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Studies on the induction of pregnancy and the number of fetuses during pregnancy in rats

Department of Companion Animal, Seoul Hoseo Occupational Training College, Seoul 07583, Korea Department of ICT, Baekseok University, Cheonan 31065, Korea Animal Biotechnology, Graduate School of Animal Life Convergence Science, Institute of Genetic Engineering, Hankyong National University, Anseong 17579, Korea Original Article J Anim Reprod Biotechnol 2020;35:232-238 pISSN: 2671-4639 • eISSN: 2671-4663 https://doi.org/10.12750/JARB.35.3.232 JARB Journal of Animal Reproduction and Biotechnology


INTRODUCTION
In mammals, the female estrus cycle refers to the periodic physiological state of estrogen secretion and LH surge followed by ovulation from the mature ovary of the ovary (Shiota et al., 1993;Seong et al., 1992). In other words, the estrus cycle in mammals is a physiological phenomenon manifested by the interaction of the hypothalamic-pituitary-ovarian cycle, which is regulated by the environment and endocrine factors (Mahoney et al., 2010).
In the case of rodents, morphological changes such as keratinization occur as the vaginal epithelial cell layer periodically changes due to the effects of estrogens secreted by follicles during the estrus. Vaginal cytology is a method that can confirm the estrus cycle through these cell layer changes (Byers et al., 2012). It is classified into four stages: proestrus, estrus, metestrus, and diestrus, based on the difference in the cell type and ratio of each cell that appears at each stage in the process of repetitive estrus cycle (Akinloye and Oke, 2010; Byers et al., 2012).
In addition, periodic estrus cycles are repeated with estrogen secreted by follicles during estrus and estrus, and progesterone secreted from the ovary luteum during late estrus and estrus (Wallen, 1990;Mahoney et al., 2010).
In addition, ovulated mature follicles are composed of granulosa cells, theca interna cells, and blood components to form a corpus luteum and secrete progesterone from luteal cells (Seong et al., 1992).
In rodents including rats, after ovulation, the newly formed corpus luteum degenerates and the next follicle develops. If stimulated by males, the corpus luteum formed after ovulation does not degenerate, but progesterone is secreted from luteal cells to maintain pregnancy (Seong et al., 1992;Shiota et al., 1993;Seong, 1996).
However, few studies have been conducted to investigate changes in the number of fetuses during pregnancy and finally the number of individuals induced to delivery in preparation for the number of luteal ovaries produced after ovulation. Therefore, this study aimed at examining the estrus cycle using vaginal smearing in rats, examining the number of fetuses during pregnancy after mating, and finally examining how fetal numbers are changing until delivery. The mating was conducted with males of the same species to change the number of fetuses by pregnancy period.

General information
We used 40 females and 10 males of mature Wisterbased rats, aged 14-16 weeks, with a weight of about 260-280 g in females and about 300-340 g in males. Contrast cycle is adjusted to artificial lighting at 14:10 (Light: 7:00-21:00, Dark: 21:00-7:00), and feed (pellet feed, rat feed) and fresh water are supplied daily without limitation.

Observation of estrus cycle by vaginal smearing
The division of the estrus cycle by vaginal smear was classified by referring to the Park et al. (2017) method.
That is, from 10 to 12 am every day, the subject was ob-

Induction of pregnancy
In the afternoon of proestrus, the male and female were combined in a male cage to induce pregnancy. The next morning, an individual whose vaginal sperm was identified was designated as the 0th day of pregnancy.

Changes in the number of fetuses during pregnancy
On the 8th and 10th, 12th, 14th, 16th, and 18th days of pregnancy and at the time of delivery (Day 21), 3-4 animals were killed immediately by cervical dislocation. The number of fetuses in the uterus was checked, and some subjects were measured by dividing the weight between male and female at birth and after weaning.

Statistics processing
The number of fetuses per gestation period was calculated as mean ± standard error (Mean ± SEM), and the comparison of the number of fetuses per gestation period was examined for significance using the Statistical Analysis System (SAS).

Confirmation of estrus cycle by morphological characteristics of vaginal mucus cells
As shown in Fig. 1, in the proestrus (A), the vaginal membrane cells were large and rounded, and the cell nuclei were confirmed. In the estrus (B), endometrial cells were large and mostly composed of keratinocytes, and the cell nuclei were not stained, and in some cells, the cell nuclei were confirmed to be small. In addition, a lot of mucus secretions could be identified at this time. In addition, the metestrus (C) was generally small in cell size, and was able to identify a large number of white blood cells and red blood cells, and rarely identified keratinocytes.
In the diestrus (D), a large number of red blood cells and white blood cells were identified, and the cell nuclei of somewhat smaller vaginal epithelial cells were identified. amining vaginal epithelial cells every day in the method shown in Fig. 1. As shown in Fig. 2, it was confirmed that a normal 4 days estrus cycle was repeated in a mature unlighted wistar-based rat, and rarely, there were individuals with a 5-day sexual cycle, but statistically significant values could not be identified.
In this experiment, the proestrus and the estrus were found to be able to easily determine the sex cycle by the ratio of keratinocytes and cell nuclei. However, the sexual cycles of the metestrus and the diestrus were difficult to distinguish.

