Introduction
Currently, cryopreservation of oocytes has been applied in many species with different goals. Animal models are used due to the limited availability of human oocytes for research purposes to assisted reproduction techniques (ARTs) (Lee et al., Reference Lee, Barritt, Moschini, Slifkin and Copperman2013), in this matter many aspects of reproductive physiology in bovines are similar to those in humans, including follicle growth, the sizes of the ovaries and oocytes, embryonic genome activation and interaction with the culture medium (Shaw et al., Reference Shaw, Oranratnachai and Trounson2000). However gametes cryopreservation also has great importance in animal breeding programmes, storage of female gametes for extended periods and animal reproduction in general (Hochi et al., Reference Hochi, Kozawa, Fujimoto, Hondo, Yamada and Oguri1996; Hochi et al., Reference Hochi, Ito, Hirabayashi, Ueda, Kimura and Hanada1998; Mara et al., Reference Mara, Carta and Dattena2013).
In several mammalian species the ability of cryopreserved oocytes to achieve later embryonic development and succeed to birth is low (Massip, Reference Massip2003; Sprícigo et al., Reference Sprícigo, Morais, Ferreira, Machado, Gomes, Rumpf, Franco and Dode2014) regardless of oocyte stage (Versieren et al., Reference Versieren, Heindryckx, O'Leary, De Croo, Van den Abbeel and Gerris2011). This situation probably arises due to intracellular ice crystal formation and chilling injury caused by the freezing procedure (slow freezing technique), which leads to the destruction of intracellular communication between cumulus cells and oocytes (gap junctions) (Hochi et al., Reference Hochi, Ito, Hirabayashi, Ueda, Kimura and Hanada1998) and consequently interferes in oocyte maturation by impairing nutrient supply during IVF (Versieren et al., Reference Versieren, Heindryckx, O'Leary, De Croo, Van den Abbeel and Gerris2011; Buccione et al., Reference Buccione, Schroeder and Eppig1990; Chian et al., Reference Chian, Niwa and Sirard1994; Ka et al., Reference Ka, Sawai, Wang, Im and Niwa1997). So, to avoid this injury the vitrification technique has been tested and has acquired satisfactory results in human oocytes (Cobo et al., Reference Cobo, Romero, Pérez, de los Santos, Meseguer and Remohí2010; Rienzi et al., Reference Rienzi, Romano, Albricci, Maggiulli, Capalbo, Baroni, Colamaria, Sapienza and Ubaldi2010; Herrero et al., Reference Herrero, Martinez and Garcia-Velasco2011; Mukaida & Oka, Reference Mukaida and Oka2012), which involves cell exposure to high concentrations of cryoprotectants and ultra-rapid cooling (Rall & Fahy, Reference Rall and Fahy1985).
On this basis, the retrieval of immature oocytes in the germinal vesicle (GV) stage, their cryopreservation or in vitro maturation (IVM) followed by in vitro fertilization (IVF) (Cha & Chian, Reference Cha and Chian1998; Tucker et al., Reference Tucker, Wright, Morton and Massey1998; Lin & Hwang, Reference Lin and Hwang2006; González et al., Reference González, Devesa, Boada, Coroleu, Veiga and Barri2011; Lee et al., Reference Lee, Barritt, Moschini, Slifkin and Copperman2013) has been investigated as an option to preserve the genetic diversity in several animal species. In humans it has emerged as an option in cases in which fertility preservation is demanded. The aim of the present study was to evaluate the influence of vitrification protocol on nuclear maturation capacity of immature oocytes, as well as to assess the effect of vitrifying and thawing oocytes at different meiotic nuclear stages in relation to its survival and development capacity after IVF and parthenogenetic activation (PA).
Materials and methods
All the chemicals used in this study were purchased from Sigma-Aldrich (Oakville, ON, Canada), unless otherwise indicated. This study was performed in three experiments: Experiment 1, GV oocytes were vitrified or not and underwent IVM and the nuclear maturation was assessed by orcein staining. Experiment 2, GV oocytes and MII oocytes were vitrified or not and their viability were assessed by calcein/ethidium homodimer-1 staining. Experiment 3, MII oocytes matured before or after vitrification were submitted to IVF and PA in order to evaluate embryo development.
Bovine ovaries collection
Bovine ovaries were collected from random stages of the oestrous cycle in a slaughterhouse and transported to the laboratory in saline solution (0.9% NaCl) containing gentamycin (50 µg/ml) at 35–37°C.
