Introduction
The industry of bovine in vitro produced (IVP) embryos has grown substantially worldwide in the past decades, with a little over a million IVP embryos produced in 2019, which represented nearly 70% of the total bovine embryos produced worldwide (Viana, Reference Viana2020). Out of IVP embryos transferred to recipients, ∼56% were transferred fresh and 44% were transferred following cryopreservation procedures (Viana, Reference Viana2020). Despite the gradual increase in the number of cryopreserved IVP embryos transferred to recipients, IVP embryos are still considered less cryotolerant than their in vivo-derived counterparts (Abe et al., Reference Abe, Yamashita, Satoh and Hoshi2002; Seidel, Reference Seidel2006; Sudano et al., Reference Sudano, Paschoal, Rascado, Magalhães, Crocomo, de Lima-Neto and Landim-Alvarenga2011; Gómez et al., Reference Gómez, Carrocera, Martín, Pérez-Jánez, Prendes, Prendes, Vázquez, Murillo, Gimeno and Muñoz2020). Therefore, the development of reliable methods to improve cryosurvival of IVP embryos is necessary to optimize the efficiency of IVP procedures and to enhance the feasibility of using IVP embryos in breeding programmes of dairy and beef herds (Ribeiro et al., Reference Ribeiro, Galvão, Thatcher and Santos2012; Leme et al., Reference Leme, Carvalho, Franco and Dode2020).
The lower cryotolerance of bovine IVP embryos seems to be related to suboptimal in vitro culture conditions (Lonergan et al., Reference Lonergan, Rizos, Gutierrez-Adan, Fair and Boland2003). The phenomenon is associated with increased apoptosis (Bain et al., Reference Bain, Madan and Betts2011) and DNA fragmentation in IVP embryos compared with in vivo-produced embryos (Velez-Pardo et al., Reference Velez-Pardo, Morales, Del Rio and Olivera-Angel2007). In addition, the elevated intracytoplasmic lipid content of IVP embryos appears to increase the sensitivity to oxidative stress and to compromise embryo quality and the response to freezing (Guérin et al., Reference Guérin, El Mouatassim and Ménézo2001; Seidel, Reference Seidel2006). Oxidative stress in embryo culture is characterized by an imbalance between oxidants and antioxidants induced by an increase in oxidative agents or a decrease in antioxidants (Rocha-Frigoni et al., Reference Rocha-Frigoni, Leão, Nogueira, Accorsi and Mingoti2014). Excessive amounts of free radicals can promote changes in proteins and lipids, resulting in DNA damage that further compromises the survival of IVP embryos to culture conditions and cryopreservation (Ray et al., Reference Ray, Huang and Tsuji2012; Jamil et al., Reference Jamil, Debbarh, Aboulmaouahib, Aniq Filali, Mounaji, Zarqaoui, Saadani, Louanjli and Cadi2020; Lin and Wang, Reference Lin and Wang2020). Oxidative modifications of cellular components represent the main stress inducer of embryos in culture and are especially important after cryopreservation because of the resulting cryo-injuries (Deleuze and Goudet, Reference Deleuze and Goudet2010). Nevertheless, it is noteworthy that free radicals are also important messengers in cell signalling (Ray et al., Reference Ray, Huang and Tsuji2012). In fact, many studies have shown a key role of physiological concentration of free radicals in plasma membrane ion transport, pH changes, redox potential, oocyte development and fertilization competence, and regulation of mitotic divisions during early embryonic development, factors that ultimately determine the success in embryo development (Jamil et al., Reference Jamil, Debbarh, Aboulmaouahib, Aniq Filali, Mounaji, Zarqaoui, Saadani, Louanjli and Cadi2020; Lin and Wang, Reference Lin and Wang2020). Therefore, the success of IVP production and cryopreservation depends greatly on the redox homeostasis of embryos, which work in an interdependent and dynamic balance between free radicals and antioxidant compounds (Lin and Wang, Reference Lin and Wang2020).
Antioxidants have a critical role in preventing excessive formation of free radicals and consequent oxidative damage (Sies, Reference Sies1997). Antioxidant precursors, especially of low molecular weight, such as thiol compounds, have been used in IVP, showing favourable effects on embryo development (Mori et al., Reference Mori, Otoi, Wongsrikeao, Agung and Nagai2006; de Castro e Paula and Hansen, Reference de Castro e Paula and Hansen2008). For instance, the antioxidant β-mercaptoethanol (βME), a precursor of glutathione (GSH), has a wide range of biological actions in embryo development (Rodríguez-González et al., Reference Rodríguez-González, López-Bejar, Mertens and Paramio2003; Mori et al., Reference Mori, Otoi, Wongsrikeao, Agung and Nagai2006). β-Mercaptoethanol has been shown to reduce 2,2′-azobis(2-amidinopropane)dihydrochloride (AAPH)-initiated peroxyl radicals, to inhibit the oxidation of free sulfhydryl residues, to chelate metal ions and divalent cations, and to remove hydroxyl radicals when in solution (Cornell and Crivano, Reference Cornell and Crivaro1972; Rashidipour et al., Reference Rashidipour, Karami-Mohajeri, Mandegary, Mohammadinejad, Wong, Mohit, Salehi, Ashrafizadeh, Najafi and Abiri2020). Supplementation of βME in different IVP stages improved developmental competence of IVP embryos through the maintenance of oxidative homeostasis and the protection of blastomeres against the effects of oxidative lesions, increasing blastocyst rates (Takahashi et al., Reference Takahashi, Nagai, Hamano, Kuwayama, Okamura and Okano1993; Caamaño et al., Reference Caamaño, Ryoo and Youngs1998; Geshi et al., Reference Geshi, Yonai, Sakaguchi and Nagai1999; de Matos and Furnus, Reference de Matos and Furnus2000; Feugang et al., Reference Feugang, de Roover, Moens, Léonard, Dessy and Donnay2004). In addition, βME added to the culture medium after vitrification resulted in beneficial effects on bovine IVP embryos, increasing blastocyst survival, hatching rates, and total cell numbers (Nedambale et al., Reference Nedambale, Du, Yang and Tian2006). In vitrified buffalo IVP blastocysts, βME modulated the expression of genes associated with embryo quality and antagonistic mechanisms to apoptosis (Moussa et al., Reference Moussa, Yang, Zheng, Li, Yu, Yan, Huang and Shang2019). Nevertheless, the effects of antioxidant precursors, such as βME, on the process of cryopreservation of IVP embryos still need more attention (Nedambale et al., Reference Nedambale, Du, Yang and Tian2006; Hosseini et al., Reference Hosseini, Forouzanfar, Hajian, Asgari, Abedi, Hosseini, Ostadhosseini, Moulavi, Safahani Langrroodi, Sadeghi, Bahramian, Eghbalsaied and Nasr-Esfahani2009; Rocha-Frigoni et al., Reference Rocha-Frigoni, Leão, Nogueira, Accorsi and Mingoti2014). Timing of supplementation and inclusion levels according to predicted needs of the IVP system are important factors to be further investigated. Therefore, the objective of this study was to evaluate the effects of timing and concentration of βME supplementation on in vitro embryo culture medium on the quality, viability, and cryotolerance of bovine IVP embryos.
