Evaluation of in vitro production rates of bovine embryos using melatonin-

In this study, we aimed to evaluate the rate of bovine embryo production by using 50 ng/mL melatonin supplementation in in vitro culture medium. For this, oocytes from slaughterhouse ovaries were matured in vitro in TCM-199 medium with Earle’s balanced salt solution + 10% SFB, FSH, and LH in an atmosphere of 5% CO2. Twenty-four hours after IVM, the oocytes underwent in vitro fertilization in human tubal fluid under the same conditions as above, for 18 h. Semen was fractionated by Percoll gradient centrifugation and the concentration of sperm was adjusted to 1 × 10/mL. Probable zygotes were then divided into two groups: the control group grown in drops of 90 μL SOFaa medium + 0.6% BSA + 2.5% SFB, in an atmosphere of 5% CO2, 90% N2, and a melatonin group (Mel), similarly cultured in 90 μL drops of SOFaa medium + 0.6% BSA + 2.5% SFB + 50 ng/mL melatonin. Cleavage rates were assessed on day 3 (D3). On D7, blastocyst formation rates were evaluated. Eight routines were performed (320 oocytes per routine). Data were analyzed with ANOVA, followed by Tukey’s range test using a general linear model. The level of statistical significance was set at 5%. There were no differences in the rates of Research, Society and Development, v. 10, n. 6, e19010615544, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i6.15544 2 cleavage or blastocyst formation between the control and melatonin groups (P > 0.05). Thus, under the conditions used in this study, supplementation with melatonin did not yield benefits in increasing the rate of in vitro bovine embryo production.


Introduction
In Brazil, 375,503 bovine embryos were produced in 2017, of which 345,528 were generated in vitro, accounting for 34.8% of global production. In vitro embryo production has consolidated itself as the technique of choice for the production of bovine embryos, accounting for the highest percentage of embryos produced, not only in beef Zebu breeds, but also in other market segments (Taurine and Zebu cattle, beef, and milk). The availability of sexed semen from mid-2005, associated with progressive improvements in the efficiency of in vitro production embryo, boosted its adoption in dairy breeds (Pontes et al., 2011;Vianna, 2011). As a consequence, the national market, characterized in 2007 by the predominance of Zebu and beef breeds, registered in 2017 a greater production of embryos in Taurine breeds or their crosses, and in dairy breeds. Thus, the profile of activity in Brazil has become similar to that observed in other regions of the world (Gonçalves & Vianna, 2019).
Thus, in vitro production embryo has become a valuable technology for assisted reproduction in cattle breeding systems. However, even with the remarkable innovations developed involving this system, the greatest obstacles to the full success of this biotechnology are low rates of post-transfer pregnancy and the greater sensitivity of embryos to cryopreservation (Maziero et al., 2016). This leads us to believe that in vitro production embryo systems induce embryonic changes that prevent full-term pregnancy. Other authors have described morphological changes between embryos in vivo and in vitro, such as increases in cytoplasmic lipid droplets and mitochondrial alterations (Sudano et al., 2011).
Although the rate of blastocyst production is influenced by the origin and quality of the oocytes used in in vitro production, it is known that the quality of embryos produced is directly related to embryonic culture conditions (Rizos et al, 2002;Lonergan et al., 2006;Maziero et al., 2020). This is due to changes in oxidative profiles due to fluctuating concentrations of oxygen during the production process. It is known that intrauterine oxygen concentrations are 2-8%, whereas atmospheric levels are 20%. When embryos are grown in an atmosphere with high oxygen content, oxidative stress conditions increase, causing an imbalance between pro-oxidant levels (ROS, reactive oxygen species, free radicals) and antioxidant abundance (Rocha-Frigoni et al., 2013).
Hence, to reduce the deleterious effects of ROS on in vitro production embryo, antioxidants are added to embryo culture media in vitro (Lira et al., 2020). Melatonin is a lipophilic molecule derived from tryptophan, present in vertebrates, invertebrates, bacteria, single-celled organisms and plants. Melatonin is produced by the pineal gland (main site), cerebellum, retina, skin, gastrointestinal cells, Harder gland, thymus, peripheral mononuclear cells, placenta, ovary, testicle, bone marrow, liver, hippocampus, and platelets (Mayo et al., 2018). Melatonin is not stored, but directly secreted both to the cerebrospinal fluid and the cardiovascular system. Thus, it can migrate freely through morphophysiological barriers such as the blood-brain barrier and placenta; therefore, it can be distributed throughout the entire body (Iwasaki et al., 2005). In mammals, melatonin performs a series of biological functions such as regulation of circadian cycles and seasonal signaling for reproduction, and has antioxidant and immunomodulatory effects (Mayo et al., 2018).
The antioxidant action of melatonin is a good alternative to the in vitro production of bovine embryos. When used during the embryo maturation and cultivation processes, there are decreases in ROS and DNA fragmentation in oocytes and blastocysts (Asgari et al., 2012;Takada et al., 2010;Takada et al., 2012). However, few results have been described in the literature, especially regarding melatonin concentration to be used. Therefore, in this study, we sought to evaluate the effects of adding 50 ng/mL melatonin to culture systems with in vitro production embryo mineral oil on embryonic production.

