Introduction
Embryo culture is an important aspect of assisted reproductive technology (ART) because it allows the zygote to mature, divide, and reach a stage where it can be transferred to the uterus (Cagnone and Sirard, Reference Cagnone and Sirard2016). It is important to optimize the composition of embryo culture media and settings for incubation during in vitro development to improve the quality of these embryos (Lan et al., Reference Lan, Lin, Chang, Lin, Tsai and Kang2019). Suboptimal conditions for cultivation often decrease embryonic output during early development due to inadequate control of homeostasis (Summers and Biggers, Reference Summers and Biggers2003). Despite the widespread use of embryo culture medium, in vitro culture (IVC) continues to develop embryos of lower viability relative to natural conception (Cagnone and Sirard, Reference Cagnone and Sirard2016), therefore it is essential to improve IVCs to reduce the effect of ART on embryo viability.
Oocyte maturation appears to be of key importance for oocyte competence. Follicular fluid (FF) prepares an extremely important microenvironment for oocyte development. Follicular fluid reflects metabolic and hormonal processes that occur in the maturing oocyte microenvironment prior to ovulation and is a predictor of outcome parameters such as fertilization, embryo cleavage, and pregnancy rates of in vitro fertilization (IVF) (Wiener-Megnazi et al., Reference Wiener-Megnazi, Vardi, Lissak, Shnizer, Reznick, Ishai, Lahav-Baratz, Shiloh, Koifman and Dirnfeld2004). Consequently, certain biochemical characteristics of the FF that surrounds the oocyte play a critical role in determining oocyte quality and the subsequent potential for fertilization and development of the embryos.
Reactive oxygen species (ROS) develop inside the follicle, particularly during the ovulation phase (Sugino, Reference Sugino2005), and it is presumed that oxidative stress is responsible for poor oocyte quality (Sugino, Reference Sugino2006). Early mammalian embryos cultured in vitro increase the level of oxygen free radicals and it is believed to play a crucial role in IVF success (Sellens et al., Reference Sellens, Stein and Sherman1981; Agarwal et al., Reference Agarwal, Said, Bedaiwy, Banerjee and Álvarez2006b). ROS sources in the culture medium originate from embryo metabolism and the culture environment (Agarwal et al., Reference Agarwal, Gupta and Sikka2006a). However, certain levels of ROS are required for sperm function, normal ovarian follicle activity, oocyte maturation, normal sperm–oocyte interactions, and sperm capacitation, in addition to compaction, blastocyst development, and implantation (Riley and Behrman, Reference Riley and Behrman1991; Agarwal and Gupta Reference Agarwal and Gupta2005; Agarwal et al., Reference Agarwal, Gupta and Sikka2006a; Gupta et al., Reference Gupta, Banerjee and Agarwal2006). It has been reported that minimal oxidative stress levels are beneficial for adequate growth and development of the embryo (Agarwal and Gupta, Reference Agarwal and Gupta2005). However, excessive amounts of oxidative stress may have adverse effects on sperm DNA, fertilization, and embryo quality and they may block or retard early embryonic development, damage the embryo cell membrane, alter the mitochondria, and result in apoptosis (Noda et al., Reference Noda, Matsumoto, Umaoka, Tatsumi, Kishi and Mori1991; Gupta et al., Reference Gupta, Banerjee and Agarwal2006; Askoxylaki et al., Reference Askoxylaki, Siristatidis, Chrelias, Vogiatzi, Creatsa, Salamalekis, Vrantza, Vrachnis and Kassanos2013; Lan et al., Reference Lan, Lin, Chang, Lin, Tsai and Kang2019). Embryos in commercial culture media produce ROS at different levels, depending on the media composition (Martín-Romero et al., Reference Martín-Romero, Miguel-Lasobras, Domínguez-Arroyo, González-Carrera and Álvarez2008; Shih et al., Reference Shih, Lee, Liu, Tsao, Huang and Lee2014). Therefore, IVC could be optimized by using antioxidants to reduce the effects of ART on embryo survival and quality.