Materials and methods
Chemicals and experimental conditions
All reagents were from Sigma-Aldrich Chemical (St. Louis, MO, USA), unless stated otherwise. All procedures were performed in a laboratory at room temperature.
In vitro embryo production
In vitro production of bovine embryos was performed according to our established standard procedures (Ribeiro et al., Reference Ribeiro, Gerger, Ohlweiler, Ortigari, Mezzalira, Forell, Bertolini, Rodrigues, Ambrósio, Miglino, Mezzalira and Bertolini2009) with a few modifications. Bovine ovaries obtained at a local slaughterhouse were transported to the laboratory at 30°C in phosphate-buffered saline (PBS) solution supplemented with penicillin (100 IU/ml) and streptomycin (0.05 mg/ml) between 2 and 4 h after collection. Ovaries were washed in PBS, and cumulus–oocyte complexes (COCs) were aspirated from 3–8 mm diameter follicles with an 18G needle attached to a 10-ml syringe and pooled into 50-ml conical tubes. Only grades I and II COCs (Stojkovic et al., Reference Stojkovic, Machado, Stojkovic, Zakhartchenko, Hutzler, Gonçalves and Wolf2001) were selected under a stereomicroscope (×15 magnification) for in vitro maturation (IVM). Selected COCs were transferred in groups of 40–50 structures to four-well dishes (Nunclon™, Roskilde, Denmark; cat. no. 176740) containing 400 μl of in vitro maturation (IVM) medium, composed of TCM 199 with Earle’s salts, supplemented with 26.2 mM NaHCO3, 25 mM HEPES, 0.2 mM sodium pyruvate, 5 μg/ml FSH/ml (Follitropin, Bioniche, Canada), 0.5 μg/ml LH (Lutropin, Bioniche, Canada) and 10% of inactivated estrous mare serum (EMS), under mineral oil, maintained at 39°C, 5% CO2 in air and saturated humidity for 24 h.
Frozen semen from a previously tested Bos taurus bull was used for bovine IVF procedures. Sperm cells were selected by the swim-up technique in Sperm-TALP medium supplemented with 6 mg/ml bovine serum albumin (BSA), at 39°C, in a water bath. After 60 min of ascending migration, the supernatant was collected and centrifuged for 5 min at 700 g, with the pellet collected and diluted in Sperm-TALP medium to obtain an inseminating dose of 1 × 106 to 1.5 × 106 spermatozoa/ml matured COCs and sperm cells were co-incubated for 18–22 h (Day 0) at 38.5°C, in 5% CO2, and saturated humidity. Fertilization medium consisted of Fert-TALP medium supplemented with 30 μg/ml heparin, 30 μg/ml penicillamine, 15 μM hypotaurine, and 1 μM epinephrine, in four-well plates with the same number of IVM structures. After fertilization (Day 1), presumptive zygotes were gently denuded by repeated pipetting in TCM-HEPES medium.
Immediately after cell removal, presumptive zygotes were washed and cultured in four-well plates containing 40–50 structures per well in 400-μl drops of SOFaaci medium (Holm et al., Reference Holm, Booth, Schmidt, Greve and Callesen1999) supplemented with 6 mg/ml of BSA covered by mineral oil. In the first 24 h, embryos were cultured in 5% CO2 in air. Cleavage rate was evaluated on Day 2 when plates were placed into an impermeable bag (Bag-system; Vajta et al., Reference Vajta, Holm, Greve and Callesen1997), under a gas mixture containing 90% N2, 5% O2 and 5% CO2 in an incubator at 38.5°C and saturated humidity, for 7 days. On Day 7 of development, blastocyst rates and embryo kinetics were evaluated by morphology and according to Stringfellow and Givens (Reference Stringfellow and Givens2010).
Vitrification and warming procedures
Embryo vitrification was performed as previously described by Werlich et al. (Reference Werlich, Barreta, Martins, Vieira, Moraes and Mezzalira2006) based on open pulled straw technology. Heated and stretched glass capillaries were used to make micropipettes (glass micropipettes, or GMP). For vitrification, groups of three blastocysts of excellent or good quality (Grade 1) on Day 7 of development were first washed for 1 min with an equilibrium solution (extracellular solution) composed of 10% ethylene glycol (EG) and 10% propylene glycol (PRO) in PBS + 10% EMS, followed by a 20 s exposure to a vitrification solution (extracellular solution) composed of 20% EG, and 20% PRO in PBS + 10% EMS, at room temperature (25ºC). Subsequently, embryos were placed in a small droplet (< 2 μl), pulled inside the GMP by capillarity, and immediately submerged into liquid nitrogen.
Warming was performed 30–90 min after vitrification by exposing the GMPs to air for 3 s, and then immerging into a tube containing 1.2 ml warming solution (PBS + 10% FBS) with 0.3 M sucrose for 5 min at 39ºC. The GMP content was transferred to a drop containing the warming solution with 0.15 M sucrose. After 5 min, embryos were segregated homogeneously in groups, according to the experiments below, and placed in in vitro culture in SOFaaci + 5% EMS at 38.5ºC, 5% CO2 and saturated humidity for 72 h to evaluate the in vitro re-expansion and hatching rates. Blastocysts reaching the hatching stage (Be) were considered viable.