Methodology
Ovaries of cows intended for commercial slaughter were used, with eight routines were performed (320 oocytes per routine). The ovaries were transported to an in vitro embryo production laboratory located in Moreira Sales, Paraná. In the laboratory, the ovaries were washed with 0.9% sodium chloride solution at a temperature of 37°C to 38°C. After washing, ovarian follicles with a diameter between 2 and 8 mm were aspirated with the aid of a 10 mL syringe fitted with 21G needles.
The follicular fluid was then held in 15 mL tubes to form a sediment at 37.5°C for 10 min. Oocyte selection was performed in HEPES medium (TCM 199 HEPES), obtaining only oocyte cumulus complexes (COCs) with three or more layers of cumulus cells and homogeneous cytoplasm. To carry out fertilization, a straw of bull semen with known fertility was thawed. Viable sperm were selected using a continuous Percoll density gradient (45% and 90%). The oocytes were fertilized with a dose of 1 × 10 6 sperm/mL and incubated for 18 h under the conditions described above. Research, Society and Development, v. 10, n. 6, e19010615544, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i6.15544 4 After IVF, the zygotes were mechanically stripped with the aid of a pipette and transferred to in vitro culture. The groups were divided into control and melatonin groups. Drops with 15 oocytes of SOFaa medium (90 µL/drop) were placed in Petri dishes, divided into a control group (without melatonin supplementation) and a melatonin group (supplemented with 50 ng/mL melatonin) and incubated under mineral oil at 38.5°C, 5% CO2 in air, and 100% humidity. On D4.5, cleavage was verified, and on D7, the rate of blastocyst formation was evaluated.
For statistical analyses, ANOVA using PROC GLM of SAS (SAS Inst. Inc., Cary, NC, USA) was used. Sources of variation in the model including treatment and replicates were considered as fixed and random effects, respectively. Data are presented as means ± standard deviations. For all analyses, a statistical significance level of 5% was adopted.

Results
The cleavage rates assessed on D3 are shown in Table 1. There was no difference between the groups, with the control group presenting 86.83 ± 3.2 % and the melatonin group exhibiting a cleavage rate of 87.20 ± 4.8 %. For the rate of embryo formation, there was also no difference between groups, control group: 44.4 ± 2.1 % of blastocysts and melatonin group: 41.5 ± 1.5 % of blastocysts, as shown in Table 2.

