Ovarian superstimulation with FSH in Wagyu breed bovine donors impacts folicular dynamics but does not improve the amount of embryos

The increase of the world population generates the need to raise the production of food of vegetal and animal origin. In Brazil, livestock farming has evolved, demonstrating the important role of the country in the production of food. In this way, researchers of this productive chain have been looking for technologies related to production and reproduction, above all, to the use of reproduction biotechnologies, seeking to increase the production of different bovine breeds. Thus, the objective of this research was to investigate the efficacy of ovarian FSH super stimulation in bovine Wagyu oocyte donor females on follicular dynamics and in vitro embryo production. Twelve Wagyu animals, aged 12 to 24 months, randomly distributed in a crossover design were used in two groups: Group 1= animals not stimulated with FSH and Group 2= animals stimulated with FSH. The follicular, oocyte and embryonic variables were evaluated. It was observed that ovarian overstimulation in Wagyu oocyte donor cows with FSH improved the mean and large follicles but reduced the rate of oocyte recovery and, despite the best percentage of viable oocytes, there was no improvement in the amount of embryos produced in vitro.


Introduction
The United Nations Organization (UNO, 2017) estimates an increase in world population, reaching 8.6 billion people in 2030 and 9.8 billion in 2050. Therefore, to meet the global food need for the population by 2050, there should be a 60% increase in production. In this way, researchers of several production chains, including beef, have studied technologies related to management, sanitation, nutrition and reproduction (Colombo et al., 2017;Cavalieri et al., 2019), seeking to increase the number of animals and / or the stocking rate of pastures, and thus increase the production of meat by area.
Animal reproduction biotechnologies can contribute positively to this scenario. With the advent of artificial insemination (AI), producers were able to use bulls with high genetic value, and with the development of superovulation (SOV) and embryo transfer (ET) schemes, cattle breeding may increase the number of calves from females of high genetic standard (Hasler, 2014). Ultrasound guided follicular aspiration has led to an increase in the number of gestations of animals with high genetic value. Also, studies evaluating several hormonal protocols have been developed to improve the reproductive indices of animals (Hasler, 2014).
A protocol that has been studied is the SOV using FSH (Follicle Stimulating Hormone) followed or not by coasting, which is a period of absence of FSH, in bovine donors, aiming to contribute even more to the in vitro production of embryos.
According to Vieira et al. (2014), treatments using stimulation with FSH can promote the development of multiple follicles in the ovaries and improve embryo yield in Holstein breeders and thus, make embryo production more effective in vitro (IVEP) by ovum pick-up (OPU). However, the results of the research differ on the use of FSH, with or without coasting, in the different subspecies (Bos taurus taurus and Bos taurus indicus) and breeds (Sartori et al., 2016).
Studies involving the beneficial effects of FSH superstimulation, such as the greater proportion of medium and large size follicles associated with the use and subsequent gonadotrophin (coasting) withdrawal, aiming to improve oocyte competence and increase gestation rate are fundamental, especially in Wagyu cattle whose research is scarce.
Wagyu cattle include four Japanese breeds: Black, Brown, Shorthorn and Polled (Gotoh et al., 2018) and the Black breed has adapted in several regions of Brazil, presenting economic importance due to the good yield, production and commercialization of meat noble with extreme marbling. This breed presents positive production characteristics such as, medium size, sexual precocity, reaching sexual maturity in up to 11 months for females and males, good maternal ability and ease of delivery, longevity, hardiness and docile temperament (Uchida et al., 2002).
Thus, the objective of this research was to investigate the efficiency of ovarian FSH super stimulation in bovine Wagyu oocyte donor females on follicular dynamics and in vitro embryo production.

