PMSG stimulates follicular growth and ovulation[15]. Some experimental studies showed that the estrus rate of gilts injected with 725–1000 IU PMSG was 70–100%[16], whereas those injected with 363–600 IU PMSG was only 25–52%[17, 18]. Therefore, a dose of 1000 IU PMSG was selected for pretreatment in this study. The follicle average diameter of gilts that were pretreated with PMSG before FTAI reached a maximum after five days. At the same time, E2, LH, and FSH also increased sharply on the fifth day, which led to ovulation between 5–6 days after PMSG treatment. Thus, PMSG pretreatment was shown to induce an estrus cycle in gilts and synchronize follicular development and ovulation time preliminarily. Based on the reproductive cycle of gilts[19], we assumed that the ovaries of gilts were in the luteal phase on the 10th day after ovulation (15–16 days after PMSG pretreatment), and in the follicular phase on the 15th day after ovulation (20–21 days after PMSG pretreatment). Therefore, FTAI treatment was selected after 15 and 20 days of PMSG pretreatment to explore the influence of different ovarian states on the FTAI effect in replacement gilts.
When the FTAI procedure was started, the average size of follicles in the PMSG-15D group was below 3 mm, and no developing or mature follicles were present, indicating that the ovarian state of gilts in the PMSG-15D group was in line with the expectation that the ovarian follicle would be in the luteal phase after 15 days of PMSG treatment. The follicle sizes of the gilts in the PMSG-20D and EST groups were similar and larger than those in the PMSG-15D and CON groups, indicating that developing follicles appeared on the ovaries of gilts in the PMSG-20D group and that the ovarian state in the PMSG-20D group was in the follicular stage.On the third day after ALT treatment, the follicles of the four groups were synchronized to the same level and the diameter of the follicles was less than 3 mm, indicating that ALT played a role in effectively preventing the development of small and medium follicles, to control synchronous estrus[20]. PMSG has follicle-stimulating activity and promotes follicular development[21]. After the PMSG injection, follicular diameter tended to increase with time. GnRH effectively improves LH secretion and promotes the final maturation and ovulation of dominant follicles. After intramuscular injection of GnRH, the follicular diameter of gilts in the four groups increased to the maximum, and the follicle reached maturity. On the second day after mating, the follicular diameter of all gilts dropped below 3 mm again, marking the end of ovulation.
Although the follicular development trend of the four groups was similar, the follicular development level was different at different time points. The study found that PMSG-15D had the largest follicle diameter at the time of PMSG injected compared with other groups. This is consistent with the results of Soede[22]. When gilts were fed with Regumate® (trade name of ATL) for 18 days during the pre-follicular phase, the average follicular size of the treated gilts increased by 0.5 mm compared to the control group, indicating that follicular selection and development had been differentiated after treatment with ALT. There is a continuous growth and atresia of ovarian follicles during days 7 to 15 of the estrous cycle, without evidence of follicular dominance, resulting in a stable size and number of small follicles on the ovaries. Moreover, ALT was found to have less inhibitory effect on follicular growth than endogenous progesterone (with or without ALT)[23, 24]. Thus the application of ALT may not adversely affect the follicles during luteal phase.
After the effects of PMSG and GnRH, the differences in follicular development between the gilts in each group were significantly increased. The maximum follicular diameter before ovulation in the PMSG-15D group was significantly larger than that in the EST group and the PMSG-20D group (Table 1). It can be seen that the gilts in the PMSG-15D group (that is, the initial state is in the luteal phase) undergoing FTAI, showed the best growth ability in terms of the degree of follicular development. Soede found that the size of follicles before ovulation was positively correlated with luteal weight on the fifth day after ovulation[25].Similarly, a positive correlation was also observed in cows and ewes between the size of the follicle before ovulation and the size of the corpus luteum[26]. In cattle, the oocytes from larger follicles showed faster embryonic developmental ability than those from smaller follicles, which demonstrated the relationship between follicular size and oocyte capacity[27–29]. From these results, it can be inferred that larger follicles may have more capable oocytes, and may form larger or more functional luteal bodies after ovulation, potentially improving the subsequent fertility.