Most vitamins act as antioxidants and prevent oxidative stress from mammalian cells (Sinbad et al., Reference Sinbad, Folorunsho, Olabisi, Ayoola and Temitope2019). Antioxidant vitamins help reduce oxidant damage by acting as waste electron sinks (Thiyagarajan and Valivittan, Reference Thiyagarajan and Valivittan2009). Vitamin B12 (B12), or cobalamin, is a water-soluble vitamin that functions as a coenzyme in various biochemical reactions such as methionine synthesis and the metabolism of branched amino acids (Van De Lagemaat et al., Reference Van De Lagemaat, De Groot and Van Den Heuvel2019). Cyanocobalamin is the form of B12 that is typically used in vitamin supplements (Hamedani et al., Reference Hamedani, Tahmasbi and Ahangari2013) and it plays a role in cell replication and DNA synthesis (Mello et al., Reference Mello, Hyde, Elsea and Whitaker2018). Cyanocobalamin is currently used as a treatment for human infertility (Gaskins et al., Reference Gaskins, Chiu, Williams, Ford, Toth and Hauser2015). Additionally, research has shown that B12 is a crucial nutrient for fetal development (Reese Pepper and Black, Reference Reese Pepper and Black2011). Bovine and sheep studies have shown that administration of cobalamin during the time of oocyte growth and post-fertilization development to the blastocyst stage plays an important role in the offspring’s health (Sinclair et al., Reference Sinclair, Allegrucci, Singh, Gardner, Sebastian, Bispham, Thurston, Huntley, Rees and Maloney2007; Kwong et al., Reference Kwong, Adamiak, Gwynn, Singh and Sinclair2010). Zacchini and colleagues reported that cobalamin supplementation during in vitro maturation (IVM) improved the developmental competence of sheep oocytes (Zacchini et al., Reference Zacchini, Toschi and Ptak2017). Roy and colleagues demonstrated that cobalamin treatment prior to IVM improved the developmental competence of porcine oocytes (Roy et al., Reference Roy, Fang, Hassan, Shin and Cho2017). It was also shown that cyanocobalamin supplementation had positive effects during the thawing of frozen boar semen on spermatozoa, IVF, and embryonic development (Mello et al., Reference Mello, Hyde, Elsea and Whitaker2018).
Of note, only the optimal concentration of cobalamin supplementation can prevent the active forms of oxygen generation and membrane lipid peroxidation and scavenge against ROS (Hamedani et al., Reference Hamedani, Tahmasbi and Ahangari2013). The effect of cyanocobalamin has not been evaluated in the development of mouse embryos through its supplementation in maturation and embryo culture medium; therefore, the aim of this study was to evaluate effects of cyanocobalamin supplementation in various concentrations on in vitro mouse maturation, fertilization and embryo development, and to determine the optimum concentration of this vitamin.
Materials and methods
Adult female (6–10-week-old) and male (8–12-week-old) NMRI mice were maintained according to the Guidelines for the Care and Use of Laboratory Animals. All mice were kept in cages with standard conditions of 12 h : 12 h, light : dark and a temperature 22 ± 2°C.
Effect of cyanocobalamin on in vitro maturation (IVM)
Ovulation was induced in the female mice with intraperitoneal (i.p.) injections of 5 IU pregnant mare serum gonadotropin (PMSG; Sigma-Aldrich, Saint Louis, MO, USA). At 48 h after the injection, cumulus–oocyte complexes (COCs) at the germinal vesicle (GV) stage (Fig. 1 Ca) were aspirated from the ovaries by puncturing the follicles with a sterile 28-gauge needle. The COCs were washed three times in maturation medium droplets that included αMEM (Sigma-Aldrich, Saint Louis, MO, USA) supplemented with 7.5 IU/ml recombinant human follicular stimulating hormone (rhFSH, Sigma-Aldrich, Saint Louis, MO, USA) and 100 IU/ml human chorionic gonadotropin (hCG, Sigma-Aldrich, Saint Louis, MO, USA). The immature oocytes were divided into five groups according to the dose of cyanocobalamin (Sigma-Aldrich, Saint Louis, MO, USA) administered [0 (control) and 100, 200, 300 and 500 pM]. The oocytes in each group were exposed to cyanocobalamin for 24 h, after which we assessed viability in metaphase M II (Fig. 1 Cb) with an inverted microscope.