Selection of cumulus–oocyte complexes (COCs)
COCs were aspirated with a syringe and needle from 2–8 mm antral follicles washed in holding medium (HM) consisting of HEPES-buffered TCM-199 and only COCs with compact, with no more than three layers of cumulus cells and an oocyte with homogeneous cytoplasm (immature oocytes), were used in the experiments. For each replicate of the experiments these COCs were randomly assigned to the three study groups: control group (immature oocytes submitted to IVM), GV group (immature frozen oocytes followed by post thawed IVM), and MII group (fresh IVM followed by oocyte vitrification).
In vitro maturation (IVM)
The basic maturation medium was TCM-199 supplemented with 0.2 M sodium pyruvate, 0.5 µg/ml follicle-stimulating hormone (FSH), 5 µg/ml luteinizing hormone (LH), 1 µg/ml estradiol, 100 IU/ml penicillin, and 0.1 mg/ml streptomycin. Maturation was accomplished in 35 mm Petri dishes covered with 3 ml of M8410 mineral oil at 38.5° C, in an atmosphere with 5% CO2 in air for a period of 22 h. COCs were cultured in a volume of 70 μl culture medium.
Oocyte vitrification and warming
All of the vitrification solution was used at room temperature (25–27°C), only the thawing solution (TS) step was used at 37°C. HM supplemented with 20% synthetic serum substitute (SSS) was used for handling oocytes during vitrification and warming. For vitrification, the oocytes were washed (three times) in an equilibrium solution containing 7.5% (v/v) ethylene glycol (EG) and 7.5% (v/v) dimethylsulfoxide (DMSO) in HM for a total of 9 min. Then, the oocytes were transferred to vitrification solution containing 15% EG, 15% DMSO, and 0.5 M sucrose for 1 min. Under a stereomicroscope (Nikon-SMZ 650), the oocytes were loaded onto the film strip of a Cryotop device (three oocytes/device) and were immediately submerged into liquid nitrogen for storage. Warming was performed by immersing directly the Cryotop device in 1 ml of TS containing 1 M sucrose (1 min). After that, the oocytes were transferred to HM with 0.5 M sucrose (3 min) and washed twice in original HM for 5 min. The thawed oocytes were then incubated in TCM-199 medium at 38.5°C, in 5% CO2 and a 95% humidified air atmosphere for 2 h. Those oocytes vitrified at GV stage were then submitted to IVM for 22 h.
Assessment of meiotic stage of bovine oocytes by orcein staining
After 22 h of IVM, before or after vitrification/warming, the COCs were denuded in a 0.03% hyaluronidase solution by repeated pipetting. The nuclear status of the oocytes was assessed microscopically (×1000 magnification), after fixation (methanol:acetic acid 3:1) and orcein staining (2%). Oocytes were classified as follows: (i) immature: oocytes with a germinal vesicle visible (GV stage); or (ii) mature: metaphase II oocytes with two groups of unequally spread chromosomes, the polar body set clustered together and the haploid set of the oocyte spread out (MII stage) (Süss et al., Reference Süss, Wuthrich and Stranzinger1988) (Fig. 1 a–c).
(a–c) Representative images of nuclear maturation (metaphase II stage) obtained from immature oocytes vitrified and submitted to in vitro maturation (IVM), in which black arrows indicate two groups of unequally spread chromosomes, the polar body set clustered together and the haploid set of the oocyte spread out – MII stage (a ×10 magnification; b, ×40 magnification; and c, ×100 magnification). Images of oocytes under viability assessment by dead/live technique staining (×40 magnification) where calcein/green fluorescence indicates viable oocytes and dead oocytes/red fluorescence. (d, e) Oocytes from the GV control group; (f, g) oocytes from GV group; (h, i) oocytes from MII control group; and (j, k) oocytes from the MII group. Representative images of embryo development to blastocyst stage in day 8 by in vitro fertilization (IVF) of oocytes from (l) control group; (m) GV group; and (n) MII group; and by parthenogenetic activation (PA) of oocytes from (o) the control group, (p) the GV group and (q) the MII group. Scale bar = 50 µm.
