Several genetic and physiological factors increase the risk of DNA damage in mammalian oocytes. Two critical events are: (i) meiosis progression, from maturation to fertilization, due to extensive chromatin remodelling during genome decondensation; and (ii) aging, which is associated with a progressive oxidative stress. In this work, we studied the transcriptional patterns of three genes, RAD51, APEX-1 and MLH1, involved in DNA repair mechanisms. The analyses were performed by real-time quantitative PCR (RT-qPCR) in immature and in vitro matured oocytes collected from 17 ± 3-month-old heifers and 94 ± 20-month-old cows. Batches of 30–50 oocytes for each group (three replicates) were collected from ovarian follicles of slaughtered animals. The oocytes were freed from cumulus cells at the time of follicle removal, or after in vitro maturation (IVM) carried out in M199 supplemented with 10% fetal calf serum, 10 IU luteinising hormone (LH)/ml, 0.1 IU follicle-stimulating hormone (FSH)/ml and 1 μg 17β-oestradiol/ml. Total RNA was extracted by Trizol method. The expression of bovine GAPDH gene was used as the internal standard, while primers for bovine RAD51, APEX-1 and MLH1 genes were designed from DNA sequences retrieved from GenBank. Results obtained indicate a clear up-regulation of RAD51, APEX-1 and MLH1 genes after IVM, ranging between two- and four-fold compared with germinal vesicle (GV) oocytes. However, only RAD51 showed a significant transcript increase between the immature oocytes collected from young or old individuals. This finding highlights RAD51 as a candidate gene marker for discriminating bovine immature oocytes in relation to the donor age.

Sperm plasma membrane (PM) proteins that demonstrate affinity for egg PM preparations have the potential to be biologically important during sperm-egg binding and/or fusion. In this study four such proteins have been identified. To provide quantitative evidence for possible biological function, the large natural variation among different porcine sperm populations with regard to their ability to interact with the egg was compared with the relative binding of egg PM material to individual proteins. An aliquot from each of 24 porcine ejaculates was evaluated by the zona-free hamster ova bioassay and the remainder processed to yield sperm PM vesicles. Aliquots of sperm PM were solubilised, separated by SDS-PAGE, western blotted and probed with partially purified, biotinylated egg PM protein. Bound egg PM proteins were visualised on western blots by an avidin/biotin/horseradish peroxidase system and analysed by scanning laser densitometry. Four sperm PM proteins (62, 39, 27 and 7 kDa estimated molecular mass) were the predominant binders of egg PM. The amount of egg PM bound to the 62 kDa protein was significantly correlated with the ability of sperm from the 24 ejaculates to penetrate zona-free hamster ova (percentage of ova penetrated, p = 0.01, R = 0.65; number of penetrated sperm per ovum, p = 0.02, R = 0.63)

Cryopreservation has not been used successfully to preserve fish embryos, although chilling techniques have been used with good results. The aim of this study was to chill Piaractus brachypomus embryos at – 10°C for various storage times. Embryos at the following ontogenetic stages were used: blastoderm – 1.2 hours post-fertilization (hpf); epiboly – 5 hpf; blastopore closure – 8 hpf; and appearance of the optic vesicle – 13 hpf. One hundred embryos were selected from each ontogenetic stage and chilled at – 10°C for 6 or 10 h. The results were analysed using analysis of variance (ANOVA) and Tukey's test at a 5% significance level. A significantly greater number of completely developed live larvae were observed following embryonic treatment with a cryoprotectant solution that contained 17.5% sucrose and 10% methanol. There was no survival for embryos cooled at – 10°C in initial developmental stages (1, 2 and 5 h hpf). Furthermore, higher survival rates were observed when embryos were treated at more advanced developmental stages (8 and 13 hpf). Therefore, P. brachypomus embryos at the blastopore-closure (8 hpf) or appearance-of-optic-vesicle (13 hpf) stages should be used for embryo chilling protocols and chilling should be performed using a 17.5% sucrose with a 10% methanol solution at – 10°C for up to 6 h. The best results were obtained with 13-hpf and 8-hpf embryos and cooling at 6 h of storage.

Embryos from certain mouse strains are arrested at the 2-cell stage in cell culture (‘2-cell block’), whereas those from other strains develop to the blastocyst stage under the same conditions. It was previously shown that blocking embryos can be rescued in culture by aggregation with an excess of 2-cell stages of a non-blocking strain such as CBA × C57BL/6 F2. Here we have employed a LacZ transgene in a blocking strain (NMRI) to follow the fate of rescued blastomeres up to the blastocyst stage. We found that rescued blastomeres can participate in both inner cell mass and trophoblast formation, thus completely overcoming the 2-cell block.