Figure 1. (A) Effect of cyanocobalamin on nuclear maturation during in vitro maturation in mouse oocytes **P < 0.001, ***P < 0.0001. (B) Effect of cyanocobalamin on in vitro fertilization success **P < 0.01, ***P < 0.001. Data are expressed as mean ± SEM. (C) Different stages of mouse oocyte after in vitro maturation and fertilization. (Ca) Germinal vesicle (GV, mature oocyte). (Cb) Extruding the first polar body of mouse oocyte. (Cc) Fertilized mouse oocyte.
Effect of cyanocobalamin on IVF and development of embryos
The matured oocytes (Fig. 1 Cb) were assessed by IVF. Sperm were collected from the cauda epididymides of the fertile NMRI males and allowed to capacitate for 1 to 1.5 h at 37°C. The sperm were subsequently diluted in human tubal fluid (HTF; Millipore) to a final concentration of 0.7–1.3 × 106 sperm/ml. The collected matured oocytes in the control and 200 pM cyanocobalamin groups were incubated with spermatozoa for 4 h and then washed to remove the excess spermatozoa. The oocytes were cultured overnight in separate dishes that contained a drop of modified potassium simplex optimized medium (KSOM Sigma-Aldrich, St. Louis, MO, USA) supplemented with the 100, 200, 300, and 500 pM concentrations of cyanocobalamin. At 24 h after fertilization, we determined the percentage of 2-cell embryos to evaluate the fertilization rate. The embryos were subsequently cultured at 37°C and 5.5% CO2 until they reached the blastocyst stage.
Superovulation
Each female mouse was superovulated by an i.p. injection of 8 IU PMSG, followed by an injection of 7.5 IU hCG 48 h later.
Two-cell embryo collection for the in vitro study
In vivo embryos at the 2-cell stage were obtained from superovulated female mice that were individually mated with fertile males of the same strain. The following morning, successful mating was confirmed by the presence of a vaginal plug in the female mice. This was considered to be gestation day 1. The 2-cell embryos were flushed and collected with M2 (Sigma-Aldrich, St. Louis, MO, USA) medium supplemented with bovine serum albumin (BSA) on day 2 post-coitum (p.c.), 48 h after hCG administration. The 2-cell embryos that had normal morphology, which consisted of equal-sized blastomeres with no apparent fragmentation as observed under an inverted light microscope, were divided into five groups – control and 100, 200, 300 and 500 pM of cyanocobalamin.
Statistical analysis
The data were analyzed by Tukey’s post-hoc test using one-way analysis of variance (ANOVA), and are presented as mean ± standard deviation (SD). The differences in the values of maturation, fertilization, and developmental rates were considered to be significant at P < 0.05. For Windows, all computations were performed using Statistical Package for the Social Sciences (SPSS) 25. All data are presented as mean ± SD in the graphs from a minimum of three independent experiments.
Results
Effect of cyanocobalamin on nuclear maturation of mouse oocytes
Figure 1(A) shows the effect of cyanocobalamin during IVM on nuclear maturation of mouse oocytes. The mature oocytes were evaluated at the MII stage, which was morphologically characterized by germinal vesicle breakdown (GVBD) and the release of a distinct first polar body (Fig. 1 Cb).
