intramuscularly after sponge removal. The experimental group (<i>n</i>&#x2009;=&#x2009;69) received an intramuscular treatment of 17&#x2009;&#x3bc;g GnRH agonist triptorelin 48&#x2009;h after sponge removal on Day 0, while the control group (<i>n</i>&#x2009;=&#x2009;41) received 1&#x2009;mL of sterile saline solution. All ewes underwent a single vaginal insemination 58&#x2009;h after the withdrawal of the progesterone sponge. The difference in pregnancy rates between the two groups was calculated. Metabolomic analysis was performed on plasma samples collected on Day 7 after the treatment of GnRH agonist. Gonadotropin-releasing hormone (GnRH) treatment significantly reduced the pregnancy rate in the experimental group compared with the control group (72.2 vs. 82.9%, <i>p</i>&#x2009;&lt;&#x2009;0.05). Metabolomic analysis indicated that GnRH treatment affected metabolites involved in collagen synthesis and prostaglandin synthesis in the endometrial tissue, which includes a marked decrease in hydroxyproline amino acid content and a significant increase in corticosterone and prostaglandin D2 lipids and unsaturated fatty acids. In summary, the injection of GnRH agonist Triptorelin 48&#x2009;h after progesterone sponges removal reduces the pregnancy rate of Huyang ewe following artificial insemination. It also affects the metabolite levels related to endometrial collagen and prostaglandin synthesis, harming embryo implantation.