Viability assessment
Denuded oocytes were assessed by calcein AM/ethidium homodimer-1 (CaAM/EthD-1) staining, based on the simultaneous determination of live cells using calcein (AM) for the detection of intracellular esterase activity and ethidium homodimer-1 (EthD-1) to label the nucleus acids of non-viable cells with plasma membrane disruption (Cortvrindt & Smitz., Reference Cortvrindt and Smitz2001; Martinez-Madrid et al., Reference Martinez-Madrid, Dolmans, Langendonckt, Defrère, Van Eyck and Donnez2004; Sanfilippo et al., Reference Sanfilippo, Canis, Ouchchane, Botchorishvili, Artonne, Janny and Brugnon2011; Merdassi et al., Reference Merdassi, Mazoyer, Guerin, Saad, Salle and Lornage2011). Fresh and thawed oocytes were submerged in DPBS containing 2 μM AM and 5 μM EthD-1 (Molecular Probes, Invitrogen-Life Technologies, Germany) for 15 min, washed in DPBS and mounted on glass slides for examination under a fluorescence microscope (AxioScope A1, Zeiss, Germany). The labelled oocytes were classified for fluorescent staining as follows: (i) as live oocytes (viable) when the cytoplasm was stained positively with AM (green fluorescence); or (ii) as dead oocytes (unviable) when chromatin was labelled with EthD-1 (red fluorescence) (Fig. 1 d–k).
In vitro fertilization and parthenogenetic activation
COCs submitted to IVM, before or after vitrification/warming, were selected for either IVF or PA. For IVF the COCs were washed in HM supplemented with 50 µg/ml gentamycin, 0.2 M sodium pyruvate and 5% FCS. Semen for all replicates was collected from a single male and stored at liquid nitrogen (LN2). Sperm were obtained by a swim-up method (Parrish et al., Reference Parrish, Susko-Parrish, Winer and First1988), sperm were washed with Sperm-TALP HEPES medium for 1 h at 38.5°C. Following this step, the supernatant sperm were then centrifuged at 500 g for 10 min and motile sperm were selected. The fertilization medium was FERT-TALP (Gordon, Reference Gordon1994), supplemented with 20 µg/ml heparin and 6 mg/ml bovine albumin essential fatty-acid free (BSA; Sigma A-7030). Sperm concentration was approximately 2 × 106 spermatozoa/ml and the sperm were co-incubated with 20 COCs for 18 h in a humid atmosphere containing 5% CO2 at 38.5°C. For PA, COCs were denuded by incubation with 0.03% hyaluronidase solution for 5 min and by repeated pipetting. Then, the oocytes at metaphase II with the presence of a first polar body were activated using 10 μM calcium ionophore for 5 min followed by 2 mM 6-DMAP for 3 h at 38.5°C, in the presence of 5% CO2 in air and high humidity. Activated oocytes were subsequently cultured in embryo medium (CR2aa medium) (Rosenkrans & First, Reference Rosenkrans and First1994) under mineral oil at 38.5°C.
Assessment of embryonic development in vitro
After IVF or PA, the presumptive zygotes were mechanically denuded (partially – up to two or three layers of cumulus cells) and cultured in 50 µl drops of embryo medium (CR2aa medium) (Rosenkrans & First, Reference Rosenkrans and First1994) supplemented with 5% FCS and 1 mg/ml BSA under mineral oil, in a moist atmosphere containing 5% CO2 at 38.5°C. The embryo medium was replaced by fresh medium every 48 h. Embryo cleavage rate was recorded on day 3 after insemination (day 0 = day of insemination) and was calculated as the number of cleaved embryos (2- to 8-cell stage) divided by the number of COCs placed in culture. Further, embryo development was assessed on day 8, when blastocyst development rate was calculated by the number of blastocyst stage embryos divided by the number of COCs placed in culture. In addition, the proportion of oocytes that developed to the hatched blastocyst stage was recorded 240 h after insemination or activation. For the in vitro embryo production experiment, five replications of IVF and seven of PA were performed in different experiments for IVF or PA.
Statistical analysis
Data regarding oocyte nuclear maturation, oocyte viability and embryonic development (cleavage and blastocyst production) were analyzed by the chi-squared (Q2) test. The hatching rate was evaluated by Fisher's exact test. The results are shown as percentages, and the level of significance was set at P < 0.05.