The 100 pM (43.33%, P < 0.001) and 200 pM (79.33%, P < 0.0001) cyanocobalamin groups had significantly more MII oocytes compared with the control group. Treatment with 100 pM cyanocobalamin significantly reduced oocyte maturation compared with the 200 pM cyanocobalamin group (P < 0.0001). Although the 300 pM (24%) and 500 pM (19.33%) cyanocobalamin groups had fewer immature oocytes than the control group (28%), these findings were not significant (Fig. 1 A).
IVF and development of mouse embryos
The success rate for IVF (Fig. 1 Cc) was measured by the number of embryos that entered the 2-cell stage. As shown in Fig. 1(B), high IVF success rate was observed in the 200 pM cyanocobalamin treatment group compared with other groups (P < 0.01).
Table 2 and Fig. 2(A) show that the morula (Fig. 2 Bb) rates were significantly different between the 100 pM cyanocobalamin (29.33%) group and the control group (P < 0.005). The rate of embryo development to the blastocyst stage (Fig. 2 Bc) was greater in the 100 pM cyanocobalamin group (37.33%) compared with the control group (32.67%), however this finding was not significant.
Figure 2. (A) Effect of cyanocobalamin during early embryogenesis after in vitro fertilization. Each point represents the mean ± SEM of four repeats. P-values are shown in Table 1. (B) Bright field photographs of embryos cultured under different concentrations of cyanocobalamin.
The results of our study demonstrated that mouse embryos cultured in the medium that contained 200 pM cyanocobalamin (49%) developed faster and had increased development to the blastocyst stage compared with mouse embryos cultured in the control group (32.67%; P < 0.0001). The 200 pM cyanocobalamin group compared with the 100 pM cyanocobalamin group had the lowest percentage of 2-cell (6%; P < 0.01) and degenerated embryos (8.67%; P < 0.01). In the 200 pM cyanocobalamin group, we observed the development of the 4-cell (2.3%), 8-cell (6%, P < 0.05), and morula (28%, P < 0.01) stages. This finding suggested that the 100 pM cyanocobalamin group delayed embryonic cleavage when compared with the 200 pM group (Table 1 and Fig. 2 A).
Table 1. Assessment of the effect of different concentrations of cyanocobalamin on the percentage of early mouse embryos in vitro
*: P < 0.05, **: P < 0.01, ***: P < 0.005 and ****: P < 0.0001: significant differences within the same column with control. Data are presented as mean ± SEM.
There were significant differences between the 300 pM cyanocobalamin and control groups in terms of the 2-cell (29.67 % vs 14%), 8-cell (1% vs 12.33%), morula (7% vs 20%), blastocyst (16.33% vs 32.67%), and degenerated (40.33% vs 16.67%) embryos (P < 0.0001) (Table 1 and Fig. 2 A, Bd). The 500 pM cyanocobalamin group had similar 8-cell, morula, and blastocyst rates (0%; P < 0.0001) (Table 1 and Fig. 2 A, Be). However, the 300 and 500 pM cyanocobalamin groups yielded similar results (29.67% and 28.9%, respectively) in terms of blocking to the 2-cell stage (Table 1 and Fig. 2A). However, the 500 pM cyanocobalamin group had a significantly higher 2-cell block and degenerated embryos compared with the other groups (P < 0.0001) (Table 1 and Fig. 2 A).
There were no significant differences in the rate of the 4-cell stage between the four different treatment groups compared with the control group at the end of the culture period (Table 1 and Fig. 2 A).
The effect of cyanocobalamin on 2-cell mouse embryo development in vitro
The developmental rates of 2-cell embryos in medium that contained different concentrations of cyanocobalamin are shown in Table 2 and Fig. 3. The embryos cultured in 100 pM cyanocobalamin had a significantly greater frequency of development to the morula stage (29%) compared with the control group (23%, P < 0.05) and blastocyst stage (52%) compared with the control group (39.5%, P < 0.0001) (Table 2 and Fig. 3 A, Ba, Bb). We also observed a significant decrease in 2-cells blocked in the 100 pM group (3%) compared with the control group (14.5%, P < 0.0001), in the 8-cell embryos (9%) compared with the control group (14%), and degenerated embryos (7%) compared with the control group (8%) (Table 2 and Fig. 3 A).