Estimation of total cell number in IVP blastocysts
Estimation of the total cell numbers in embryos was performed according to Ribeiro et al. (Reference Ribeiro, Gerger, Ohlweiler, Ortigari, Mezzalira, Forell, Bertolini, Rodrigues, Ambrósio, Miglino, Mezzalira and Bertolini2009). Hatched embryos from each treatment group were segregated by hatching day and by morphological classification, being fixed in 99% ethanol at 4ºC for at least 12 h. For the estimation of cell number, fixed embryos were exposed to a solution of 15 μg/ml bisbenzimide (Hoechst 33342) in 99% ethanol for 10 min protected from direct light. Embryos were then placed in a minimum volume in a 10-μl glycerol drop on a slide, which was covered with a coverslip. Finally, embryos were examined under UV light (450–490 nm excitation, 500–540 nm emission) at ×400 magnification in an inverted fluorescence microscope (Zeiss AxioVert 135®, Oberkochen, Germany).
Experiment I: Effect of β-mercaptoethanol (βME) added to the post-warming in vitro culture (PWC) medium on blastocyst survival after vitrification
The effects of adding 50 or 100 μM βME to the PWC on the survival and hatching rates after 72 h were evaluated. Day-7 grade-1 vitrified blastocysts (n = 191) were segregated randomly to three groups, as follows: (i) 0-βME group, control group without βME supplementation in the PWC medium; (ii) 50-βME group, PWC medium supplemented with 50 μM βME; and (iii) 100-βME group, PWC medium supplemented with 100 μM βME. Embryos were in vitro cultured for up to 72 h, at 38.5ºC, 5% CO2, and saturated humidity, with evaluation of re-expansion and hatching rates every 24 h. β-Mercaptoethanol was added to fresh culture medium on the day of blastocyst warming, and its content was not replenished during the 72 h culture to prevent disturbance in the culture conditions and to make the proposed intervention more practical for consideration in commercial laboratories.
Experiment II: Effect of βME supplementation prior to and after vitrification on embryo survival and in vitro development
The effects of adding 100 μM βME to the medium prior to and/or after vitrification on the cryotolerance and viability of vitrified IVP bovine blastocysts were evaluated (Figure 1). In vitro culture medium was either supplemented or not with 100 μM βME from Day 1 to Day 7 of embryo development. Resulting Day-7 blastocysts were vitrified and, after warming, in vitro cultured in medium either supplemented or not with 100 μM βME for up to 72 h. Thus, four experimental groups were formed in a 2 × 2 factorial, as follows:
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CTRL–CTRL: control IVC and control PWC
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CTRL–βME: control IVC and βME-supplemented PWC
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βME–CTRL: βME-supplemented IVC and control PWC
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βME–βME: βME-supplemented IVC and βME-supplemented PWC.
Figure 1. Treatment design of Experiment II. In total, 3735 COCs were matured and fertilized in vitro and then randomly allocated to be cultured in control medium (Control IVC) or medium containing 100 μM βME (βME IVC) from Day 1 to Day 7 of embryo development. Resulting Day-7 blastocysts were vitrified and, after warming, randomly allocated to post-warming culture (PWC) in control medium (control PWC) or medium containing 100 μM βME (βME PWC) for 72 h, forming four experimental groups in a 2 × 2 factorial design. CTRL–CTRL: control IVC and control PWC; CTRL–βME: control IVC and βME-supplemented PWC; βME–CTRL: βME-supplemented IVC and control PWC; βME–βME: βME-supplemented IVC and βME-supplemented PWC.
The main effect of βME supplementation in IVC (βME–IVC) was evaluated as (CTRL–CTRL + CTRL–βME) vs. (βME–CTRL + βME–βME). The main effect of βME supplementation in PWC (βME–PWC) was evaluated as (CTRL–CTRL + βME–CTRL) vs. (CTRL–βME + βME–βME).
Cleavage rates were evaluated on Day 2, and blastocyst rates and embryo kinetics on Day 7. Re-expansion rates were evaluated 12 h after embryo warming, and hatching rates were evaluated at 24, 48, and 72 h of PWC. In addition, the morphological quality and total cell numbers were evaluated in hatched blastocysts after 72 h of PWC. Similar to Experiment I, β-mercaptoethanol was added to fresh culture medium on the day that embryos were put into culture, and its content was not replenished during the culture periods to prevent disturbance in the culture conditions and to make the proposed interventions more practical for consideration in commercial laboratories.
Statistical analyses
Data regarding cleavage and blastocyst rates, embryo morphological quality after IVP, and re-expansion and hatching rates after 12 h and 72 h of PWC, respectively, were compared between groups within each experiment by chi-squared (χ2) test, for P < 0.05. Total cell numbers in hatched embryos between groups were subjected to analysis of variance, with pairwise comparisons using Tukey’s test (P < 0.05). Statistical analyses were performed using Minitab® (State College, PA, USA).
Results
Experiment I: Effect of β-mercaptoethanol (βME) supplementation to the in vitro culture medium after vitrification on subsequent embryo survival
According to Table 1, the supplementation with 100 μM βME in PWC improved hatching rates of vitrified Day-7 blastocysts when compared with the control (0-βME) group (66.7% vs. 47.7%, P < 0.05), and both groups did not differ from the 50-βME group (52.4%). Based on such results, after five replications, the 100 μM βME concentration was chosen to be used in Experiment II.
Table 1. Bovine IVP blastocyst hatching rates following vitrification, after 72 h of post-warming in vitro culture (PWC) in medium supplemented with 0 (control, 0-βME), 50 µM (50-βME) or 100 µM βME (100-βME)
a,bNumbers in the column with distinct superscripts differ (P < 0.05).
Experiment II: Effect of βME on embryo cryotolerance and post-cryopreservation viability
Results summarizing the effects of 100 μM βME supplementation during the IVC and/or the PWC are presented in Table 2 and Figures 1–4. In total, 3735 COCs used for bovine IVP of embryos, and after seven replications, the supplementation of 100 μM βME to the IVC did not affect cleavage rates on Day 2 of development, which averaged 80.6%. However, compared with the control group, βME-IVC negatively interfered with subsequent embryo development to the blastocyst stage (Table 2) and caused a delay in embryo kinetics on Day 7 of development (Figure 2). Compared with the control, βME-IVC resulted in a greater proportion of embryos on stages 5 and 6, and a smaller proportion of embryos on stages 7–9 (Figure 2). Embryo quality classification, however, was not affected by treatment (Table 2).