Expression of mRNAs and proteins of ZO-1 and occludin was analyzed in pig oocytes and parthenogenetic diploid embryos during preimplantation development using real-time RT-PCR, western blotting and immunocytochemistry. All germinal vesicle (GV) and metaphase (M)II oocytes and preimplantation embryos expressed mRNAs and proteins of ZO-1 and occludin. mRNA levels of both ZO-1 and occludin decreased significantly from GV to MII, but increased at the 2-cell stage followed by temporal decrease during the early and late 4-cell stages. Then, both mRNAs increased after compaction. Relative concentration of zo1α− was highest in 2-cell embryos, while zo1α+ was expressed from the morula stage. Occludin expression greatly increased after the morula stage and was highest in expanded blastocysts. Western blotting analysis showed constant expression of ZO-1α− throughout preimplantation development and limited translation of ZO-1α+ from the blastocysts, and species-specific expression pattern of occludin. Immunocytochemistry analysis revealed homogeneous distribution of ZO-1 and occludin in the cytoplasm with moderately strong fluorescence in the vicinity of the contact region between blastomeres, around the nuclei in the 2-cell to late 4-cell embryos, and clear network localization along the cell-boundary region in embryos after the morula stage. Present results show that major TJ proteins, ZO-1 and occludin are expressed in oocytes and preimplantation embryos, and that ZO-1α+ is transcribed by zygotic gene activation and translated from early blastocysts with prominent increase of occludin at the blastocyst stage.

We have investigated the possibility that mitotic nuclei originating from preimplantation stage embryos and placed in the oocyte cytoplasm can undergo remodelling that allows them to undergo meiosis in the mouse. To address this question, we have used enucleated germinal vesicle (GV) ooplasts as recipients and blastomeres from the 2-, 4- or 8-cell stage as nuclear donors. We employed two methods to obtain ooplasts from GV oocytes: cutting and enucleation. Although efficiency of the reconstruction process was higher after enucleation than after cutting (90% and 70% respectively), the developmental potential of the oocytes was independent of how they had been produced. Nuclei from the 2-, 4-, or 8-cell stage embryos supported maturation in about 35%, 55% and 60% of cases, respectively. The time between nuclear envelope breakdown and the first meiotic division was shortened by up to 5 h in reconstructed oocytes, a period equivalent to the mitotic division of control blastomeres. About one-third of oocytes reconstituted with blastomere nuclei divided symmetrically instead of extruding a polar body; however, in the majority of them metaphase plates were found, suggesting that reconstructed oocytes (cybrids) underwent a meiotic rather than mitotic division. The highest percentage of asymmetric divisions accompanied by metaphase plates was found in cybrids with 8-cell-stage blastomere nuclei, suggesting that the nuclei from this stage appear to conform best to the cytoplasmic environment of GV ooplasts. Our results indicate that the oocyte cytoplasm is capable of remodelling blastomere nuclei, allowing them to follow the path of the meiotic cell cycle.

This study aimed to achieve high frequencies of nuclear maturation and penetrability through the zona pellucida of hamster oocytes cultured under protein-free conditions. Completion of nuclear maturation by cumulus-intact, immature oocytes (79% metaphase II stage) was depressed (37% p < 0·05) by adding four amino acids (glutamine, isoleucine, methionine and phenylalanine) reported necessary for nuclear maturation of cumulus-free oocytes. Following in vitro maturation, cumulus cells were removed and oocytes were inseminated with capacitated sperm, but after 6 h sperm:egg co-incubation, only 24% of in vitro matured oocytes were penetrated compared with 60% of in vivo matured oocytes (p < 0·05). Time required for zona lysis by α-chymotrypsin was not significantly different among in vitro and in vivo matured oocytes and 1-cell embryos. Addition to the maturation medium of soybean trypsin inhibitor or fetuin, both known to inhibit the zona reaction in vitro, did not improve penetrability of in vitro matured oocytes, implying that in hamsters, unlike other rodent species, a premature zona reaction is unlikely to be responsible for inhibiting sperm penetration. When oocytes were incubated with 20% periovulatory oviductal fluid (OF) for another 3 h after maturation, penetration was significantly improved (60% vs 37% with and without OF, respectively; p < 0·05), but was not equivalent to penetration of in vivo matured follicular oocytes similarly treated with OF (84%, p < 0·05)However, zona penetration was further improved by increasing sperm concentration from 1·0 × 104 (66%) to 5·0 or 10·0 × 104 sperm/ml (89%, p < 0·05). This study shows that nuclear maturation of hamster oocytes can occur in chemically defined medium, and indicates that a deficiency in the zona of in vitro matured oocytes can be overcome by preincubation with OF and insemination at high sperm conccentration.

