High survival rate of bovine oocytes matured in vitro following vitrification

Improving pregnancy rates associated with the use of cryopreserved human oocytes would be an important advance in human assisted reproductive technology (ART). Vitrification allows glasslike solidification of a solution without ice crystal formation in the living cells. We have attempted to improve the survival rates of oocytes by a vitrification technique using bovine models. In vitro matured oocytes with or without cumulus cells were vitrified with either 15.0% (v/v) ethylene glycol (EG) + 15% (v/v) dimethylsulfoxide (DMSO) + 0.5 M sucrose or 15% (v/v) EG + 15% (v/v) 1,2-propanediol (PROH) + 0.5 M sucrose, using 'Cryotop' or 'thin plastic sticker', respectively. The oocyte survival rates after vitrifying-warming, and the capacity for fertilization and embryonic development were examined in vitro. The rate of embryonic development to blastocyst was significantly higher (P<0.05) in the oocytes vitrified with 15% (v/v) EG + 15% (v/v) PROH + 0.5 M sucrose than in the oocytes vitrified with 15% (v/v) EG + 15% (v/v) DMSO + 0.5 M sucrose (7.4% +/- 4.1 vs. 1.7% +/- 3.0, respectively). Oocytes vitrified without cumulus cells had a higher survival rate after thawing and a superior embryonic developmental capacity compared with oocytes vitrified with cumulus cells. Prolonged pre-incubation time after thawing adversely affected the rates of embryonic cleavage and development. These results indicate that in vitro matured bovine oocytes can be vitrified successfully with the mixture of the cryoprotectants, EG + PROH, the absence of cumulus cells for vitrification does not affect oocyte survival rate after warming, and vitrified and warmed oocytes do not require pre-incubation before in vitro fertilization.     

Bovine oocytes grown in serum-free medium acquire fertilization competence

We previously found that bovine oocytes 90-99 microm in diameter in early antral follicles grew to nearly their final size in serum-free medium, with some of the oocytes acquiring the nuclear competence to reach the second metaphase. In the present study, we examined the competence of the fertilization and pre-implantational development of the oocytes grown in serum-free medium. Bovine early antral follicles, 0.4-0.7 mm in diameter, were collected mechanically using fine forceps, embedded in collagen gels, and cultured in serum-free medium for 16 days. Grown oocytes which were enclosed by granulosa cells and did not show disintegrated ooplasm were recovered as normal oocytes, were transferred to the maturation medium, and then inseminated with spermatozoa. Ten to 12 h after insemination, 28% (41/145) of the oocytes were penetrated by spermatozoa. Of the penetrated oocytes, 18 (12%) formed a female and a male pronuclei, and 10 (7%) had a female pronucleus and an enlarged sperm head. Among the abnormally penetrated oocytes (13/41), 10 were penetrated by multiple spermatozoa and 3 were penetrated by a spermatozoon at the first metaphase stage. Of the 106 inseminated oocytes grown under serum-free conditions, 8 oocytes had cleaved and developed to the 2-cell stage 48 h after insemination, and 3-4-cell embryos and 5-8-cell embryos were observed after 72-96 h. However, no embryo developed to the blastocyst stage within 8 days. These results indicate that bovine oocytes grown in serum-free medium can be fertilized, but acquire insufficient embryonic development competence under the employed culture conditions.     

An efficient method for the sanitary vitrification of bovine oocytes in straws

Background

At present, vitrification has been widely applied to humans, mice and farm animals. To improve the efficiency of vitrification in straw, bovine oocytes were used to test a new two-step vitrification method in this study.

Results

When in vitro matured oocytes were exposed to 20% ethylene glycol (EG20) for 5 min and 40% ethylene glycol (EG40) for 30 s, followed by treatment with 30% glycerol (Gly30), Gly40 or Gly50, a volume expansion was observed in Gly30 and Gly40 but not Gly50. This indicates that the intracellular osmotic pressure after a 30 s differs between EG40 and ranged between Gly40 (approximately 5.6 mol/L) and Gly50 (approximately 7.0 mol/L). Since oocytes are in EG40 just for only a short period of time (30 s) and at a lower temperature (4°C), we hypothesize that the main function of this step in to induce dehydration. Based on these results, we omitted the EG40 step, before oocytes were pretreated in EG20 for 5 min, exposed to pre-cooled (4°C) Gly50, for 30 s, and then dipped into liquid nitrogen. After warming, 81.1% of the oocytes survived, and the surviving oocytes developed into cleavage stage embryos (63.5%) or blastocysts (20.0%) after parthenogenetic activation.