Results
Assessment of meiotic stage of bovine oocytes
In total, 114 oocytes from the control group and 152 oocytes from GV group were destined to meiotic stage analysis. For this assessment orcein staining was applied. As the oocyte position during fixation is crucial for meiotic stage identification – the meiotic spindle shall be visible and the chromosomes aligned in the metaphasic plate – an expressive number of oocytes were considered not analysable [GV control group = 30 (26%) and GV group = 70 (46%)]. Among the analysable oocytes, 90% (n = 76) of the immature oocytes cryopreserved before IVM (GV group) presented meiotic nuclear maturation after IVM and 92.7% (n = 76) of the immature oocytes not submitted to cryopreservation (GV control group) were considered matured (MII) (P = 0.78; Experiment 1).
Viability assessment
Immature oocytes vitrified before IVM showed similar viability rate between the GV group 56% (19/34) and the GV control group 77% (37/48) (P = 0.055). However, the MII group showed lower viability 27% (11/41) in relation to the MII control group 84% (32/38) (P < 0.0001). Thus, higher viability rate was observed in immature oocytes (GV group) in comparison to in vitro-matured oocytes (MII group) (P = 0.017) (Table 1; Experiment 2).
Oocyte viability rate of immature and mature oocytes cryopreserved and matured in vitro
Treatment groups: GV control group (immature oocytes subsequent to IVM without cryopreservation); GV group (immature oocytes vitrified and then submitted to IVM); MII control group (oocytes matured in vitro and then vitrified); MII group (immature oocytes subsequent to IVM without cryopreservation). Values with the same superscripts letters are significantly different (P < 0.05).
Assessment of embryonic development in vitro by IVF
When analyzing the capacity of oocytes for embryonic development in vitro by IVF, no difference was found in cleavage rate between oocytes from GV group 28% (11/39) and MII group 26% (12/46) (P = 1.0). However, cleavage rates in both groups were significantly lower than those observed in immature oocytes submitted to IVM without vitrification (MII control group) 80% (169/211) (P < 0.001). The embryo development in MII control group (fresh) in relation to blastocyst formation was 26% (54/211) and hatching 41% (22/54) while in both vitrified groups (GV group and MII group) no blastocyst formation or hatching was observed (Table 2).
Embryo development after in vitro fertilization of immature and mature cryopreserved oocytes
hpi, hours post insemination. a,b,c,d Values with the same superscripts letters are significantly different (P < 0.05).
Assessment of embryonic development in vitro by PA
To examine the influence of the fertilization capacity of the spermatozoa, PA was performed. The cleavage rates were significantly lower in both vitrified groups, GV group 28% (14/50) and MII group 22% (14/63) (P = 0.59). However, cleavage rates were statistical different in the MII control group 58% (118/205) in relation to the GV group (P < 0.002) and MII group (P < 0.001). Development to the blastocyst stage was significantly lower in the GV group 2% (1/50) while in the MII control group the cleavage rate was 24% (50/205) (P < 0.0001). However, in MII group there was no blastocyst formation (P < 0.001). No significant difference was founded between the vitrified groups (GV group and MII group) in relation to blastocyst formation (P = 0.65; Experiment 3) (Table 3). In this way, the hatching rates in GV group were 100%, which represents one hatched blastocyst in 50 cultured embryos (Table 3) while in the MII control group the hatching rate was 18% (9/50) (P = 0.04). In MII group there was no hatching after PA (P = 0.003) (Table 3 and Fig. 1 l–q).
Embryo development after parthenogenetic activation of immature and mature cryopreserved oocytes
hpi, hours post insemination. a– f Values with the same superscripts letters are significantly different (P < 0.05).
Discussion
Different from human oocytes that showed high survival rates and embryo development after vitrification in comparison to slow freezing (Katayama et al., Reference Katayama, Stehlik, Kuwayama, Kato and Stehlik2003; Kuwayama et al., Reference Kuwayama, Vajta, Kato and Leibo2005; Kuwayama, Reference Kuwayama2007), bovine oocytes have repeatedly been tested for vitrification with poor results. In this short study, we demonstrated that bovine oocyte vitrification by the Cryotop method significantly reduced oocyte viability and developmental competence despite the nuclear maturation stage at the time of cryopreservation. However, immature oocytes (GV stage) were shown to be more resistant to the cryopreservation process than mature oocytes due to the ability to mature and its viability by Dead-Live technique was not compromised. The immature oocytes, after vitrification and IVM, were able to progress from the GV stage to the MII stage. Similar data were obtained by Sripunya et al. (Reference Sripunya, Liang, Panyawai, Srirattana, Ngernsoungnern, Ngernsoungnern, Ketudat-Cairns and Parnpai2014), in a study in which surviving bovine oocytes vitrified by Cryotop method were lately subjected to IVM for 24 h and showed a 58% rate of MII, while oocytes in vitro-matured without vitrification showed an 81% MII rate (Sripunya et al., Reference Sripunya, Liang, Panyawai, Srirattana, Ngernsoungnern, Ngernsoungnern, Ketudat-Cairns and Parnpai2014). It should be noted that several are the mechanisms involved in the correct progression of meiosis; among them the activities of the cytoplasmic proteins of the maturation promoting factor (MPF) complex and of the proteins of the mitogen-activated protein kinase (MAPK) family, which have been demonstrated to be preserved after IVM and the cryopreservation process (Kano et al., Reference Kano, Takenaka, Yamamoto, Nagayama, Nishida and Murata2000; Lefebvre et al., Reference Lefebvre, Terret, Djiane, Rassinier, Maro and Verlhac2002).