Table 2. Cleavage and blastocyst rates, and embryo quality by morphological evaluation after in vitro culture (IVC) of bovine IVP embryos in IVC medium supplemented with 0 (CTRL–IVC) or 100 µM βME (βME–IVC)
a,bNumbers in the same column with distinct superscripts differ (P < 0.05).
Figure 2. Developmental kinetics of Day-7 bovine blastocysts after IVC from Day 1 to Day 7 in medium supplemented (βME–IVC) or not (CTRL–IVC) with 100 µM βME. Classification of stage of development: 5 = early blastocyst; 6 = blastocyst; 7 = expanded blastocyst; 8 = hatching blastocyst; 9 = hatched blastocyst. a,bWithin stage of development, columns with distinct superscripts differ (P < 0.05).
Data on the effect of βME on embryo re-expansion and hatching rates in all groups are presented in Table 3 and Figure 3. The use of βME during IVC, both prior to (βME–CTRL), and prior to and after (βME–βME) vitrification of Day-7 blastocysts increased re-expansion rates compared with the CTRL–CTRL group. However, re-expansion rates in the CTRL–βME group were lower than the βME–βME group and did not differ from the CTRL–CTRL and the βME–CTRL groups. Compared with the CTRL–CTRL group, total hatching rates were higher after βME supplementation after (CTRL–βME), and prior to and after (βME–βME) vitrification. Supplementation of βME during the IVC period alone (βME–CTRL) resulted in similar hatching rates to CTRL–CTRL, which was also lower compared with the CTRL–βME and βME–βME groups (Table 3). On a daily basis, such differences were pronounced only 48 h after warming, as shown in Figure 3(a). When the main effect of βME supplementation in IVC was evaluated (CTRL–CTRL + CTRL–βME vs. βME–CTRL + βME–βME), βME supplementation improved the rate of blastocyst re-expansion but did not affect the rate of blastocyst hatching after vitrification (Figure 3b). The opposite was seen for the main effect of βME supplementation in PWC (CTRL–CTRL + βME–CTRL vs. CTRL–βME + βME–βME), in which βME supplementation did not affect the rate of blastocyst re-expansion but enhanced the rate of blastocyst hatching after vitrification (Figure 3c).
Table 3. Re-expansion and hatching of vitrified bovine blastocysts produced in vitro supplemented with 0 or 100 µM of βME during the in vitro culture (IVC) and/or during the post-warming culture (PWC)
a,b,cNumbers in the same column with distinct superscripts differ (P < 0.05).
1 Experimental group: CTRL–CTRL = control IVC and control PWC; CTRL–βME = control IVC and βME-supplemented PWC; βME–CTRL = βME-supplemented IVC and control PWC; βME–βME = βME-supplemented IVC and βME-supplemented PWC.
2 Re-expansion was evaluated at 12 h and total hatching was evaluated at 72 h after rewarming of vitrified bovine blastocysts.
Figure 3. Effect of 100 µM β-Mercaptoethanol (βME) added to in vitro culture (IVC) and/or post-warming culture (PWC) medium on daily blastocyst hatching rates through 72 h after vitrification according to factorial arrangement of treatments: CTRL–CTRL: control IVC and control PWC; CTRL–βME: control IVC and βME-supplemented PWC; βME–CTRL: βME-supplemented IVC and control PWC; βME–βME: βME-supplemented IVC and βME-supplemented PWC (A). Blastocyst hatching rates at 72 h according to main effects of βME on IVC (B) and the main effects of βME on PWC (C). a,bWithin time of evaluation, columns with distinct superscripts differ (P < 0.05).
As for total cell number in hatched blastocysts, those from βME–βME group had a greater number of cells than those in CTRL–CTRL (181.2 ± 7.4 vs. 139.0 ± 9.9 cells, respectively), with the total cell counts in embryos in CTRL–βME (165.2 ± 6.7 cells) and in the βME–CTRL (159.9 ± 9.2 cells) groups being intermediate and similar to all groups (Figure 4a). Regarding the main effects of the factorial treatments, a greater number of cells in hatched blastocysts was observed for βME supplementation in both IVC (Figure 4b) and PWC (Figure 4c).
Figure 4. Effect of 100 µM β-mercaptoethanol (βME) added to in vitro culture (IVC) and/or post-warming culture (PWC) medium on total cell number in hatched blastocysts 72 h after vitrification according to factorial arrangement of treatments. CTRL–CTRL: control IVC and control PWC; CTRL–βME: control IVC and βME-supplemented PWC; βME–CTRL: βME-supplemented IVC and control PWC; βME–βME: βME-supplemented IVC and βME-supplemented PWC (A). Total cell number in hatched blastocysts according to the main effects of βME on IVC (B) and the main effects of βME on PWC (C). a,bWithin time of evaluation, columns with distinct superscripts differ (P < 0.05).
Discussion
This study evaluated the effects of adding βME in the IVC medium on the development, quality, viability, and cryotolerance of bovine embryos prior to and after vitrification. Although the effects of supplementing IVC medium with βME on the developmental ability and quality of IVP mammalian bovine embryos have been examined previously (Takahashi et al., Reference Takahashi, Nagai, Hamano, Kuwayama, Okamura and Okano1993, Reference Takahashi, Kuwayama, Hamano, Takahashi, Okano, Kadokawa, Kariya and Nagai1996; Caamaño et al., Reference Caamaño, Ryoo and Youngs1998; de Matos and Furnus, Reference de Matos and Furnus2000; Feugang et al., Reference Feugang, de Roover, Moens, Léonard, Dessy and Donnay2004; Mori et al., Reference Mori, Otoi, Wongsrikeao, Agung and Nagai2006; Nedambale et al., Reference Nedambale, Du, Yang and Tian2006; Moussa et al., Reference Moussa, Yang, Zheng, Li, Yu, Yan, Huang and Shang2019), no previous study has compared the effect of βME supplementation both prior to and after the vitrification process on the viability of IVP embryos. Therefore, we tested the hypothesis that βME added to the culture medium of bovine embryos, before and/or after cryopreservation, would increase the cryotolerance to vitrification. Our results showed that adding 100 μM βME in the IVC period improved cryotolerance, as indicated by increased re-expansion rates and total cell number of hatched blastocysts after vitrification, whereas adding 100 μM βME in the PWC increased hatching rates and total cell number of hatched blastocysts after vitrification.