The results showed that the variation range of follicle size in PMSG-15D group at PMSG treatment and pre-ovulation was smaller than that in other groups, and the difference value was only 0.94 mm and 0.81 mm, indicating that the gilts in PMSG-15D group had a better synchronism in follicle development after FTAI treatment. The results also showed that the distribution of ovulation time of gilts in the four groups was different. All the gilts in PMSG-15D group completed ovulation within 36 hours, and the distribution of ovulation time was more concentrated than that in CON group, EST group and PMSG-20D group, indicating that the ovulation synchrony was better in gilts pretreated with PMSG for 15 days. The reason for the difference in follicular development and ovulation time was closely related to the hormone level in gilts. To verify the hormone level change, blood samples were collected at 42 hours after stopping feeding ALT.The level of FSH in the PMSG-15D group was the lowest, and it had the highest uniformity (Fig. 9).This may be due to that FSH of gilts in PMSG-15D group were not only inhibited by exogenous ATL supplementation, but also inhibited by endogenous progesterone secreted in luteal stage[24].Subsequently, follicular development and ovulation in gilts were further improved by PMSG and GnRH treatment.
Successful insemination of gilts depends primarily on the ovulation and inseminationtime[30]. In theory, sperm show optimal fertility at 24 hours after insemination[31, 32]. However, if insemination is done too late, the oocytes would be degraded[33], which negatively impacts the sow fertility, including reductions in pregnancies, fertilization rates[34], and embryo survival[35]. Since the artificial insemination of FTAI was performed in the afternoon and morning on the 24th and 25th day, respectively, the optimal ovulation time was concentrated on the 25th day. This experiment found that the interval between GnRH injection to ovulation of PMSG-15D group was significantly shortened, and the ovulation rate (94.5%) was the highest on the 25th day, indicating that gilts in PMSG-15D group ovulated earlier than the other groups, and were more suitable for the current FTAI procedure. However, we also found that 18.9% of gilts in the PMSG-20D group ovulated after 25 days (Fig. 6), indicating that the ovulation time was too late.
In the EST group, the average follicle diameter was not the largest when treated with PMSG, but the percentage of gilts with an average follicle diameter above 3 mm was the highest. Therefore, the time from follicular development to ovulation in the EST group of gilts did not need to be too long after stoppong feeding of ATL, leading to a significant shorter intrval between GnRH injection to ovulationthan the other groups. However, gilts in the EST group had smaller preovulatory follicles, poor ovulation synchronization, and low reproductive efficiency.The gilts in EST group was in the follicular stage at the beginning of ATL treatment, and the follicles on the ovary were mainly composed of large follicles[23]. Previous study had found a relatively low but constant concentration of LH during ALT treatment may cause the growth of large follicles[36]because large follicles are more sensitive to LH than small follicles[37]. In cows, ALT treatment for synchronization the estrous cycle had been found to prolong the lifespan of the dominant follicle, preventing it from becoming atretic[36]. Thus, if the large follicle was not atresia and degraded during ALT treatment, a portion of the follicle will continue to develop until ovulate after stopping feeding ALT. This may lead to excessive maturation of pig oocytes, which may negatively affect the maturation and fertilization of oocytes[38].
In addition,Almeida found that the ovulation time depends on the duration of estrus, and there was a strong correlation between estrus duration and ovulation interval (r = 0.57, P = 0.0001)[39]. In this experiment, the CON, PMSG-15D, and PMSG-20D groups all showed a positive correlation between estrus duration and the interval from estrus to ovulation, while only the EST group showed no correlation (Fig. 7). Therefore, although the EST group ovulated earlier, it did not follow the correlation rule and presented with the worst follicular development, poor synchronization of ovulation time, and other factors explained the poor reproductive performance of the gilts in the EST group in this experiment.
Ultrasonic monitoring results of the PMSG-15D group showed that follicular development and ovulation time were superior to those of other groups since the initial state ovary was in the luteal phase. In subsequent FTAI, the reproductive performance of the PMSG-15D group improved, including the pregnancy rate (80.77%) and litter size (10.44 ± 2.83) (Table 3). However, the pregnancy rate and litter size of the PMSG-15D group did not increase significantly. This may be mainly because litter size is a compound variable influenced by many factors, such as follicular mass[26], ovulation number[40], and uterine volume[41]. Hazeleger found that sows with feed restriction had smaller follicles and lower ovulation rates, indicating that follicle diameter before ovulation could reflect the number of ovulations on the ovaries[42]. Furthermore, the ovulation rate is moderately heritable and positively correlated with the litter size of gilts. Therefore, the lack of significant improvement in the litter rate in the PMSG-15D group was likely due to the increased ovulation rate and the embryo crowding caused by the limited uterine volume of gilts[43–45].