Table 2. Assessment of the effect of different concentrations of cyanocobalamin on the development of mouse 2-cell embryos in vitro
*: P < 0.05, **: P < 0.01, ***: P < 0.005 and ****: P < 0.0001: significant differences within the same column with control. Data are presented as mean ± SEM.
Figure 3. (A) Effect of different concentrations of cyanocobalamin on the development of mouse two-cell embryos in vitro. Each point represents the mean ± SEM of three repeats. P-values are shown in Table 2. (B) Morphology of embryos resulted from different concentrations of cyanocobalamin.
Treatment with 200 pM cyanocobalamin during early embryogenesis increased the blastocyst rate (70%) of embryos compared with the control group (39.5%; P < 0.0001). There were no significant differences in the percentages of morula (24%) and degenerated (2.5%) embryos compared with the control group (Table 2 and Fig. 3 A, Bc). The blastocyst rate of embryos increased in the 200 pM cyanocobalamin group (70%) during late embryogenesis when compared with the 100 pM cyanocobalamin group (52%; P < 0.0001). The lowest percentage of blocked 2-cell (1% vs 14.5%) and 8-cell (2.5% vs 14%) embryos was observed in the 200 pM cyanocobalamin group (P < 0.0001) (Table 2 and Fig. 3 A).
The percentage of the 2-cell blocked (46.5% vs 14.5%) and degenerated (25% vs 8%) embryos were significantly higher in the 300 pM cyanocobalamin group compared with the culture medium alone (P < 0.0001). In the 300 pM cyanocobalamin group, there were significant differences in the embryos that reached the 8-cell (4%), morula (13.5%), and blastocyst (7%) stages compared with the control group (P < 0.0001) (Table 2 and Fig. 3 A, Bd).
There were significantly more embryos in the 2-cell block and degenerated in the 500 pM cyanocobalamin group compared with the control and the other treatment groups (P < 0.0001). There were no embryos observed in the 8-cell, morula, and blastocyst stages in the 500 pM cyanocobalamin group (Table 2 and Fig. 3 A, Be). No significant differences existed in the rate of embryos that reached the 4-cell stage between the four different treatment groups compared with the control group at the end of the culture period (Table 2 and Fig. 3 A).
Discussion
Frequently, in routine IVF, a few morphologically mature MII oocytes obtained from mature follicles remain unfertilized or, if fertilized, they result in low quality embryos under the same culture conditions (Rizzo et al., Reference Rizzo, Raffone and Benedetto2010). Follicular fluid constitutes the actual pre-fertilization environment of the mature oocyte and may affect IVF outcome parameters of fertilization, embryo cleavage, and pregnancy rates (Agarwal et al., Reference Agarwal, Saleh and Bedaiwy2003). Follicular fluid, as well as granulosa cells, growth factors and steroids hormones include leukocytes, cytokines and macrophages, all of which can produce ROS (Attaran et al., Reference Attaran, Pasqualotto, Falcone, Goldberg, Miller, Agarwal and Sharma2000). ROS may be produced by the environment, impaired oocyte metabolism, or both (Rizzo et al., Reference Rizzo, Raffone and Benedetto2010). Pre-treatment with cobalamin has a positive effect on the microenvironment of the maturing oocyte by reducing total homocysteine (Hcy) concentrations in the pooled FF and increasing follicular diameter (Rizzo et al., Reference Rizzo, Raffone and Benedetto2010). Studies have shown that Hcy mediates ROS accumulation (Van De Lagemaat et al., Reference Van De Lagemaat, De Groot and Van Den Heuvel2019). The potential antioxidant properties of cobalamin can be attributed to direct scavenging of ROS and reduction of Hcy-induced oxidative stress (Van De Lagemaat et al., Reference Van De Lagemaat, De Groot and Van Den Heuvel2019). Glutathione plays a significant role in oocyte maturation (Luberda, Reference Luberda2005) and is essential for fertilization, preimplantation, and embryonic development (Nakamura et al., Reference Nakamura, Fielder, Hoang, Lim, McConnachie, Kavanagh and Luderer2011). The results of studies indicate that elevated levels of GSH in matured mouse and hamster oocytes are needed to form the male