The integrity of the plasma membrane is an important cryobiological property that affects oocyte viability after vitrification because it influences membrane permeability to water and to cryoprotective agents (CPAs), which if unbalanced can lead to excessive osmotic edema and induce rupture and lysis of the membrane and consequent oocyte death (Kuwayama, Reference Kuwayama2007). The vitrification of immature oocytes, followed by IVM, did not damage the cellular esterase metabolism of the oocytes (which were able to metabolize calcein) or the integrity of the plasma membrane (as inferred by the non-penetration of ethidium bromide), once their viability was similar to the control group oocytes. In contrast, oocyte viability was impaired in mature oocytes, reducing from 77% in the control group to 27% in the vitrified mature group; this change may indicate that oocyte vitrification in the MII stage might have impaired, at least in part, the oocyte metabolism and promoted disruption of the plasma membrane. Opposite results were demonstrated by Chian et al. (Reference Chian, Kuwayama, Tan, Tan, Kato and Nagai2004). Based on the integrity of the oocyte membrane and the zona pellucida, they showed high survival rates (91.8%) of bovine oocytes submitted to IVM and subsequently to vitrification and thawing (Chian et al., Reference Chian, Kuwayama, Tan, Tan, Kato and Nagai2004). In the same way, Jin et al. (Reference Jin, Kawai, Hara, Takeda, Seki, Nakata, Matsukawa, Koshimoto, Kasai and Edashge2011) showed that the plasma membranes of bovine oocytes were highly permeable, facilitating CPA movement during freezing and thawing and preventing in part the damage caused by the formation of intracellular ice, by CPA toxicity and by excessive osmotic edema (Jin et al., Reference Jin, Kawai, Hara, Takeda, Seki, Nakata, Matsukawa, Koshimoto, Kasai and Edashge2011).
Several studies have been conducted in order to better vitrify bovine oocytes at different meiotic stages, however the optimal meiotic stage is still not clear. Regardless of the protocol, the devices and cryoprotectants agents adopted the vitrification process impairs the capacity of embryo formation (rates lower than 5%) from bovine oocytes (Otoi et al., Reference Otoi, Yamamoto, Koyama, Tachikawa, Murakami, Kikkawa and Suzuki1997; Shaw et al., Reference Shaw, Oranratnachai and Trounson2000; Men et al., Reference Men, Monson and Rutledge2002; Chian et al., Reference Chian, Kuwayama, Tan, Tan, Kato and Nagai2004; Zhou et al., Reference Zhou, Al Naib, Sun and Lonergan2010; Prentice et al., Reference Prentice, Singh, Dochi and Anzar2011; Sripunya et al., Reference Sripunya, Liang, Panyawai, Srirattana, Ngernsoungnern, Ngernsoungnern, Ketudat-Cairns and Parnpai2014; Sprícigo et al., Reference Sprícigo, Morais, Ferreira, Machado, Gomes, Rumpf, Franco and Dode2014)). Prentice et al. (Reference Prentice, Singh, Dochi and Anzar2011) obtained lower cleavage rates (93% versus 42%) and blastocyst formation rates (31% versus 0.4%) in oocytes vitrified by the Cryotop method when compared with non-vitrified bovine oocytes (Prentice et al., Reference Prentice, Singh, Dochi and Anzar2011), similar results were also described by Sripunya et al. (Reference Sripunya, Liang, Panyawai, Srirattana, Ngernsoungnern, Ngernsoungnern, Ketudat-Cairns and Parnpai2014) and by Arcarons et al. (Reference Arcarons, Morató, Spricigo, Ferraz and Mogas2015).