Methodology
The experiment was carried out at School Farm -BIOTEC, at the University Unicesumar, Maringá, Paraná, Brazil Twelve Wagyu females, aged 12 to 24 months, mean weight of 350 kg, oocyte donors from a commercial program, with no abnormalities in the reproductive tract, as determined by gynecological and ultrasonographic examination, were used.
The animals were kept in brachiaria pickets (Brachiaria brizantha cv MG-5), with mineral supplementation and water ad libitum, being submitted to hygienic-sanitary management adopted in the property.
The animals were randomly assigned to a crossover design in two groups: At the time of aspiration, the ovarian follicles were identified and the diameter (mm) determined by ultrasound (ALOKA SSD-500) and classified by size into three categories: 3 to 5 mm (small follicles), 6 to 8 mm (mean follicles) and equal to or larger than 9 mm (large follicles) (Ferreira et al. 2004).
Ultrasound (ALOKA SSD-500®) with 5 mHz microconvex transducer (UST 974-5) was used to perform the follicular aspiration procedure, adjusted to a specific follicular suction guide for the bovine reproductive system. A 20G needle connected to a 50 mL Falcon tube was used through an aspiration system (Cook VBOA 18L®). The vacuum pressure was  At the time of follicular aspiration, a low epidural anesthesia with 5 mL of 2% lidocaine (Pearson®, São Paulo/SP, Brazil) was performed to reduce peristaltic movements and discomfort in the animal. After administration of lidocaine, the animal had a vulva washed and cleaned with paper towel. The transducer was inserted into the vaginal sac and the manipulation of the ovaries through the rectum was performed to better visualize them on the ultrasound screen. The follicles were placed in the puncture line indicated on the ultrasound screen and aspirated, at random, not following order of size, through the needle and the vacuum pump, the same procedure was performed on all visible follicles in both ovaries. After aspiration the vacuum system was cleaned with the oocyte receiving medium and the used needles were discarded.
For washing and oocyte selection, the aspirated material was transferred to the embryo collection filter and washed with the same medium used for aspiration. The remaining sediments in the filter were transferred to Petri dish and the search, counting and classification of the oocytes as feasible and not viable. In the present study, the presence of cumulus and homogeneous cumulus and ooplasm were considered viable (grade I, II and III), those with heterogeneous and apoptotic vesicles were unfeasible (grade IV (De Loos et al., 1989).
The oocytes were placed in 90μL microdrople of maturation medium covered with mineral oil.
After 15 hours of IVF, the zygotes were in vitro cultured (IVC) in SOF (Synthetic Oviduct Fluid) medium supplemented with SFB (Nutricell®, Campinas, SP, Brazil), incubated with a gas atmosphere containing 20% CO2 in air, with maximum humidity.
After 48 hours of IVC, the cleavage rate was evaluated and the culture medium was renewed. In this period, when the cleavage occurred, the embryonary development with two, four and eight cells was observed.
The effect of FSH overestimation on the follicular, oocyte and embryonary variables was analyzed by the generalized linear model, including the main effect of the treatment (Super Stimulation with FSH vs. Control) and other sources of variation. In the presence of a significant effect, the means were compared by the Tukey's test. Differences in oocyte, viable oocyte and blastocyst recovery rates (FSH Super Stimulation vs. Control) were analyzed by the binary logistic regression model including the same treatment effect and sources of variation. In both models, all possible effects and interactions were considered. For descriptive analysis, the quantitative data are presented as mean (M) and standard error (SE) and qualitative data as percentage (%). All statistical analyzes were performed in the Minitab® statistical program, version 18.1. The level of significance to reject H0 (the null hypothesis) was 5%, so a level of significance ≤ 0.05 was considered to indicate an effect of categorical variables and their interactions.