pronucleus after fertilization and for early embryo development (Gardiner and Reed, Reference Gardiner and Reed1994; Zuelke et al., Reference Zuelke, Jeffay, Zucker and Perreault2003). There is significantly less glutathione in matured in vitro oocytes compared with those matured in vivo (Luberda, Reference Luberda2005). Cobalamin can indirectly stimulate ROS scavenging by maintaining glutathione levels (Van De Lagemaat et al., Reference Van De Lagemaat, De Groot and Van Den Heuvel2019). Oxidative stress causes a negative impact on fertilization and the in vitro preimplantation developing embryo (Lopes et al., Reference Lopes, Lane and Thompson2010). During IVF, gametes and embryos are in culture media (Martín-Romero et al., Reference Martín-Romero, Miguel-Lasobras, Domínguez-Arroyo, González-Carrera and Álvarez2008) that contain serum or serum synthetic replacements (SSR), albumin, vitamins, and other elements such as buffers or heavy metal chelators. Consequently, the medium itself can be a source of ROS during handling and culture (Burton et al., Reference Burton, Hempstock and Jauniaux2003). Several researchers have reported that high intracellular ROS concentrations have adverse effects during maturation and early cleavage, and result in changes in chromosome segregation during meiosis, disrupted fertilization, 2-cell embryo blockage, and low pregnancy levels (Martín-Romero et al., Reference Martín-Romero, Miguel-Lasobras, Domínguez-Arroyo, González-Carrera and Álvarez2008). During the IVC of preimplantation embryos, the absence of maternal antioxidants also leads to a breakdown in the balance between ROS production and clearance, and results in developmental arrest. Therefore, antioxidants are commonly monitored during the cultivation of preimplantation embryos to remove excessive ROS (Ye et al., Reference Ye, Xu, Liu, Pang, Lian, Zhong, Su and Wang2017).
The present study showed that exposure of mouse oocytes to 200 pM cyanocobalamin improved progression of maturation in mouse oocytes. We also observed an increased fertilization rate following IVF in the 200 pM cyanocobalamin group, which was associated with quantitative improvement in blastocysts and the rate of blastocysts from matured oocyte in vivo. Minimizing oxidative stress is especially important when IVF is performed for assisted reproduction (Tsunoda et al., Reference Tsunoda, Kimura and Fujii2014).
The 2-cell block identified functionally the failure of fertilized eggs to develop in vitro after the 2-cell stage in the medium (Biggers, Reference Biggers2004). Arrest of embryos developed in vitro is commonly seen in several strains of mice and other species (Betts and Madan, Reference Betts and Madan2008). The increased levels of ROS in mice were related to the 2-cell embryo block (Nasr-Esfahani et al., Reference Nasr-Esfahani, Aitken and Johnson1990). The 2- to 4-cell embryos had immature (undifferentiated) mitochondria and elevated intracellular ROS levels (Betts and Madan, Reference Betts and Madan2008). An immature mitochondrial electron transport system can also result in increased ROS production, which leads to the formation of superoxide (Kimura et al., Reference Kimura, Tsunoda, Iuchi, Abe, Totsukawa and Fujii2010). As mentioned earlier, one of the most important potential antioxidant properties of cobalamin is direct scavenging of ROS, specifically superoxide (Van De Lagemaat et al., Reference Van De Lagemaat, De Groot and Van Den Heuvel2019). We found that the rates of 2-cell block and degenerated embryos were substantially reduced in the presence of 200 pM cyanocobalamin. Subsequently, the current study found that doses higher that 200 pM cyanocobalamin (300 and 500 pM) significantly decreased early embryo developmental potential and increased the amount of degenerated embryos. As well, treatment with 100 pM cyanocobalamin showed a slight increase in the embryo development rate compared with the control. When the concentration of cyanocobalamin was increased to 200 pM, we observed a protective effect of cyanocobalamin on embryo development. These data suggested that cyanocobalamin has a dose-dependent antioxidant feature.