These poor results in reproductive outcomes, especially in the cleavage rates, may be, at least partially, a result of the zona pellucida hardening that occurs after oocytes cryopreservation (Carroll et al., Reference Carroll, Depypere and Matthews1990; Gook et al., Reference Gook, Osborn, Bourne and Johnston1994) With this concern, in the present study, PA was performed in an attempt to overcome the influence of the fertilization capacity of the spermatozoa. However, despite PA of the vitrified GV and MII oocytes, there was no improvement in cleavage rates or blastocyst formation. Thus, these data suggest that, in addition to ZP impairment, there must have been further damage that bypassed this structural event and severely compromised the reproductive capacity of the oocytes. As an example, if inadequate cytoplasm maturation occurs, processes such as protein synthesis (Sirard et al., Reference Sirard, Richard and Mayes1998), molecular modifications (Kubelka et al., Reference Kubelka, Motlík, Schultz and Pavlok2000), migration and reorganization of organelles (Stojkovic et al., Reference Stojkovic, Machado, Stojkovic, Zakhartchenko, Hutzler, Gonçalves and Wolf2001) will be impaired. According to Meirelles et al. (Reference Meirelles, Caetano, Watanabe, Ripamonte, Carambula, Merighe and Garcia2004) transcript and protein stock in the oocyte cytoplasm is extremely important for ensuring ideal cytoplasm maturation and initial embryonic development (Meirelles et al., Reference Meirelles, Caetano, Watanabe, Ripamonte, Carambula, Merighe and Garcia2004). Thus, the lower cleavage rates in the groups submitted to cryopreservation compared with controls indicated that the first stages of embryonic development were partially impaired by vitrification, although the stage of nuclear maturation among cryopreserved oocytes did not influence this impairment. It should be noted that no specific evaluation was performed in the present study regarding this topic. After these first stages of embryonic development (eight cells in cattle), maternal–zygotic transition (MZT) occurs, with activation of the embryonic genome and synthesis of new proteins (Meirelles et al., Reference Meirelles, Caetano, Watanabe, Ripamonte, Carambula, Merighe and Garcia2004). In this phase we detected a marked interference of cryopreservation in MZT, and we did not obtain blastocysts from cryopreserved oocytes submitted to IVF, regardless of the meiotic stage.
Other studies have corroborated our study and showed that although vitrification might not compromise the capacity for nuclear maturation of oocytes in GV stage, the cryopreservation process itself impairs embryonic development (Aman & Parks., Reference Aman and Parks1994; McWilliams et al., Reference Mcwilliams, Gibbons and Leibo1995; Rojas et al., Reference Rojas, Palomo, Albarracín and Mogas2004). Furthermore, in theory, oocytes in the MII stage would be more susceptible to meiotic anomalies and consequently to poor embryonic development because of the distribution of the chromosomes throughout the metaphase plate and the presence of a meiotic spindle, which are targets of the deleterious effects of the vitrification process (Brunet et al., Reference Brunet, Pahlavan, Taylor and Maro2003). In accordance, studies with vitrified mature bovine oocytes has observed that vitrification modifies the cytoskeletal components as alterations in the meiotic spindle and consequently an increase in chromosomal abnormality occurs that compromises the developmental capacity of in vitro-matured bovine oocytes (Bogliolo et al., Reference Bogliolo, Ariu, Fois, Rosati, Zedda, Leoni, Succu, Pau and Ledda2007; Morató et al., Reference Morató, Izquierdo, Paramio and Mogas2008). Besides this theoretic impact of cryopreservation on oocytes genetics, there have already been many newborn babies born from human vitrified oocyte with no reports of increased fetal malformation or neonatal complications, and this technique may be applied for human mature oocytes cryopreservation, no longer an experimental practice (Practice Committees of American Society for Reproductive Medicine, 2013). However, few data are available concerning immature oocytes, which may be considered for specific clinical situations in fertility preservation in the routine of assisted reproduction clinics.
In conclusion, we cannot define the best or more resistant meiotic stage for cryopreservation, as this procedure has a great impact on the capacity for embryonic development of vitrified bovine oocytes despite the nuclear meiotic stage. Thus, better protocols specifically designed for bovine oocytes must be developed, and only then the influence of the meiotic stage on cryotolerance may be tested.
Financial support
This study was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo–FAPESP, Brasil (2009/04102–7).