Initially we conducted a dose–response experiment to determine an optimal βME concentration in PWC conditions because of the large variation in concentrations reported in the literature, which ranged from 5 μM to 500 μM (Takahashi et al., Reference Takahashi, Nagai, Hamano, Kuwayama, Okamura and Okano1993; de Matos et al., Reference de Matos, Furnus, Moses, Martinez and Matkovic1996; Caamaño et al., Reference Caamaño, Ryoo and Youngs1998; Geshi et al., Reference Geshi, Yonai, Sakaguchi and Nagai1999; Feugang et al., Reference Feugang, de Roover, Moens, Léonard, Dessy and Donnay2004). Although the supplementation with 50 μM or 100 μM βME had no significant differences on hatching rates between one another in Experiment I, 100 μM βME promoted higher hatching rates than controls (0 μM βME), corroborating with previous studies that demonstrated that 100 μM supported adequate embryo development (Caamaño et al., Reference Caamaño, Ryoo and Youngs1998; Nedambale et al., Reference Nedambale, Du, Yang and Tian2006; Hosseini et al., Reference Hosseini, Forouzanfar, Hajian, Asgari, Abedi, Hosseini, Ostadhosseini, Moulavi, Safahani Langrroodi, Sadeghi, Bahramian, Eghbalsaied and Nasr-Esfahani2009).
In general, data on the effects of βME on embryo development are inconsistent regarding the optimal concentration of βME used in the culture medium (Hosseini et al., Reference Hosseini, Forouzanfar, Hajian, Asgari, Abedi, Hosseini, Ostadhosseini, Moulavi, Safahani Langrroodi, Sadeghi, Bahramian, Eghbalsaied and Nasr-Esfahani2009). Geshi et al. (Reference Geshi, Yonai, Sakaguchi and Nagai1999) showed that 10 μM βME in a co-culture system improved embryo development. Caamaño et al. (Reference Caamaño, Ryoo and Youngs1998) noted that 10 and 100 μM of βME in the culture medium had no difference in blastocyst development (25.3% and 21.3%, respectively), but blastocyst rates in both groups were higher than in the controls without the GSH precursor (10.7%). In contrast, supplementation of 100 μM βME resulted in lower development rates of bovine embryos compared with 50 μM βME (Mori et al., Reference Mori, Otoi, Wongsrikeao, Agung and Nagai2006). The latter corroborated with Takahashi et al. (Reference Takahashi, Nagai, Hamano, Kuwayama, Okamura and Okano1993), who reported that lower concentrations of βME (e.g. 50 μM) promoted better embryo development than higher concentrations, which had detrimental effects on the hatching capacity of embryos. Interestingly, Feugang et al. (Reference Feugang, de Roover, Moens, Léonard, Dessy and Donnay2004) showed that 50 μM in the culture medium had no protective effects on embryos, but supplementation with 100 μM βME increased hatching rates and total cell numbers in surviving blastocysts. In the same study, 100 μM βME reduced apoptosis and stimulated glutathione synthesis in Day-7 IVP embryos. Conversely, studies in other species, such as the pig, demonstrated that 50 μM and 100 μM βME supplementation had no effect on embryo development, total cell number, apoptosis index, and cryotolerance (Castillo-Martín et al., Reference Castillo-Martín, Yeste, Pericuesta, Morató, Gutiérrez-Adán and Bonet2015). According to Hosseini et al. (Reference Hosseini, Forouzanfar, Hajian, Asgari, Abedi, Hosseini, Ostadhosseini, Moulavi, Safahani Langrroodi, Sadeghi, Bahramian, Eghbalsaied and Nasr-Esfahani2009), the apparent inconsistency among studies could be attributed to the culture conditions or developmental stages in which βME has been added. In fact, differential effects of βME as an antioxidant on embryo development seem to be dependent on the species, breed, supplementation dose, cell type, and bioavailability on the culture medium (Nikseresht et al., Reference Nikseresht, Toori, Rahimi, Fallahzadeh, Kahshani, Hashemi, Bahrami and Mahmoudi2017). Although apoptosis was not evaluated in our study, the use of βME in the PWC medium had beneficial effects by increasing embryo survival, manifested by higher hatching rates and total cell numbers in hatched blastocysts.
Zona pellucida (ZP) thickness is considered a morphological marker associated with the likelihood of hatching and implantation outcomes in transferred frozen–thawed embryos (Balakier et al., Reference Balakier, Sojecki, Motamedi, Bashar, Mandel and Librach2012; Nada et al., Reference Nada, El-Noury, Al-Inany, Bibars, Taha, Salama, Hassan and Zein2018). Although ZP thickness of blastocysts was not measured in our study, higher hatching rates under the 100-µM βME supplementation might be related to structural changes in the ZP mediated by thiol-disulfide exchange reactions, in which the thiol group reduces disulfide bonds and induces expansion in ZP glycoproteins (Takeo et al., Reference Takeo, Horikoshi, Nakao, Sakoh, Ishizuka, Tsutsumi, Fukumoto, Kondo, Haruguchi, Takeshita, Nakamuta, Tsuchiyama and Nakagata2015; Truong and Gardner, Reference Truong and Gardner2017). Nonetheless, the higher total cell numbers of blastocysts supplemented with βME indicated improvements in cell proliferation that could lead to faster growth and expansion of blastocysts, promoting mechanical thinning of the ZP and successful hatching of the blastocyst (Goud et al., Reference Goud, Goud, Joshi, Puscheck, Diamond and Abu-Soud2014; Khanmohammadi et al., Reference Khanmohammadi, Movahedin, Safari, Sameni, Yousefi, Jafari and Zarbakhsh2016; Giorgi et al., Reference Giorgi, Ferriani and Navarro2021), a phenomenon already reported by others that tested supplementation with the antioxidant N-acetyl-cysteine (Giorgi et al., Reference Giorgi, Ferriani and Navarro2021).