Hormesis refers to a biphasic dose response to an environmental agent with a low-dose stimulus or beneficial effect and a high-dose inhibitory or harmful effect (Kendig et al., Reference Kendig, Le and Belcher2010; Assadollahi et al., Reference Assadollahi, Mohammadi, Fathi, Hassanzadeh, Erfan, Soleimani, Banafshi, Yousefi and Allahvaisi2019). It has been shown that vitamins, including B12, are hermetic nutrients (Hayes, Reference Hayes2007). The results of the present study agree with findings of previous studies. In our study, we observed that oocytes and embryos were healthier in the 200 pM cyanocobalamin dose. According to previous studies, the optimal amount of cobalamin is 200 pM (Hannibal et al., Reference Hannibal, Lysne, Bjørke-Monsen, Behringer, Grünert, Spiekerkoetter, Jacobsen and Blom2016; Green et al., Reference Green, Allen, Bjørke-Monsen, Brito, Guéant, Miller, Molloy, Nexo, Stabler, Toh, Ueland and Yajnik2017). Zacchini and colleagues reported that 200 pM of cobalamin supplementation during IVM improved the developmental competence of sheep oocytes (Zacchini et al., Reference Zacchini, Toschi and Ptak2017). Excessive antioxidants are ineffective and often have harmful effects (Tsunoda et al., Reference Tsunoda, Kimura and Fujii2014). It appears that high doses of antioxidants serve as enzyme inhibitors and mutagens by inhibiting topoisomerases, proteasome synthesis, or the synthesis of fatty acids (Crespo et al., Reference Crespo, García-Mediavilla, Almar, González, Tuñón, Sánchez-Campos and González-Gallego2008, Sameni et al., Reference Sameni, Javadinia, Safari, Amjad, Khanmohammadi, Parsaie and Zarbakhsh2018).
Improving the formulation of the embryo culture medium leads to an increase in the ability of mammalian embryos to survive in vitro and preimplantation (Farin et al., Reference Farin, Crosier and Farin2001). The culture system and the compounds used in it could affect the quality of the embryo, while the quality of the oocyte is considered to be the main determinant of blastocyst function (Rizos et al., Reference Rizos, Ward, Duffy, Boland and Lonergan2002). However, the environment to which the embryos are exposed in the preimplantation stage plays an important role in determining blastocyst quality (Pereira et al., Reference Pereira, Dode and Rumpf2005).
In conclusion, the present study demonstrated that supplementation of 200 pM cyanocobalamin in IVC medium enhanced nuclear maturation of oocytes, IVF, and embryo development in mice. However, higher concentrations of cyanocobalamin (300 and 500 pM) in IVC negatively affected oocyte maturation, IVF, and embryo development. Therefore, our findings suggest that 200 pM cyanocobalamin might improve in vitro production systems of mouse embryos.
Acknowledgements
This study, as a MSc. thesis, was funded by grants provided from Kurdistan University of Medical Sciences (mo. IR.MUK.REC.1398/292). We express our appreciation to all members of the Cellular and Molecular Research Center for their helpful consultation and deliberation during this work.
Financial support
Kurdistan University of Medical Sciences (No. IR.MUK.REC.1398/292).
Conflicts of interest
The authors declare they have no competing financial interests.
Ethical standards
The experiments were conducted with the approval of the Ethics Committee of the Kurdistan University of Medical Sciences.