Conclusions

These results demonstrate that in a two-step vitrification procedure, the permeability effect in the second step is not necessary. It is possible that the second step is only required to provide adequate osmotic pressure to condense the intracellular concentration of CPAs to a level required for successful vitrification.     

An efficient method for the sanitary vitrification of bovine oocytes in straws

Background：At present,vitrification has been widely applied to humans,mice and farm animals.To improve the efficiency of vitrification in straw,bovine oocytes were used to test a new two-step vitrification method in this study.Results：When in vitro matured oocytes were exposed to 20%ethylene glycol（EG20） for 5 min and 40%ethylene glycol（EG40） for 30 s,followed by treatment with 30%glycerol（Gly30）,Gly40 or Gly50,a volume expansion was observed in Gly30 and Gly40 but not Gly50.This indicates that the intracellular osmotic pressure after a 30 s differs between EG40 and ranged between Gly40（approximately 5.6 mol/L） and Gly50（approximately 7.0 mol/L）.Since oocytes are in EG40 just for only a short period of time（30 s） and at a lower temperature（4℃）,we hypothesize that the main function of this step in to induce dehydration.Based on these results,we omitted the EG40 step,before oocytes were pretreated in EG20 for 5 min,exposed to pre-cooled（4℃） Gly50,for 30 s,and then dipped into liquid nitrogen.After warming,81.1%of the oocytes survived,and the surviving oocytes developed into cleavage stage embryos（63.5%） or blastocysts（20.0%） after parthenogenetic activation.Conclusions：These results demonstrate that in a two-step vitrification procedure,the permeability effect in the second step is not necessary.It is possible that the second step is only required to provide adequate osmotic pressure to condense the intracellular concentration of CPAs to a level required for successful vitrification.     

不同冷冻载体对牛未成熟卵母细胞玻璃化冷冻效果的影响

为了研究不同冷冻载体制备方法及其对牛未成熟卵母细胞发育能力的影响,以牛未成熟卵母细胞为实验材料,分别探究开放式拉长细管(OPS,open pulled straw)和玻璃微管(GMP,glass micropipette)的制备方法,并以OPS、GMP和冷冻叶片为冷冻载体玻璃化冷冻牛未成熟卵母细胞,解冻后经体外成熟培养和体外受精,统计形态正常率、成熟率、卵裂率及囊胚率。结果显示:以简易小酒精灯为热源,以细管为原材料可以制备出适用的OPS冷冻载体;以酒精喷灯为热源,以细玻璃管为原材料可以制备出适用的GMP冷冻载体。OPS和GMP组形态正常率分别为(74.3%±1.8)%和(72.5%±2.6)%;无显著差异(P0.05),上述二组均显著低于冷冻叶片组(82.1%±1.3)%,P0.05,但三组间成熟率、卵裂率和囊胚率均无显著差异(P0.05)。研究表明,分别以简易小酒精灯和酒精喷灯为热源,以细管和细玻璃管为原材料可以成功制备出OPS和GMP载体;以OPS、GMP和冷冻叶片为冷冻载体均可成功地玻璃化冷冻牛未成熟卵母细胞。     

Effect of medium change on the development of in vitro matured and fertilized bovine oocytes cultured in medium containing amino acids

Bovine in vitro matured and fertilized oocytes were cultured for 153 hr in groups of 3 or 30 in 30 microl of modified synthetic oviduct fluid medium supplemented with amino acids. The concentration of ammonium in culture medium at 153 hr of culture was significantly decreased by medium change at 72 hr of culture. However, regardless of embryo density, medium change had no beneficial or detrimental effect on the development of bovine embryos. Increase in the development to blastocysts and production of ammonium were observed when embryos were cultured in groups of 30. These results indicated that the ammonium concentration detected in this culture system has a negligible effect on the development of bovine embryos to blastocysts.     