Results from Experiment II indicated that the 100 μM βME supplementation during the IVC had no influence on cleavage rates (80.2% for controls vs. 81.1% for βME), noting that the addition of βME only on Day 1 was not enough to interfere with the first cleavage. In similar experimental designs, Hosseini et al. (Reference Hosseini, Forouzanfar, Hajian, Asgari, Abedi, Hosseini, Ostadhosseini, Moulavi, Safahani Langrroodi, Sadeghi, Bahramian, Eghbalsaied and Nasr-Esfahani2009) and Rocha-Frigoni et al. (Reference Rocha-Frigoni, Leão, Nogueira, Accorsi and Mingoti2014) also found no interference from βME in cleavage rates. However, in contrast with our findings, results reported by Hosseini et al. (Reference Hosseini, Forouzanfar, Hajian, Asgari, Abedi, Hosseini, Ostadhosseini, Moulavi, Safahani Langrroodi, Sadeghi, Bahramian, Eghbalsaied and Nasr-Esfahani2009) indicated that βME supplementation in the IVC medium, either during 1–8 days and/or 9–10 days of embryo culture, improved the overall developmental competence and quality of bovine IVP embryos. Such findings are consistent with those reported for 4-cell and 8-cell (Geshi et al., Reference Geshi, Yonai, Sakaguchi and Nagai1999) and 8-cell to 16-cell (Caamaño et al., Reference Caamaño, Ryoo and Youngs1998) stage embryos, and beyond the morula stage (Feugang et al., Reference Feugang, Van Langendonckt, Sayoud, Rees, Pampfer, Moens, Dessy and Donnay2003).
Healthy in vitro embryo development depends on keeping the redox homeostasis of blastomeres, which requires a balance between ROS and antioxidant compounds (Bain et al., Reference Bain, Madan and Betts2011; Jamil et al., Reference Jamil, Debbarh, Aboulmaouahib, Aniq Filali, Mounaji, Zarqaoui, Saadani, Louanjli and Cadi2020; Lin and Wang, Reference Lin and Wang2020). Low or moderate levels of ROS are critical for the signal transduction for certain biologically active factors such as nuclear factor κB (NF-κB) (Jamil et al., Reference Jamil, Debbarh, Aboulmaouahib, Aniq Filali, Mounaji, Zarqaoui, Saadani, Louanjli and Cadi2020), which are associated with the regulation of maternal-to-embryonic transition and cell differentiation (Schreck et al., Reference Schreck, Rieber and Baeuerle1991). In this study, 100 μM βME during IVC interfered negatively with blastocyst rates and developmental kinetics. Tsuzuki et al. (Reference Tsuzuki, Saigoh and Ashizawa2005) suggested that βME may remove some divalent cations that are needed for embryonic development prior to the 2-cell stage. Similar results were reported by Rocha-Frigoni et al. (Reference Rocha-Frigoni, Leão, Nogueira, Accorsi and Mingoti2014) who obtained reduced blastocyst rates in bovine embryos supplemented with 100 μM βME compared with unsupplemented controls (33.4% vs. 48.7%, respectively). In agreement with our findings, it is possible that higher βME concentrations (100 μM) during IVC result in suboptimal ROS levels and negatively affect important cellular processes such as the NF-κB signalling. This concept is supported by evidence suggesting that fluctuations in the oxidants:antioxidants ratio should affect first bovine embryo cleavage, and also affect embryo kinetics and development to the blastocyst stage (Lopes et al., Reference Lopes, Lane and Thompson2010).
Despite the negative effects of βME supplementation during IVC observed in our study, most studies in the literature have demonstrated improvements in embryo development with supplementation of different βME doses in the culture medium (Takahashi et al., Reference Takahashi, Nagai, Hamano, Kuwayama, Okamura and Okano1993; Caamaño et al., Reference Caamaño, Ryoo and Youngs1998; Geshi et al., Reference Geshi, Yonai, Sakaguchi and Nagai1999; Hosseini et al., Reference Hosseini, Forouzanfar, Hajian, Asgari, Abedi, Hosseini, Ostadhosseini, Moulavi, Safahani Langrroodi, Sadeghi, Bahramian, Eghbalsaied and Nasr-Esfahani2009). The inconsistency of our findings in comparison with those reported by others may be related to the fact that blastocyst rates in the control group were rather low in those studies (7.1%, 10.7%, 15.4%, and 17.1%, respectively), which suggests that βME supplementation during IVC may be beneficial under unfavourable culture conditions that result in redox unbalance, becoming less helpful when blastocyst rates exceed 30–40% (Rizos et al., Reference Rizos, Ward, Duffy, Boland and Lonergan2002), as seen in this study.
Vitrification can impair the developmental competence of embryos (Moussa et al., Reference Moussa, Yang, Zheng, Li, Yu, Yan, Huang and Shang2019; Leme et al., Reference Leme, Carvalho, Franco and Dode2020) and this effect can be evaluated by embryo developmental kinetics (Leme et al., Reference Leme, Carvalho, Franco and Dode2020). In the current study, despite the reduction in blastocyst development by Day 7, embryos supplemented with βME during IVC had improved cryotolerance to vitrification based on the increased rate of re-expansion after warming (βME-IVC = 85.7 vs. Control = 72.5). In addition, supplementation with βME in the PWC medium showed a significant increase in blastocyst hatching rates during the first 72 h of post-warming culture (βME-IVC = 60.7 vs. Control = 39.1%). Despite the independent results of βME supplementation in IVC and in PWC, the combination of βME supplementation in both IVC and PWC further enhanced cryotolerance of blastocysts to vitrification, resulting in an 87.5% re-expansion and 63.8% hatching. Furthermore, hatched blastocysts exposed to βME before or after vitrification had greater number of cells at 72 h post-warming compared with controls, which further supported the positive effects of βME in cryotolerance to vitrification. In contrast with our study, Hosseini et al. (Reference Hosseini, Forouzanfar, Hajian, Asgari, Abedi, Hosseini, Ostadhosseini, Moulavi, Safahani Langrroodi, Sadeghi, Bahramian, Eghbalsaied and Nasr-Esfahani2009) reported no differences in re-expansion rates when supplementing culture medium with 100 μM βME prior to vitrification (Days 1–7 of IVC). Nevertheless, Nedambale et al. (Reference Nedambale, Du, Yang and Tian2006) demonstrated that supplementation with 100 μM βME to the medium after blastocyst vitrification and warming significantly increased blastocyst re-expansion and survival for the first 6 h, hatching rates, and total cell numbers. Similar effects were obtained in hatched bovine blastocysts on Day 8 (Feugang et al., Reference Feugang, de Roover, Moens, Léonard, Dessy and Donnay2004) and on Day 9 (Van Soom et al., Reference Van Soom, Yuan, Peelman, de Matos, Dewulf, Laevens and de Kruif2002) when embryos were cultured with βME and cysteine, respectively. These findings suggest that the viability of vitrified blastocysts can be improved if warmed and briefly cultured in medium supplemented with antioxidants prior to embryo transfer.