Discussion
The results obtained in this study showed that the addition of 50 ng/mL melatonin to bovine embryo culture medium did not increase the rate of embryo production, which was similar to the control group. Contradictory results have been previously reported with the use of melatonin in the culture medium at different doses and treatment durations. The different embryo culture systems are also questionable, as melatonin has lipophilic characteristics, which can interfere with its action in systems involving mineral oil, as reported earlier by Assis (2014). It is known that the presence of mineral oil in the system can lead to impaired melatonin action.
In cattle, one of the first studies that tested melatonin in embryonic culture used a dose of 10 -4 M, with an increase in the rate of blastocyst formation in ambient atmospheric conditions with 20% O2 (68.9% melatonin vs. 50.8% control) (Papis et al., 2007). Studies in swine have yielded improvements in in vitro embryo production with the use of melatonin (1 nM) in embryonic culture in a system with mineral oil, increasing the cleavage and blastocyst rates. However, the genes involved in antioxidant or pro-action were not evaluated as oxidants (Rodrigues et al., 2008).
Many different free radical scavengers have been investigated for potential protection against oxidative stress in mammalian embryos subjected to in vitro culture systems. Starting with extracellular enzymes such as superoxide dismutase or catalase, other systems including taurine/hypotaurine, thioredoxin, pyruvate or low molecular weight thiols such as βmercaptoethanol, glutathione, and cysteine were tested (Guérin et al., 2001). Zhao et al. (2015) showed the effect of adding 10 -9 M melatonin to IVM and the potential for development of Other authors have shown that the addition of melatonin at a concentration of 10 -6 M protects oocytes against damage caused by thermal shock (Paula, 2007). Assis (2014) found that melatonin exhibits stable behavior under embryo culture conditions with or without oil, with antioxidant action at a dose of 50 ng/mL. There was also an increase in the production and quality of embryos in vitro with culture medium supplemented with melatonin and subjected to induced oxidative stress.
When used in the in vitro production of embryos during embryo maturation and culture, melatonin decreases EROS and DNA fragmentation in oocytes and blastocysts (Rodrigues et al., 2007); Asgari et al., 2012). However, there is still no well-established optimal melatonin dose because the results of cleavage and formation of blastocysts are similar to those of control groups.
According to Takada et al. (2010) supplementation with a physiological concentration of melatonin (10 -9 M) in in vitro maturation medium with or without a combination of gonadotropins has no effect on nuclear maturation or on rates of cleavage and blastocyst formation. These rates ranged from 88% to 90%, 85% to 88%, and 42% to 46%, respectively. These data corroborate the present study, in which the use of melatonin in the culture medium did not enhance the embryonic development index. In contrast, the positive effects of melatonin on nuclear maturation and embryonic development were reported by Kang et al. (2009) in pigs and by Manjunatha et al. (2009) in buffalos. Tsantarioutou et al. (2007) did not observe differences in bovine cleavage rates or in blastocyst production frequency when melatonin was added to the IVM medium, which was similar to the findings of the present study. According to a number of authors, embryo culture conditions are well established in cattle (Papis et al., 2007;Parrish et al., 1994;Holm et al. 1999;Sagirkaya et al., 2007;Adona et al., 2008;Corrêa et al., 2008). Some of the obstacles to the full success of in vitro embryo production are events that occur in the oocytes during the IVM period. Oocyte maturation is a process in which cells acquire an intrinsic capacity for gradual development until reaching activation of the embryonic genome (Maziero et al., 2016).
Cebrian-Serrano et al. (2013) evaluated the effects of adding melatonin to the IVM medium of bovine oocytes subjected to thermal shock in terms of production rates and quality of blastocysts. These authors found that 10 -4 M melatonin was alleviate bovine oocytes from harmful effects of heat stress. In addition, Lira et al. (2020) found that the 10 -4 M concentration resulted in better embryonic quality. However, in our study, where melatonin was added to the IVC medium only, we did not observe any improvement in the rate of embryonic production by D7.

Conclusion
In conclusion, the addition of 50 ng/mL melatonin to the culture medium of bovine embryos did not improve the rate of blastocyst formation. Thus, the beneficial effects of melatonin in increasing the in vitro production of bovine embryos should be further studied and, in particular, the ideal concentration required in embryonic culture media for improved in vitro production embryos outcomes should be deduced.