High survival of bovine mature oocytes after nylon mesh vitrification,as assessed by intracytoplasmic sperm injection

Intracytoplasmic sperm injection (ICSI) is an alternative technique to in vitro fertilization (IVF) for producing transferable blastocysts, especially in combination with cryopreserved oocytes, when the IVF system does not work sufficiently. The present study was conducted to directly compare the efficacy of producing bovine blastocysts by ICSI and IVF from vitrified-warmed and fresh oocytes. Denuded oocytes with a detectable first polar body were vitrified-warmed using a nylon mesh device. In the non-vitrified control group, blastocyst yields 8 days after IVF and ICSI were 32.0 and 26.8%, respectively. Oocyte vitrification and subsequent IVF resulted in an impaired blastocyst yield (15.0%); however, such a loss of efficacy due to vitrification was not observed in the ICSI group (blastocyst yield, 25.2%). The alignment of cortical granules beneath the oolemma was comparable between the fresh control and vitrified-warmed oocytes. Here, we report the high survival of vitrified-warmed bovine oocytes, as assessed by ICSI.     

牛有腔卵泡卵母细胞体外成熟的研究

试验从屠宰场收集了 4 8头青年黄牛、34头青年水牛的卵巢共 16 4个 ,共回收可用卵母细胞 86 4枚。水牛平均每个卵巢可回收 3.2 2枚可用卵母细胞 ,相当于黄牛 (6 .72枚 )的一半。试验结果表明 :水牛卵巢生长卵泡少 ,卵母细胞产量少、质量差 ;3种采集黄牛卵泡卵母细胞的方法 ,用抽吸加切割法平均从每个卵巢回收的可用卵数 (7.71枚 )都极显著高于抽吸法 (6 .19枚 )和切割法 (6 .4 4枚 ) (P <0 .0 1) ,但是该法手续繁杂 ,适于一次且只能收集少量卵巢时使用 ;在黄牛和水牛卵母细胞成熟培养液中用 0 .3%PVP代替 10 %FBS ,牛卵母细胞的体外成熟率无显著差异 (P >0 .0 5 )。     

GMP玻璃化冷冻未成熟牛卵母细胞核成熟及冷冻损伤试验

本试验通过荧光染色的方法建立了未成熟牛卵母细胞在体外培养过程中第1次减数分裂的各个阶段的参考判定图谱;根据这个标准来观察毛细玻璃管(GMP)玻璃化冷冻对卵母细胞核成熟和冷冻损伤的影响。结果表明,从屠宰场废弃的卵巢表面卵泡内抽取的COCs,70%处于生发泡期,12.5%生发泡开始破裂,7.5%已开始浓缩,这说明从屠宰场获得的COCs有较高的异质性;卵母细胞在成熟培养22h时收获排出第一极体的卵母细胞,可得到丰度较高的极体-胞质染色体对称、紧密相邻的成熟卵母细胞;GMP玻璃化冷冻损伤主要有2种表现形式,首先,直接影响膜结构的完整性,包括细胞膜和核膜,这可从退化的细胞比例看出(8～24h,有21.9%～27.2%的细胞处于该阶段),其次,影响CONDENSED向MⅠ期的过渡,这可从处于CONDENSED卵母细胞的比例看出(8～24h,有24.1%～34.3%的细胞处于该阶段)。     

Ultrastructure of immature and mature human oocytes after cryotop vitrification

In vitro maturation of vitrified immature germinal vesicle (GV) oocytes is a promising fertility preservation option. We analyzed the ultrastructure of human GV oocytes after Cryotop vitrification (GVv) and compared it with fresh GV (GVc), fresh mature metaphase II (MIIc) and Cryotop-vitrified mature (MIIv) oocytes. By phase contrast microscopy and light microscopy, the oolemmal and cytoplasmic organization of fresh and vitrified oocytes did not show significant changes. GVv oocytes showed significant ultrastructural alterations of the microvilli in 40% of the samples; small vacuoles and occasional large/isolated vacuoles were abnormally present in the ooplasm periphery of 50% of samples. The ultrastructure of nuclei and mitochondria-vesicle (MV) complexes, as well as the distribution and characteristics of cortical granules (CGs), were comparable with those of GVc oocytes. MIIv oocytes showed an abnormal ultrastructure of microvilli in 30% of the samples and isolated large vacuoles in 70% of the samples. MV complexes were normal, but mitochondria-smooth endoplasmic reticulum aggregates appeared to be of reduced size. CGs were normally located under the oolemma but presented abnormalities in distribution and matrix electron density. In conclusion, Cryotop vitrification preserved main oocyte characteristics in the GV and MII stages, even if peculiar ultrastructural alterations appeared in both stages. This study also showed that the GV stage appears more suitable for vitrification than the MII stage, as indicated by the good ultrastructural preservation of important structures that are present only in immature oocytes, like the nucleus and migrating CGs.     

