Abstract
In small ruminants, laparotomy for ovarian exploration followed by oocyte collection has been progressively replaced by laparoscopic puncture of follicles, which has become an important method for obtaining oocytes in vivo. However, the superovulation protocols and collection frequency used for laparoscopic ovum pick-up (LOPU) in sheep still require further investigation. This study explored the factors influencing LOPU efficiency in sheep, including Controlled Internal Drug Release (CIDR) for estrus synchronization, FSH source and dose, and recovery intervals. The optimal superovulation protocol (using the CIDR device, a total of 16 mg of long-acting recombinant ovine FSH (LR-FSH) administered in two doses, and a one-month interval between LOPU sessions) was subsequently identified. Ovarian follicles were collected via LOPU from Hu sheep and Altay sheep for transcriptomic and metabolomic sequencing to explore interbreed differences in follicular development. The results indicated that LOPU efficiency was significantly higher in the CIDR group (p < 0.05) and with a 30-day recovery interval (p < 0.05). No significant differences in LOPU efficiency were observed between FSH sources or hormone doses. Furthermore, Hu sheep exhibited significantly higher LOPU efficiency and more antral follicles than Altay sheep. Transcriptomic analysis of follicular contents and metabolomic profiling of follicular fluid revealed that differentially expressed genes and metabolites were primarily enriched in pathways related to steroidogenesis, amino acid metabolism, and fatty acid metabolism. This study provides an optimized treatment protocol to enhance LOPU efficiency and integrates multi-omics analyses to elucidate the molecular mechanisms underlying follicular development differences among various breeds.