The fertilisation of free-spawning invertebrates, mainly sea urchins, has been studied extensively during the last hundred years. However, results obtained from in vitro experiments do not always reflect what happens in the real world. Organisms in their natural habitats have a complex set of challenges, cues and behaviours to contend with during fertilisation and early development, factors that are normally not considered in the laboratory setting. This review examines recent work on fertilisation ecology and discusses the relevance of these results to the findings gleaned from laboratory research. Emphasis is placed on stresses associated with fertilisation in situ, and how responses to environmental stresses (such as from turbulence, oxidative stress, ultraviolet radiation and pathogens) might affect the fertilisation process.

Transient increases in the concentration of cytosolic Ca2+ are essential for triggering egg activation events. Increased Ca2+ results from its rapid release from intracellular stores, mainly mediated by one or both intracellular calcium channels: the inositol trisphosphate receptor (IP3R) and the ryanodine receptor (RyR). Several regulatory pathways that tailor the response of these channels to the specific cell type have been proposed. Among its many modulatory actions, calcium can serve as an activator of a cytosolic phospholipase A2 (cPLA2), which releases arachidonic acid from phospholipids of the endoplasmic reticulum as well as from the nuclear envelope. Previous studies have suggested that arachidonic acid and/or its metabolites were able to modulate the activity of several ion channels. Based on these findings, we have studied the participation of the phospholipase A2 (PLA2) pathway in the process of Bufo arenarum oocyte activation and the interrelation between any of its metabolites and the ion channels involved in the calcium release from the intracellular reservoirs at fertilization. We found that addition of both melittin, a potent PLA2 activator, and arachidonic acid, the main PLA2 reaction metabolite, was able to induce activation events in a bell-shaped manner. Differential regulation of IP3Rs and RyRs by arachidonic acid and its products could explain melittin and arachidonic acid behaviour in Bufo arenarum egg activation. The concerted action of arachidonic acid and/or its metabolites could provide controlled mobilization of calcium from intracellular reservoirs and useful tools for understanding calcium homeostasis in eggs that express both types of receptors.

Infertility is the most common issue in the field of reproductive medicine. Many factors affect fertility status, including life-style choices such as cigarette smoking or alcohol use. The aim of this study was to investigate the effects of cigarette smoking on oocyte quality as well the quantity in young fertile women. In total, 56 young fertile women who were undergoing oocyte donation programmes were included in this study. The effects of cigarette smoking on antral follicle count, number of follicles and oocytes retrieved and morphology were assessed by an expert embryologist. The results of this study showed that cigarette smoking did not have a significant effect on the follicle count or the number of oocytes retrieved from young and fertile women. However, a significant difference was observed in the morphological assessment. In conclusion, although cigarette smoking does not seem to affect oocyte development, it had an effect on cytoplasmic anomalies and therefore may lower pregnancy chance. Therefore, it is crucial to give proper counselling to patients who are trying to become pregnant both naturally and by in vitro fertilization.

Germinal vesicle (GV)-stage horse oocytes with diffuse chromatin are meiotically incompetent and degenerate in culture, whereas horse oocytes having condensed chromatin within the GV are meiotically competent. Degeneration of incompetent oocytes in culture may be related to premature GV breakdown, which could possibly be prevented by inhibition of m-phase protein activity. We examined the effects of 6-dimethylaminopurine (6-DMAP), butyrolactone and roscovitine on GV-stage horse oocytes. Culture in the presence of 2 mM 6-DMAP for 24 h suppressed meiosis (2% MI or MII compared with 38% for untreated oocytes). The proportion of GV-stage oocytes having condensed chromatin was not different between 6-DMAP culture and directly fixed controls; however, the proportion of oocytes with diffuse chromatin was significantly lower, and more oocytes with diffuse chromatin had atypical chromatin than did controls (p < 0.01). Culture with butyrolactone at 100 mM suppressed meiosis (5% MI + II). Again, this treatment maintained GV-stage oocytes having condensed chromatin, but the proportion of oocytes with diffuse chromatin was significantly reduced compared with directly fixed controls (p < 0.05). Culture with roscovitine at 25 μM was also effective in maintaining GV-stage oocytes having condensed chromatin; however, culture with 100 μM roscovitine did not suppress meiosis or maintain oocytes in the GV stage. These results indicate that meiosis in GV-stage horse oocytes having condensed chromatin may be suppressed by inhibitors of m-phase protein activity; however, oocytes originally having diffuse chromatin appear to degenerate in culture even in the presence of these inhibitors.