From a biochemical point of view, βME is a low-molecular-weight thiol that interacts directly with a few oxidized radicals (Mori et al., Reference Mori, Otoi, Wongsrikeao, Agung and Nagai2006) and acts as a precursor to l-gamma-glutamyl-l-cysteinyl-glycine, also known as glutathione (GSH). Thiols protect cysteine from oxidation by reducing disulfide bridges in cysteine molecules, and increase its absorption by cells, thus promoting GSH synthesis (Takahashi et al., Reference Takahashi, Nagai, Hamano, Kuwayama, Okamura and Okano1993; Feugang et al., Reference Feugang, de Roover, Moens, Léonard, Dessy and Donnay2004; Rashidipour et al., Reference Rashidipour, Karami-Mohajeri, Mandegary, Mohammadinejad, Wong, Mohit, Salehi, Ashrafizadeh, Najafi and Abiri2020). Glutathione is considered the major representative of nonenzymatic antioxidants present in oocytes and embryos (Rodríguez-González et al., Reference Rodríguez-González, López-Bejar, Mertens and Paramio2003) and it is associated with the removal of ROS and detoxification of lipid peroxides (Johnson and Nasr-Esfahani, Reference Johnson and Nasr-Esfahani1994). These aspects make GSH interesting for IVP embryos because oocytes and embryos cultured in vitro accumulate more lipids than in vivo-derived embryos (Rizos et al., Reference Rizos, Ward, Duffy, Boland and Lonergan2002). Accumulation of ROS and intracellular lipids during IVP of embryos increases the peroxidation of cell membranes (de Matos et al., Reference de Matos, Furnus, Moses, Martinez and Matkovic1996), and the cellular damage caused by cryopreservation makes cells even more susceptible to the actions of ROS (Agarwal et al., Reference Agarwal, Sharma, Nallella, Thomas, Alvarez and Sikka2006). Although GSH levels in embryos were not evaluated in our study, other reports have demonstrated that βME supplementation during IVC increases intracellular GSH levels and blastocyst rates (Takahashi et al., Reference Takahashi, Nagai, Hamano, Kuwayama, Okamura and Okano1993) and reduces lipid peroxidation (de Matos et al., Reference de Matos, Furnus, Moses, Martinez and Matkovic1996). In another study, βME-induced GSH provided an available reserve until the first cleavage, improving the IVP efficiency of embryos from immature oocytes (de Matos and Furnus, Reference de Matos and Furnus2000). Despite the known role that GSH plays in the maintenance of the intracellular redox state (Feugang et al., Reference Feugang, de Roover, Moens, Léonard, Dessy and Donnay2004), the mechanism of action by which βME exerts its effect on embryos is still not completely elucidated.
In addition to βME, several other substances of distinct natures and mechanisms of actions have been proposed as antioxidant supplements to medium for the culture and cryopreservation of somatic cells and in vitro embryo production in humans and animals, both under experimental and clinical protocols (Budani and Tiboni, Reference Budani and Tiboni2020). To note, some examples include the use of resveratrol, melatonin, coenzyme Q, Vitamins (A, B complex, C, D and E), acetyl-l-carnitine, N-acetyl-cysteine, α-lipoic acid and, similar to βME, thiol compounds that stimulate GSH synthesis, such as dithiothreitol (DTT), and cysteamine. Similar to βME, and as a small molecule redox reagent used to reduce disulfide bonds and maintain monothiols in a reduced state (Cleland, Reference Cleland1964), DTT has proved to be beneficial for mouse and human oocytes and embryos when in culture, perhaps with less potentially toxicity than βME (Cleland, Reference Cleland1964; Tarín et al., Reference Tarín, Ten, Vendrell and Cano1998; Liu et al., Reference Liu, Trimarchi and Keefe1999). We are unaware of studies focusing on the toxic levels of βME on mammalian embryos. But it is important to mention that an excess of thiol compounds may impair the redox equilibrium during oocyte maturation and subsequent embryo development (Guérin et al., Reference Guérin, El Mouatassim and Ménézo2001). Liu et al. (Reference Liu, Trimarchi and Keefe1999) showed that altering the thiol redox status in mouse embryos induced cell cycle arrest and cell death. In this way, Tsuzuki et al. (Reference Tsuzuki, Saigoh and Ashizawa2005), exploring the effects of βME during IVM and IVC on bovine IVP, showed that 100 µM βME increased both the numbers of cumulus cells attached to oocytes, as well as the total cell numbers in blastocysts by modulating ATP metabolism in oocytes. In contrast, 500 µM βME decreased embryo development, which suggests that concentrations of more than 100 µM βME could be detrimental to bovine embryos. More studies are needed to clarify the possible βME toxic effects on mammalian embryo development in vitro.