Changes in the number of fetuses during pregnancy
In order to investigate the change in the number of fetuses during pregnancy, females in the state of estrus were selected by vaginal smearing and mating was performed by merging the same type of individuals into the male cages in the afternoon of the day. As a result of checking the vaginal epithelial cells the next morning, some individuals identified vaginal plugs and sperm. After that, the estrus cycle was examined until delivery to the same method (Fig. 3).
After the estrous stage of the female is confirmed as proestrus and mated with the same male. The next morning, sperm mass was found in vaginal smears (Day 0).
After 19 days of pregnancy, vaginal smears revealed a diestrus (Fig. 3). Table 1 shows the change in the number of fetuses every 2-3 days from the 8th day of pregnancy to the expected delivery day (Day 21). As shown in Table 1. The number of fetuses on the 8th and 10th day of pregnancy was 15.2 ± 0.4 and 15.4 ± 0.3, respectively. In contrast, the number of fetuses on the 12th, 16th, 18th, and 20th days of pregnancy was 12.9 ± 0.6, 12.6 ± 0.3, 12.8 ± 0.3, and 12.4 ± 0.6, respectively, and 11.9 ± 0.9 at delivery. As such, the unexpected results in this experiment were significantly (p < 0.05) decreased fetal numbers between the 10th and 12th days of pregnancy, and thereafter, there was no significant difference in fetal number until delivery. Fig. 4 shows the results of measuring the change in the weight of male and female fetuses by growth stage from birth to 21 days after delivery. The weight at birth immediately after delivery was an average of 9.2 ± 2.3 g in Table 1. Changes in the number of fetuses from early pregnancy to delivery in rats  females, but an average of 9.8 ± 3.7 g in males. There was no significant difference between sexes. After that, the growth stage showed modest growth until the 14th day after delivery, but significant (p < 0.05) growth was observed until the 20th day of the weaning period.

DISCUSSION
In the results of Fig. 1 Fig. 1. It was confirmed that the normal 4-day estrus cycle of mature wistar rats was repeated.
Rarely, there were individuals with a 5-day sexual cycle, but statistically significant numbers could not be identified. Therefore, it was found that wistar-type rats under normal feeding control, temperature, and constant light and dark conditions can confirm the correct estrus cycle by vaginal smearing.
Among rodents, in the case of wild animals, there is a difference in cell types and ratios according to the reproductive and non-reproductive seasons, and there is a slight difference according to the time of daylight (Byers et al., 2012).
In addition, in this experiment, the collection time of the vaginal mucosa cells was performed between 9 am and 12 pm every day, and it was generally possible to observe the exact estrus cycle. However, if the sampling time was irregular, there was some difference in determining the correct estrus cycle. Therefore, in order to confirm the correct estrus cycle, it is necessary to collect vaginal epithelial cells at the same time every day with appropriate specification environment.
On the other hand, dogs, which are popular as pets, show estrus every 6 months, have a long estrus cycle, and have various symptoms of estrus for each individual, making it difficult to directly determine the right time to fertilize and the exact time of ovulation (Concannon and Lein, 1989;Allen, 1992). In addition, Park et al. (2013) reported that the day when the ratio of keratinized vaginal epithelial cells in small dogs increased to more than 90% was able to predict the delivery date at the same time as the time of mating. Therefore, it is judged that the vaginal epithelial cytology method can be used industrially as a means of confirming the time for artificial insemination and insemination of dogs (Allen, 1992;Feldman and Nelson, 1996).
In the future, it is expected that it will be highly utilized for judging the mating time and proliferation of specific pets depending on the vaginal epithelial cytology method.
In order to induce pregnancy, crosses were carried out with males of the same breed. The next morning after mating, pregnancy was confirmed by checking vaginal plugs and sperm cells. In addition, as a result of examining the vaginal smear until delivery by the same method, it was confirmed that it remained in the diestrus state until delivery (Fig. 3).
Therefore, pregnancy maintenance is presumed that progesterone is secreted from the new luteal body by mating stimulation of males and plays a physiological role in protecting the fetus until delivery (Smith et al., 1975;Shiota et al., 1993). Table 1. The number of fetuses on the 8th and 10th day of pregnancy was 15.2 ± 0.4 and 15.4 ± 0.3, respectively, but the number of fetuses on the 12th, 16th, 18th, and 20th day of pregnancy was respectively. 12.9 ± 0.6, 12.6 ± 0.3, 12.8 ± 0.3, 12.4 ± 0.6, and 11.9 ± 0.9 at delivery. As described above, the unexpected result in this experiment was that the number of fetuses was significantly decreased (p < 0.05) between the 10th and 12th days of pregnancy, and thereafter, there was no significant difference in the number of fetuses until delivery.

As shown in
In rodents such as rats, the corpus luteum formed after ovulation does not secrete progesterone and immediately regresses and repeats a short estrus cycle (4-5 days) in which new follicles develop. This is because the activity of 20α-Hydroxysteroid dehydrogenase (20α-HSD) in the corpus luteum is high, and this enzyme metabolizes progesterone to biologically inactive 20α-dihydroprogesterone (20α-OHP) (Seong et al., 1992(Seong et al., , 1995Shiota et al., 1993). Therefore, to analyze the significant difference in the number of fetuses between the 10th and 12th days of pregnancy, additional studies on steroid hormones and molecular biology are considered necessary. Fig. 4 shows the results of measuring the change in the weight of male and female fetuses by growth stage from birth to 21 days after delivery. The weight at birth immediately after delivery was an average of 9.2 ± 2.3 g in females, but an average of 9.8 ± 3.7 g in males. There was no significant difference between sexes. After that, the growth stage showed modest growth until the 14th day after delivery, but significant (p < 0.05) growth was observed until the 20th day of the weaning period.
These results indicate that the individual is growing rapidly by ingesting the mother's pellet feed as the weaning period from the 3rd week after delivery.

CONFLICTS OF INTEREST
No potential conflict of interest relevant to this article was reported.