The POU family subclass V (POU-V) proteins have important roles in maintaining cells in an undifferentiated state. In Xenopus, expression of the POU-V protein Oct60 was detected in oocytes and was found to decrease in blastula- to gastrula-stage embryos. In addition, Oct60 overexpression inhibits some signals in early embryogenesis, including Activin/Nodal, BMP, and Wnt signalling. In this report, we analysed mechanisms of Oct60 promoter activation and discovered that Oct60 transcription was activated ectopically in somatic nuclei by oocyte extract treatment. Promoter assays demonstrated that Oct60 transcription was activated in oocytes specifically and that this activation was dependent on an Octamer-Sox binding motif. ChIP assays showed that the Oct60 protein binds the motif. These results suggest that Oct60 transcription is regulated by a positive-feedback loop in Xenopus oocytes.

Birds exhibit physiological polyspermy, i.e. numerous spermatozoa enter the germinal disc of an oocyte and form pronuclei during fertilisation. However, only one of them unites with the female pronucleus to form a zygote nucleus; the supernumerary spermatozoal nuclei degenerate at the early cleavage stages. To establish a factor responsible for spermatozoal degeneration, the presence of DNase activity was studied in vitro in extracts of Japanese quail oocytes using λ DNA/HindIII as a substrate. The experimental conditions were designed to reveal the presence of either DNase I or DNase II activities, separately. Degradation of the substrate DNA was evaluated by electrophoresis on agarose gels stained with ethidium bromide. High activities of DNase I and DNase II were found in the germinal discs of the largest vitellogenic oocytes. DNase I activity was estimated to be about 3 × 10−3 Kunitz units and DNase II about 4 × 10−2 Kunitz units per germinal disc. DNase I activity in an oocyte seems to increase during oogenesis since DNA degradation by the extracts from the germinal discs of the largest vitellogenic oocytes was much higher than by those from previtellogenic and small vitellogenic oocytes. The presence of high DNase I and II activities in the largest vitellogenic oocytes would point to their role in degradation of DNA from supernumerary spermatozoa entering the ovum during polyspermic fertilisation in birds. The enzymes could be a factor, or one of the factors, in the late block to polyspermy in the cytoplasm of avian eggs. It is suggested here that the DNase activities might also be responsible for poor efficiency in obtaining transgenic birds by microinjection of exogenous DNA into the fertilised chick ovum.

The goal of this study was to examine the effect of insulin-like growth factor I (IGF-I; added during post-thaw culture (48 h)) on the preimplantation viability and quality of cryopreserved bovine in vivo recovered embryos. The morula stage embryos, non-surgically recovered from superovulated dairy cows of Czech Fleckvieh cattle breed, had previously been cryopreserved by a slow freezing technique and stored in liquid nitrogen since 1989–1990. Following thawing, the embryos were cultured for 48 h either alone (no IGF-I) or in the presence of IGF-I (10 or 100 ng/ml); non-cultured embryos served as a control. Thereafter, the embryos were analyzed for cleavage to the blastocyst stage, apoptosis (TUNEL), embryo cell number and quality of actin cytoskeleton. Following post-thaw culture 41% of embryos developed to advanced blastocysts. IGF-I increased this per cent and, at a higher dose, essentially reduced the per cent of degenerated embryos. In cultured embryos, IGF-I at both doses elevated the cell number compared with non-cultured embryos. However, in comparison with embryos cultured without IGF-I, only the higher IGF-I dose resulted in elevating the embryo cell number. The TUNEL index was significantly lowered by IGF-I treatment. Thawed embryos were mostly of the grade III actin type and fewer (12%) had grade II actin, whilst no grade I actin embryos were noted. The addition of IGF-I resulted in the appearance of grade I actin embryos (8.33 and 6.9% for 10 and 100 ng/ml, respectively). These observations indicate that the addition of IGF-I during post-thaw culture can improve the quality of bovine cryopreserved embryos.

