Seasonal reproduction in sheep reduces reproductive efficiency. Melatonin (MT) plays a crucial role in reproductive processes. The purpose of this study was to assess the effects of a 5-day MT implant pretreatment on estrus synchronization and reproductive performance in sheep during seasonal anestrus. A total of 40 multiparous Mongolian sheep were selected and randomly divided into two groups. In the MT group (n = 20), the ewes received an MT implant for 5 days, and then, they were given a progesterone (P4)-containing vaginal sponge for 14 days with equine chorionic gonadotropin (eCG) administered (330 I.U. per ewe; I.M.) at sponge removal. Control (CON) ewes (n = 20) were similarly treated but did not receive MT implants. The results demonstrated that MT implantation significantly improved serum levels of total antioxidant capacity (T-AOC), superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), and glutathione peroxidase (GSH-Px), increased post-ovulatory luteal diameter and serum P4 levels, and reduced ovarian apoptosis. Compared with the CON group, the MT group showed significantly higher pregnancy (68.23% vs. 50.59%) and lambing rates (63.53% vs. 47.06%; number of lambed ewes/number of total ewes) following cervical-timed artificial insemination. Ovarian transcriptome analysis revealed 522 differentially expressed genes (DEGs) in the MT group compared with the CON group, including 355 upregulated and 167 downregulated DEGs. In addition, MT significantly enhanced proliferation and inhibited apoptosis in cultured granulosa cells (GCs) and luteal cells (LCs) in vitro. Moreover, it enhanced the antioxidant capacity of GCs and LCs probably by activating the NRF2 signaling pathway as well as stimulating steroid hormone synthesis. In conclusion, MT implantation 5 days before applying the conventional P4-eCG protocol enhances ovine reproductive outcomes during seasonal anestrus. MT implantation has a beneficial role on the growth and function of ovarian cells. These findings offer novel evidence supporting the functional role of MT in mammalian reproduction, and would be informative for optimizing estrus synchronization in sheep.

To improve the efficiency of prostaglandin F_2α (PG)-based estrus synchronization in sheep, this study assessed the effect of the gonadotropin-releasing hormone (GnRH) included in the long-interval PG treatment regimen for sheep estrus synchronization during the breeding season. In experiment 1, 30 multiparous Mongolian sheep (3–4.5 years old) were randomly divided into three groups. In the progesterone (P4)–equine chorionic gonadotropin (eCG) group (P4-eCG, n = 10), the ewes were synchronized with intravaginal P4 sponges for 14 days, and received an injection of 330 IU of eCG at sponge withdrawal. In the PG group (n = 10), the ewes received two doses of 0.1 mg PG with a 14 day interval. In the PG-GnRH-PG group (n = 10), the ewes were synchronized by two doses of 0.1 mg PG with a 14 day interval like that in the PG group, but received 50 μg of GnRH 7 days after the first injection of PG. It was found that, at the end of treatment, the number of corpus luteum on the ovaries and the concentration of the serum P4 in the PG-GnRH-PG group were significantly higher than that of the PG and P4-eCG groups. In experiment 2, 59 multiparous Mongolian sheep (3–4.5 years old) were assigned to three groups, like in experiment 1 (n = 20, 20 and 19 for the P4-eCG, PG and PG-GnRH-PG groups, respectively). The estrus of ewes in the PG-GnRH-PG group was more synchronous compared to the PG group. After insemination of the estrus ewes, the pregnancy rate was numerically but not significantly higher in the PG-GnRH-PG group than that in the P4-eCG and the PG groups. In a field test, 285 multiparous Hu sheep (3–4.5 years old) were randomly assigned to a P4-eCG group (n = 142) and PG-GnRH-PG group (n = 143). Timed artificial insemination showed no significant differences in the rates of pregnancy and lambing between the PG-GnRH-PG and P4-eCG groups. We conclude that the addition of GnRH in the long-interval PG protocol may improve the efficiency of PG-based estrus synchronization, and would represent a potential alternative to the conventional P4-eCG based protocol during the breeding season in sheep.