It is generally believed that βME supplementation in culture medium improves the developmental competence of IVP embryos, maintaining the levels of intracellular glutathione and improving in vivo development following transfer to female recipients (Truong et al., Reference Truong, Soh and Gardner2016). Similarly, βME addition to in vitro culture medium prior to (Takahashi et al., Reference Takahashi, Kuwayama, Hamano, Takahashi, Okano, Kadokawa, Kariya and Nagai1996; Caamaño et al., Reference Caamaño, Ryoo and Youngs1998) and after vitrification (Nedambale et al., Reference Nedambale, Du, Yang and Tian2006) has been shown to improve vitrified blastocyst survival rate, incubation capacity, total cells numbers, and the protection against apoptosis. In fact, findings by Moussa et al. (Reference Moussa, Yang, Zheng, Li, Yu, Yan, Huang and Shang2019) demonstrated a higher level of expression of E-cadherin, β-catenin, and Oct4 in vitrified blastocysts cultured in the presence of βME, suggesting that βME protects vitrified blastocyst against apoptosis. In turn, vitrified embryos in the absence of βME showed a reduction in the expression of E-cadherin and β-catenin, also causing significant changes in the expression of Oct4, Cdx2, and Gata3, which are essential factors for the development of blastocysts and cell differentiation into inner cell mass (ICM) and trophectoderm (TE) cells in bovine embryos (Goissis and Cibelli, Reference Goissis and Cibelli2014; Sakurai et al., Reference Sakurai, Takahashi, Emura, Fujii, Hirayama, Kageyama, Hashizume and Sawai2016). E-cadherin is a transmembrane surface molecule that involves Ca2+-dependent cells for cellular adhesion to other cells (Takeichi, Reference Takeichi1988), which is related to compaction (Fleming et al., Reference Fleming, Warren, Chisholm and Johnson1984). E-cadherin transcripts and protein were found in both the ICM and TE of expanded bovine blastocysts (Barcroft et al., Reference Barcroft, Hay-Schmidt, Caveney, Gilfoyle, Overstrom, Hyttel and Watson1998). The suppression of E-cadherin mRNA and protein resulted in lower blastocyst rates (Nganvongpanit et al., Reference Nganvongpanit, Müller, Rings, Gilles, Jennen, Hölker, Tholen, Schellander and Tesfaye2006). β-Catenin is related to cell-to-cell adhesion, acting as an intracellular signalling molecule from the cytoplasm to the nucleus (Willert and Nusse, Reference Willert and Nusse1998). Oct4 plays a role as an anti-apoptotic factor (Guo et al., Reference Guo, Mantel, Hromas and Broxmeyer2008). Therefore, decreased expression of Oct4 may disturb the balance between pro-apoptotic and anti-apoptotic factors, which in turn may lead to a higher frequency of apoptotic cells (Nedambale et al., Reference Nedambale, Du, Yang and Tian2006). Thus, βME seems to play a critical role in increasing the resistance of vitrified embryos to oxidative stress, reducing apoptosis.
In our study, the observed detrimental effects of adding βME in the 6-d IVC and the beneficial effects of adding βME on the 3-d PWC were likely to have been mediated by the activity of βME or its metabolites during the first day of culture and exposure to treatment. Although βME is highly stable in aqueous solution (Wong et al., Reference Wong, Kirkland, Schwanz, Simmons, Hamilton, Corkey and Guo2014), Stevens et al. (Reference Stevens, Stevens and Price1983) has shown that βME has a short half-life time, depending on the pH and temperature of the medium (∼10 h at pH 7.5 and at 20°C). Therefore, considering that we did not replenish the culture medium with additional βME during both culture periods, IVC and PWC, it is likely that βME bioavailability was not constant during the entire duration of the incubation periods and the effects were mediated towards the beginning of the incubation periods, when bioavailability of βME was greater. In the IVC period, specifically, embryos were cultured in 5% CO2 and ∼20% O2 in the first 24 h, and then placed in an atmosphere of 90% N2, 5% O2 and 5% CO2 after evaluation of cleavage rates on Day 2 until Day 7 and after warming of vitrified blastocysts. Thus, the βME treatment was likely to be more active in the first 24 h of IVC, when oxygen tension was higher and when the formation of free radicals was more likely to occur (de Castro e Paula and Hansen, Reference de Castro e Paula and Hansen2008). In the PWC culture, the βME treatment was likely to be more active in the first few hours after warming of vitrified blastocysts, when the oxidative stress associated with cryo-injuries is more substantial (Deleuze and Goudet, Reference Deleuze and Goudet2010; Truong and Gardner, Reference Truong and Gardner2020). Therefore, although we did not replenish βME in the culture medium to ensure that its bioavailability was more constant throughout the culture periods, we believe that the most critical points of oxidative stress in our IVP/freezing model were covered with antioxidant supplementation. Interestingly, despite the short half-life, the manifestation of biological effects of βME seemed to persist for several days. In fact, we did not observe an effect of βME in the cleavage rate on Day 2, but observed a large difference in blastocyst development on Day 7 and important changes in blastocyst cryotolerance up to Day 10. In the PWC, the βME treatment affected expansion rate in the first 24 h, but the effects on blastocyst hatching seemed to be maintained until 72 h post-warming.
In summary, our study indicated that supplementing culture medium with 100 µM βME prior to and/or after vitrification improved survival and developmental rates of bovine embryos following vitrification. An increase in cryotolerance rates was observed, with higher survival and hatching rates, better quality blastocysts, especially when βME was supplemented after warming. Even though βME supplementation during IVC reduced blastocyst yield and delayed embryo kinetics, the resulting blastocysts had greater re-expansion rates following vitrification and warming. βME supplementation during in vitro PWC, in combination or not with its use during in vitro culture prior to vitrification, increased embryo survival rates after cryopreservation, and increasing hatching rates and total cell numbers in hatched blastocysts. The positive effect of βME on cryotolerance can be maximized if added throughout the culture period. Future investigations must be performed to elucidate the effect of βME at the molecular, metabolic, and physicochemical levels on bovine IVP embryos in order to understand the mechanisms related to the acquisition of cryoresistance in embryos, and to determine the optimal dose–response effects of βME supplementation during IVC to optimize embryo development in different IVP conditions.
Acknowledgements
The authors thank Frigorífico Verdi Ltda., from Pouso Redondo, SC, Brazil, for supplying bovine ovaries.
Financial support
This study was partially supported by research grant no. 0192058 from CAPES/Brazil. K.M. was supported by a scholarship from CAPES/Brazil.
Conflict of interest
The authors declare none.
Ethics statements
Not applicable.