The aim of this study was to evaluate follicular development, morphological integrity, and antioxidant potential of preantral ovarian follicles from Bos taurus indicus females grown in vitro with alpha-lipoic acid. Ovaries (n = 24) of Bos taurus indicus (n = 12) females were collected during slaughter and fragmented. A randomly obtained fragment from each pair of ovaries was fixed in Bouin (non-cultivated control; D0). These fragments were intended for classical histology (morphology and evaluation of follicular growth), and a fragment from each pair of ovaries was frozen at −80°C (non-cultivated control; D0), and assigned for analysis of oxidative stress [thiobarbituric acid reactive substances (TBARS), nitroblue tetrazolium (NBT), and ferric reducing antioxidant power (FRAP)]. The remaining fragments were cultured in vitro for 6 (D6) or 12 (D12) days, containing only minimum essential medium (MEM) or MEM supplemented with alpha-lipoic acid (50, 100, or 250 ng/ml), on an extracellular matrix of agarose gel, in an oven at 38.5ºC. Every 2 days, 100% of the culture medium was replaced. Supplementation with 100 ng/ml was effective for maintaining follicular integrity after 6 days of culture (primordial: 51.28%; development: 36.88%; P < 0.0001). There was no difference (P > 0.05) between treatments compared with the non-cultivated control treatment (D0), using the NBT and TBARS assays. Therefore, supplementation of the in vitro culture medium of bovine preantral ovarian follicles with a concentration of 100 ng/ml of alpha-lipoic acid at 6 days of culture was effective for maintaining follicular integrity and, after 6 days, maintaining stable levels of reactive oxygen species.

This study was aimed at developing a hormonal treatment protocol in order to optimize the proportion of pronuclear-stage embryos to be used for DNA microinjection in a goat transgenic founder production programme. A total of 46 adult BELE® and 47 adult standard goats (1–5 years old) were used as donors and recipients, respectively. They were heat-synchronized using intravaginal sponges containing 60 mg medroxyprogesterone acetate for 10 days with an injection of 125 μg cloprostenol on the morning of the eighth day. Recipients were injected with 400 IU eCG at the time of sponge removal while donors received a total of 133 mg NIH-FSH-P1 (Folltropin-V) given twice daily in decreasing doses over 3 days starting 48 h before sponge removal. Ovulation was induced in donors by injecting 100 μg of GnRH at 24 h (GnRH24) or 36 h (GnRH36) after sponge removal. Embryo recovery was performed by oviduct flushing following a standard mid-ventral laparotomy procedure. The proportion of embryos in the pronuclear stage of development was higher in the GnRH36 group (90% vs 34%, p<0.01). Embryos were microinjected with a DNA expression cassette followed by transfer to the oviduct of synchronized recipients. A higher, yet not statistically significant, pregnancy rate was found in the recipients transferred with pronuclear-stage embryos compared with those transferred with 2-cell-stage embryos (64% vs 37%, chi-square p=0.06). One transgenic female founder was produced from the group of recipients transferred with pronuclear-stage microinjected embryos.

The effect of the p42/44 mitogen-activated kinase (MAPK) inhibitor, PD98059, on MAPK activation and meiosis resumption in mouse oocytes was studied. When germinal vesicle (GV)-stage denuded oocytes (DOs) were cultured continuously in 50 μM PD98059, germinal vesicle breakdown (GVBD) was postponed for 2-3 h. MAPK phosphorylation and activation was delayed as well. However, PD98059 did not impair histone H1 kinase activation. After 14 h of culture there was no significant difference in the rate of DOs reaching metaphase II (MII) arrest in either control or experimental conditions. The effect of PD98059 on MAPK inhibition was further tested in epidermal growth factor (EGF)-treated oocyte–cumulus complexes (OCCs). Exposure of GV-stage OCCs for 5 min to EGF (10 ng/ml) induced a considerable increase in MAPK phosphorylation. After OCCs were further cultured in 50 μM PD98059 a rapid dephosphorylation of MAPK was induced. Already after 1 min of treatment the non-phosphorylated form of MAPK dominated, indicating the high effectivity of PD98059. This result indicates that short EGF/PD98059 treatment of OCCs induced MAPK phosphorylation/dephosphorylation in cumulus cells only. As only a transient delay in MAPK phosphorylation and activation was observed in PD98059-treated DOs we conclude that there is also another PD98059-nonsensitive pathway(s) leading to MAPK activation in mouse oocytes. The data obtained suggest that meiosis resumption in mouse oocytes is somehow influenced by the MEK/MAPK activation pathway.