影响黄牛小腔卵泡卵母细胞体外受精及早期胚胎发育的因素

从屠宰场收集黄牛卵巢,取皮质深层卵母细胞进行体外成熟、体外受精和早期胚胎体外培养,分析了影响其效果的因素。结果表明,在成熟培养液中添加FSH(10IU/mL)、HCG(20IU/mL)和17β-E2(1mg/L)对卵母细胞受精后早期胚胎发育能力有极显著促进作用;等量牛卵泡液(BFF)与新生牛血清(NCS)对体外受精胚胎发育效果影响不显著,以15?F为宜;颗粒细胞与输卵管上皮细胞均能显著提高卵母细胞体外成熟受精后早期胚胎的发育率,颗粒细胞 输卵管上皮细胞对克服胚胎阻滞现象效果显著。     

Recovery of bovine oocytes from small vesicular follicles for in vitro maturation and fertilization

Five dairy and four beef breed, mature cows were used as oocyte donors to develop a system of multiple recovery of oocytes for in vitro maturation and fertilization. The animals were alternately treated with either 20 mg of follicle stimulating hormone (FSH) in four equal intramuscular injections or saline at 12 h intervals starting between days 9 and 13 of the oestrous cycle, and the procedure was repeated at three-week intervals for up to four collections. Eighteen collections resulted in the recovery of 124 oocytes from 181 follicles (69%). No serious side-effects were observed. Recovery was equally successful in both breeds and was not reduced in repeat attempts upon the same animal. Treatment with FSH only marginally increased the recovery rate (p<0.07) and did not affect the number of follicles aspirated (p>0.05), which varied significantly (p<0.05) between cows.From 110 oocytes matured and fertilized in vitro, 70 embryos were recovered after culture in the rabbit oviduct or with trophoblastic vesicles in vitro, of which 30 had cleaved and 5 had progressed to an advanced stage of development. Hormone treatment did not affect zygote development (p>0.05). Four non-surgical transfers of embryos obtained in these studies have resulted in two pregnancies determined ultrasonographically and the birth of a heifer calf. This suggests that the procedure for multiple oocyte recovery is safe and that it can be used sucessfully for obtaining oocytes for in vitro maturation and fertilization.     

Xenotransplantation of goat ovary as an alternative to analyse follicles after vitrification

The aim of this study was to evaluate the caprine preantral follicles enclosed on vitrified/warmed ovarian cortex grafted to nude BALB/mice during 1 month. The ovarian cortex from goats was fragmented (3 × 3 × 0.5 mm) and divided into four groups: fresh control, vitrified control, fresh transplant and vitrified transplant. Follicular morphology, development and density, fibrosis as well as apoptosis, and tissue revascularization were evaluated. It was also observed a significant decrease in morphologically normal preantral (primordial, transition, primary and secondary) follicles in both vitrified control and vitrified transplant treatments when compared with both fresh control and fresh transplant. However, fresh control and fresh transplant exhibited a similar percentage of developing follicles. Additionally, Vitrified control showed a significant increase in developing follicles in comparison with both fresh control and fresh transplant. Follicular density significantly decreased in all treatments in comparison with fresh control. We observed high fibrosis in both fresh transplant and vitrified transplant. The mRNA expression of caspase 3 was lower in both fresh transplant and vitrified transplant in comparison with vitrified control. In conclusion, xenotransplantation is an excellent strategy to maintain normal preantral follicle morphology after vitrification/warming of goat ovarian tissue. Yet, in order to ensure the survival and development of these follicles, it is essential to improve the revascularization of the graft.     

