Background: Fixed-time artificial insemination (FTAI) uses exogenous reproductive hormones to regulate the sexual cycle of sows and realize the mating of pigs at a fixed-time without estrus identification. There is a great difference in litter size following FTAI, which may be due to the ovarian status before treatment. However, the specific underlying mechanism remains unclear.Results: We selected replacement gilts (n=104) and divided them into four groups: CON (n=35), EST (n=16), pregnant mare serum gonadotrophin (PMSG)-15D (n=26), and PMSG-20D (n=27). The PMSG-15D and PMSG-20D groups were pretreated with PMSG for 15 and 20 days, respectively. The four groups were then subjected to the same FTAI treatment. Pretreatment with PMSG causes ovulation in the gilts, to artificially promote an estrus cycle. At 42 hours after ALT feeding stopped, The follicular diameter of PMSG-15D group (2.94±0.24 mm) was significantly higher (P<0.05) than the other groups.,and the proportion of gilts with follicular diameter greater than 3 mm in the PMSG-15D group was significantly higher than that in the CON group (37.50% vs 5.88%). The maximum follicular diameter before ovulation in the PMSG-15D group (6.28±0.23 mm) was significantly larger (P<0.05) than that of the PMSG-20D group (5.98±0.59 mm) and EST group (5.60±0.47 mm), indicating that the follicular development of the PMSG-15D group was better than that of the other groups. The ovulation time of the PMSG-15D group was concentrated on the 25th to 26th day of FTAI. The ovulation rate was as high as 94.5% in the optimal insemination window period and was more suitable for FTAI. Compared with the CON (52.70±12.71 h) and the PMSG-20D (47.88±13.98 h) groups, the GnRH to ovulation interval (42.33±5.87 h) was significantly shortened in the PMSG-15D group. The level of follicle-stimulating hormone of gilts in the PMSG-15D group was more uniform and significantly lower than that in the CON group (P<0.01) at 42 h after Altrenogest feeding. The pregnancy rate (80.77%), total litter size (10.44±2.83), and live litter size (10.00±2.61) of the PMSG-15D group were higher than those of the other groups.Conclusions: We feasibly optimized FTAI using PMSG pretreatments. When the ovaries of replacement gilts were in the luteal stage owing to PMSG pretreatment for 15 days, the follicular development and reproductive performance of replacement gilts were significantly improved.