Results and Discussion
Based on the results obtained, it was observed that FSH over stimulation increased mean and large follicles (p<0.005) and, consequently, improved the total number of follicles (Table 1).
Corroborating our findings, Goodhand et al. (1999) also observed that the use of FSH increased the number and the mean diameter of the follicles available for puncture. In fact, Ribeiro et al. (2011) pointed out that hormonal over stimulation increases the number of follicles with adequate diameter for aspiration, that is, greater than 6 mm.
However, despite this increase in the number of medium and large follicles, a reduction in oocyte recovery rate (81.0 vs 38.3%) (p<0.0001) was observed. Nevertheless, there are contradictory reports in the literature regarding recovery rate as a function of follicle size. Goodhand et al. (1999) found that although FSH increased the number and mean diameter of the available follicles for puncture, the number of oocytes recovered varied. Ribeiro et al. (2011) stated that hormonal overstimulation increases the number of follicles of adequate size for aspiration and recovery rate. Chagas et al. (2014) stated that small follicles present a lower rate of recovery compared to medium and large follicles. Ongaratto et al. (2015) worked with Brangus and Angus animals and found that the use of estradiol benzoate, progesterone to synchronize the follicular wave, PGF2α to avoid the presence of corpus luteum and superovulation with FSH improved the number of oocyte cumulus complexes (COCs) recovered by OPU. Other studies (Blondin et al., 2002;Oliveira et al., 2016) reported that association between FSH and coasting eliminates small follicles and increases the quantity of medium and large follicles, facilitating aspiration.
However, it is emphasized that, these data diverge partially with the results of this study, because despite the larger size of the follicles, the recovery rate was lower. Similar to the findings of this study, Lonergan et al. (1994) also observed a lower recovery of oocytes in situations with a predominance of large follicles at the time of aspiration. The low recovery rate found in this study is attributed to race and losses in the abdominal cavity. Research shows that there are differences in the rate of recovery according to the species, showing that the protocols of follicular superovulation and aspiration in Bos indicus present better results, since they present a greater number of antral follicles and, consequently, a greater number of oocytes recovered when compared to the animals (Taurus et al., 1996), as is the case of the Wagyu breed of this study. However, the greater size of the follicles in OPU may result in a greater probability of loss of COCs in the peritoneal cavity or in greater difficulty in aspirating it, although Ribeiro et al. (2011) reported that it was easier to aspirate larger follicles because of better visualization on ultrasound.
The results also showed that FSH treatment improved the percentage of viable oocytes (74.5 vs 83.6%). Regarding the number of total and viable oocytes recovered, Barbosa et al. (2013) observed the production of embryos in Nelore cows and reported that the mean number of oocytes aspirated ranged from 26.58 to 44.08. Silva et al. (2017) evaluated reproductive data from donors of Indian bovine breed oocytes and taurine submitted to ultrasonic guided follicular aspiration. The authors Research, Society andDevelopment, v. 10, n. 10, e165101018811, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i10.18811 analyzed the field data of 734 follicular aspirations and observed a mean, among the races, of 31.85±16.65 oocytes per aspiration, being 25.32±13.80 viable, with emphasis on the largest number of oocytes aspirated and viable for donors of the Nellore breed, an Indian breed.
Thus, despite the superiority in the percentage of viable oocytes observed in the FSH treated group, the number of total oocytes (G1:17.4 ± 2.9, G2: 12.2 ± 2.5) and (G1:13.0±2.3; G2:10.2±2.3) obtained in this independent treatment study were lower than those reported in the literature for other races, especially Indian races, being in compliance with the (Table 1).
In the present study, Bos indicus presented a higher number of oocytes recovered than Bos taurus (Sartori et al., 2016;Silva et al., 2017).
In addition, the results also showed that although FSH treatment improved the percentage of viable oocytes, there was no increase in the number of embryos, showing equivalent embryo production between the two groups evaluated (3.0 vs 2.4).
Unlike the findings of this research, Ribeiro et al. (2011) pointed out that hormonal overstimulation increases the number and size of follicles and increases the number of embryos. Baruselli et al. (2012) also reported an increase in PIVE in an experiment with Brangus and Angus donors treated with four equal doses of FSH, twice a day, initiated at the time of follicular wave emergence, and with OPU at 24 hours after the last FSH treatment. Other studies have shown that FSH over stimulation, associated with coasting, improves oocyte competence , blastocyst and gestation rates (Labrecque et al., 2013), increases the efficiency of IVP and percentage of in vitro blastocyst development and number of embryos per OPU session (Vieira et al., 2014). Therefore, even with the 48 hour interval between the last FSH application and the OPU used in this research and with the highest percentage of viable oocytes, it was not possible to increase the number of embryos.
It is thought that oocyte viability is only indicative of oocyte competence, based on its morphology, indicating the ability of the oocyte to ensure embryonic development after fertilization (Sirard, 1989). Furthermore, it is known that in the in vivo production of bovine embryos, approximately 80% of ovulated oocytes develop to blastocyst, whereas in PIVE, only 40% reach this stage (Lonergan;Fair, 2008). Researchers (Rizos et al., 2002;Bertagnolli et al., 2004) show that the in vitro maturation (IVM) environment influences this low rate. Experiments have found that oocytes obtained from in vivo maturation after stimulation with FSH and GnRH are more competent to develop blastocysts than in vitro maturates (Van De Leemput et al., 1999;Humblot et al., 2005), suggesting that that IVM should determine suboptimal conditions for the development of immature oocytes (Pereira et al., 2010).

Conclusion
Ovarian overstimulation in Wagyu oocyte donor cows with FSH improved the mean and large follicles but reduced the rate of oocyte recovery and, despite the best percentage of viable oocytes, there was no improvement in the number of embryos produced in vitro.