Improved monospermic fertilization and subsequent blastocyst formation of bovine oocytes fertilized in vitro in a medium containing NaCl of decreased concentration

The objective of this study was to evaluate whether changes in NaCl concentration in a fertilization medium could improve normal fertilization and preimplantation development of bovine oocytes. In vitro matured bovine oocytes were inseminated with frozen-thawed semen for 18 hr in a Tyrode's medium with albumin, lactate and pyruvate (TALP), to which 114 (TALP-114), 96 (TALP-96) or 78 (TALP-78) mM NaCl was added. Presumptive zygotes were cultured for 192 hr in a modified TALP containing 90 mM NaCl, 1.5 mM glucose, 0.3% (w/v) BSA, minimal essential medium (MEM) essential and nonessential amino acids, and insulin-transferrin-selenium complex. Lower polyspermy rate was obtained by the insemination in TALP-96 (7.8 +/- 2.3%) than by the insemination in TALP-114 (25.6 +/- 1.4%), without decrease in male pronucleus (MPN) formation. Fertilization in TALP-78 also yielded decreased polyspermic fertilization (3.8 +/- 1.5%), but significant decrease in MPN formation was found (63.1 +/- 3.1%). In preimplantation development, more blastocysts developed from oocytes inseminated in TALP-96 (24.1 +/- 1.7%) than from oocytes inseminated in TALP-114 (16.8 +/- 1.4%). TALP-78, however, did not improve preimplantation development beyond the 8-cell stage compared with TALP-114. Mean cell number of blastocyst was higher when oocytes were fertilized in TALP-96 (137.0 +/- 4.5) than in TALP-114 (123.1 +/- 5.1) and in TALP-78 (102.3 +/- 4.5). These results demonstrate that insemination of bovine oocytes in a TALP with decreased NaCl concentration (96 mM) improves blastocyst formation and embryo viability. Decrease in NaCl concentration below 96 mM, however, may delay or inhibit MPN formation, and inhibits subsequent development in vitro.     

Xenografting of bovine secondary follicles into ovariectomized female severe combined immunodeficient mice

Xenografting of ovarian tissue into immunodeficient mice has been used as a model to study the dynamics of follicular development and provides an alternative method for the production of mature oocytes. In a previous experiment, we demonstrated that xenografted bovine secondary follicles developed to the antral stage in severe combined immunodeficient (SCID) mice. In the present study, we examined the development of bovine secondary follicles (140-190 microm in diameter) grafted into ovariectomized mice in comparison with intact female mice as a control. At 4 weeks after grafting, several antral follicles ranging from 350 to 550 microm (457.6 +/- 50.8 microm) in diameter were found in the control mice, while a single large (larger than 2.5 mm) antral follicle and other small follicles were observed in every ovariectomized mouse. At 6 weeks after grafting, the mean diameter of morphologically normal follicles had further increased in the control group (591.8 +/- 132.0 microm). In ovariectomized mice, however, the mean diameter of follicles decreased (4 weeks: 864.2 +/- 988.2 microm; 6 weeks: 496.5 +/- 137.6 microm), since the single large antral follicle observed at 4 weeks had degenerated by 6 weeks. In control mice, more than 70% of follicles were morphologically normal and formed an antrum, and most of the follicles contained morphologically normal oocytes which grew to 122.5 +/- 2.2 microm. In ovariectomized mice, morphologically normal oocytes also grew larger than before grafting, but their survival rate was significantly lower than that in control mice. These results suggest that ovariectomy of host mice alters the developmental pattern of xenografted bovine secondary follicles to accelerate a single follicle to develop in the graft.     

The application of apoptotic inhibitor in apoptotic pathways of MII stage porcine oocytes after vitrification

Apoptosis is one of the main drivers of the decline in developmental potential of porcine oocytes after vitrification. However, which apoptotic pathways are engaged after vitrification remains poorly understood. To distinguish among the possible apoptotic pathways induced by vitrification of MII stage porcine oocytes, this study detected activity and expression levels of several key proteins and genes in both the death receptor and mitochondrial pathways using in situ fluorescence staining and real‐time PCR (RT‐PCR) following the addition of specific inhibitors of either the death receptor or the mitochondrial apoptotic pathway (Z‐IETD‐FMK or Z‐LEHD‐FMK, respectively) into the incubation solution. Survival and parthenogenetic developmental ability were also examined. The results showed the following: (i) compared with the vitrified group, the activities of pan‐caspase, caspase 3, caspase 8 and caspase 9 as well as the early apoptotic rate were significantly lower in the Z‐IETD‐FMK and Z‐LEHD‐FMK groups (p < .05); (ii) After vitrification, mitochondrial ΔΨm decreased from 1.33 for fresh oocytes to 0.90 for vitrified oocytes, while the addition of Z‐IETD‐FMK or Z‐LEHD‐FMK following vitrification increased mitochondrial ΔΨm to 1.09 and 1.05, respectively (p < .05); (iii) Relative expression levels of apoptotic‐related genes from both the death receptor pathway (caspase 8 and TNF‐α) and the mitochondrial pathway (caspase 9, Bcl‐2 and CuZnSOD) changed substantially after the addition of Z‐IETD‐FMK or Z‐LEHD‐FMK into the incubation solution; (iv) Survival, cleavage and blastocyst rates were higher in the Z‐IETH‐FMK or Z‐LEHD‐FMK groups than in the vitrified group (p < .05). In summary, both the death receptor and the mitochondrial apoptotic pathways participate in the apoptosis of MII stage porcine oocytes after vitrification.     