Superovulatory response is characterized by a high degree of variability and unpredictability. The aim of the present experimental study was to examine whether the amount of maternal body fat can influence the efficiency of ovarian stimulation with gonadotropins. Female mice of two body condition types, normal and obese, produced in a standardized two-generation model, were subjected to ovarian stimulation using eCG and hCG followed by natural mating. Produced ova and embryos were recovered on day 1 and day 4 of pregnancy respectively, and several quantitative, qualitative and developmental parameters were evaluated in them. The overall response of mouse females with normal and elevated amounts of body fat to superovulation was similar: They produced almost the same numbers of ova and embryos on average. Conversely, a higher number of immature oocytes, non-fertilized mature oocytes and lower-stage zygotes were collected from fat females. In both groups, the majority of fertilized oocytes was able to cleave and reach the higher stages of development. However, in the group of fat mice, a lower number of blastocysts was collected, and these blastocysts showed increased incidence of apoptotic cell death. In conclusion, although the response of normal and fat mice to superovulatory treatment was similar, the quality and developmental capacities of produced ova were lower in the group of fat donors.

The role of activin-A in follicular development and on the mRNA expression levels of different genes in goat secondary follicles was evaluated. Goat secondary follicles (≥150 μm) were cultured for 18 days under control conditions or with the addition of either 50 or 100 ng/ml activin-A (Experiment 1). The mRNA levels for the genes that code for activin-A, ActR-IA, ActR-IB, ActR-IIA, ActR-IIB, follicle stimulating hormone receptor (FSH-R) and P450 aromatase were measured in each condition (Experiment 2). We observed that after 6 days of culture, the antrum formation rate was higher in cultures with added activin-A than in the cultured control (P < 0.05). The addition of 50 ng/ml activin-A increased the follicular growth rate in the final third of the culture (days 12–18), resulting in a higher percentage of meiosis resumption (P < 0.05). On day 6, the addition of activin-A (50 ng/ml) increased the levels of ActR-IA mRNA compared with the cultured control (P < 0.05). After 18 days, the addition of 50 ng/ml activin-A significantly increased the levels of its own mRNA compared with the non-cultured control. Moreover, this treatment reduced the mRNA levels of P450 aromatase in comparison with the cultured control (P < 0.05). Higher levels of P450 aromatase mRNA were found for both activin-A treatments compared with the non-cultured control (P < 0.05). No difference in estradiol levels was detected among any of the tested treatments. In conclusion, the addition of activin-A to culture medium stimulated early antrum formation as well as an increase in the daily follicular growth rate and the percentage of meiosis resumption.

In the present study, a porcine system was supplemented with sorbitol during in vitro maturation (IVM) or in vitro culture (IVC), and the effects of sorbitol on oocyte maturation and embryonic development following parthenogenetic activation were assessed. Porcine immature oocytes were treated with different concentrations of sorbitol during IVM, and the resultant metaphase II stage oocytes were activated and cultured in porcine zygote medium-3 (PZM-3) for 7 days. No significant difference was observed in cumulus expansion and the nuclear maturation between the control and sorbitol-treated groups, with the exception of the 100 mM group, which showed significantly decreased nuclear maturation and cumulus expansion. There was no significant difference in the intracellular reactive oxygen species (ROS) levels between oocytes matured with 10 or 20 mM sorbitol and control groups, but 50 and 100 mM groups had significantly higher ROS levels than other groups. The 20 mM group showed significant increases in intracellular glutathione and subsequent blastocyst formation rates following parthenogenetic activation compared with the other groups. During IVC, supplementation with sorbitol significantly reduced blastocyst formation and increased the apoptotic index compared with the control. The apoptotic index of blastocysts from the sorbitol-treated group for entire culture period was significantly higher than those of the partially sorbitol-exposed groups. Based on these findings, it can be concluded that the addition of a low concentration of sorbitol (20 mM) during IVM of porcine oocytes benefits subsequent blastocyst development and improves embryo quality, whereas sorbitol supplement during IVC has a negative effect on blastocyst formation.