Effect of vitrification at different meiotic stages on epigenetic characteristics of bovine oocytes and subsequently developing embryos

Vitrification by the Cryotop method is frequently used for bovine oocyte cryopreservation. Nevertheless, vitrified oocytes still have reduced developmental competency compared with fresh counterparts. The objective of this study was to compare the effect of vitrification either at the germinal vesicle (GV) stage or at the metaphase II (MII) stage on epigenetic characteristics of bovine oocytes and subsequently developing embryos. Our results demonstrated that vitrification of oocytes at each meiotic stage significantly reduced blastocyst development after in vitro fertilization (IVF). However, vitrification at the GV stage resulted in higher blastocyst development than did vitrification at the MII stage. Irrespective of the meiotic stage, oocyte vitrification did not affect 5-methylcytosine (5mC) immunostaining intensity in oocyte DNA. However, at both stages, it caused a similar reduction of 5mC levels in DNA of subsequently developing blastocysts. Oocyte vitrification had no effect on the intensity of H3K9me3 and acH3K9 immunostaining in oocytes and subsequent blastocysts. The results suggest that irrespective of meiotic stage, oocyte vitrification alters global methylation in resultant embryos although such alteration in the oocytes was not detected. Oocyte vitrification might not influence histone acetylation and methylation in oocytes and resultant embryos. Vitrification at the immature stage was more advantageous for blastocyst development than at the mature stage.     

Oxygen tension and medium supplements for in vitro maturation of bovine oocytes cultured individually in a chemically defined medium

The effects of adding cysteamine, EGF, and glucose as an energy substrate under low oxygen tension during in vitro maturation (IVM) were examined to find ways of improving the individual in vitro production (IVP) system in individually cultured bovine oocytes. The basic medium was mSOFaa containing 1 mg/ml polyvinyl alcohol. Immature oocytes were individually cultured in an IVM medium with 10 ng/ml EGF, 100 microM cysteamine, or EGF plus cysteamine under 20% or 5% O(2). Cleavage and blastocyst rates were significantly higher (P<0.05) in IVM culture was under 20% O(2) than in culture under 5% O(2). Under 5% O(2), neither EGF nor cysteamine improved embryonic development. The proportion of matured oocytes was significantly higher (P<0.05) in the presence of 1.5 mM glucose under 20% O(2) (68.6%), and 5.5 mM (66.7%) and 10 mM (65.5%) glucose under 5% O(2). The presence of 5.5 mM glucose significantly (P<0.05) increased the maturation rate compared with the absence of glucose, irrespective of addition of EGF and cysteamine. The addition of cysteamine alone in the maturation medium significantly (P<0.05) increased the intracellular GSH concentration in the oocytes. Also, under 5% O(2) cysteamine and/or EGF significantly (P<0.05) improved the proportions of penetrated oocytes, cleavage and blastocyst formation, which were similar levels to those of oocytes matured under 20% O(2). After vitrification, the re-expanding and hatching rates of blastocysts derived from the individual IVP system containing cysteamine under 5% O(2) were significantly (P<0.05) higher than those of blastocysts derived from the individual IVP system without cysteamine under 5% O(2) and the group IVP system under 20% O(2). The present study showed that a high glucose level (5.5 or 10 mM) was optimal in IVM culture under low (5%) oxygen tension. The addition of EGF and/or cysteamine to the maturation medium had no positive effect on nuclear maturation, but improved fertilizability, developmental competence and cryoresistance following vitrification, probably due to increased GSH synthesis during the IVM process.