The present study investigated the effects of vitrification of porcine oocytes either at the immature Germinal Vesicle (GV) stage before in vitro maturation (GV-stage oocytes) or at the pronuclear stage after in vitro maturation and fertilization (zygotes) on DNA integrity in relevance with their subsequent embryo development. Vitrification at the GV stage but not at the pronuclear stage significantly increased the abundance of double-strand breaks (DSBs) in the DNA measured by the relative fluorescence after γH2AX immunostaining. Treatment of GV-stage oocytes with cryoprotectant agents alone had no effect on DSB levels. When oocytes were vitrified at the GV stage and subjected to in vitro maturation and fertilization (Day 0) and embryo culture, significantly increased DSB levels were detected in subsequent cleavage-stage embryos which were associated with low cell numbers on Day 2, the upregulation of the RAD51 gene at the 4-8 cell stage (measured by RT-qPCR) and reduced developmental ability to the blastocyst stage when compared with the non-vitrified control. However, total cell numbers and percentages of apoptotic cells (measured by TUNEL) in resultant blastocysts were not different from those of the non-vitrified control. On the other hand, vitrification of zygotes had no effect on DSB levels and the expression of DNA-repair genes in resultant embryos, and their development did not differ from that of the non-vitrified control. These results indicate that during vitrification GV-stage oocytes are more susceptible to DNA damages than zygotes, which affects their subsequent development to the blastocyst stage.

Vitrification and warming can trigger premature meiosis in immature porcine oocytes. Our aim was to compare the efficacies of two meiotic inhibitors, dibutyryl-cAMP and roscovitine for the meiosis synchronization during in vitro maturation (IVM) of porcine oocytes vitrified at the germinal vesicle (GV) stage. We first compared the efficacy of 1 mM dibutyryl-cAMP and 25 μM roscovitine on meiotic arrest during the first 22 h of IVM. Dibutyryl-cAMP could maintain the GV stage in 83.5% of oocytes; however, roscovitine was even more effective (96.6%), whereas only 17.4% of the oocytes remained at the GV stage without these additives. Temporal meiotic arrest for 22 h by roscovitine did not reduce the percentage of oocytes reaching the Metaphase II stage during subsequent IVM. However, after parthenogenetic stimulation or in vitro fertilization, subsequent embryo development to the blastocyst stage was compromised after roscovitine treatment, whereas dibutyryl-cAMP improved the percentage of blastocyst development. In conclusion, dibutyryl-cAMP could derogate but not completely prevent premature meiosis in vitrified oocytes, whereas roscovitine could more efficiently prevent it. However, for embryo production, the use of roscovitine was disadvantageous, whereas the use of dibutyryl-cAMP was beneficial.

The present study was conducted to examine the feasibility of in vitro embryo production and transfer technologies for producing piglets of Agu, an Okinawan indigenous pig breed. After collection of oocytes from surgically dissected ovaries, they were subjected to in vitro maturation. After in vitro maturation/fertilization, a total of 616 putative embryos were transferred into four commercial Western pig recipients, one of which became pregnant and farrowed a total of eight Agu piglets. These results demonstrate that in vitro embryo production using ovaries from Agu females is useful for breeding management and conservation of indigenous breeds.

This study was conducted to examine whether the nuclear to cytoplasmic (N/C) ratio had any influence on the timing of embryo compaction and blastocoel formation, as well as formation rate and quality of blastocyst. First, we produced embryos with increased N/C ratio by removal of approximately one-third of the cytoplasm and with decreased N/C ratio by doubling the oocyte cytoplasm with an enucleated oocyte. The initiation of compaction and cavitation in reduced cytoplasm group was significantly earlier (P < 0.05) compared with the control and doubled cytoplasm groups. The rate of blastocysts in the reduced cytoplasm and doubled cytoplasm groups was significantly lower (P < 0.05) compared with the control group. Blastocyst quality in terms of total cell number in the reduced cytoplasm group was significantly lower (P < 0.05) compared with the doubled cytoplasm group, but not different from the control group. Next, we produced embryos with various N/C ratios by oocyte fusion combined with cytochalasin D treatment. The onset of compaction and cavitation in the 2N/2C group (decreased N/C ratio) was significantly delayed (P < 0.05) or had the tendency to be delayed (P = 0.064), respectively, compared with the control group (2N/1C). A significantly higher rate of blastocyst was observed in the 4N/2C group compared with the 1N/1C group (P < 0.05) but not different from the remaining groups. These results demonstrated that an increase in N/C ratio caused an earlier occurrence of morula compaction and blastocyst formation in both in vitro fertilization (IVF) and parthenogenetically activated pig embryos.

Male pronucleus (MPN) formation is a very important physiological event during fertilization, which affects in vitro production of transferrable embryos. The aim of this study was to find out the correlation between the number of penetrated sperm and the occurrence of failure of MPN formation in porcine oocytes. In vitro matured porcine oocytes were fertilized in vitro with frozen epididymal sperm. Two different frozen sperm lots were tested in this study, which were different in terms of polyspermy rates. The numbers and the status of penetrated sperm in oocytes were evaluated 10 h after insemination. Under high polyspermy condition, the polyspermy rate was 83.5% with an average mean of 3.5 sperms per penetrated oocyte, whereas the percentage of polyspermy was 65.5% with an average mean of 2.4 sperms per penetrated oocyte under moderate polyspermic condition. Correlation analysis revealed a negative correlation between the number of penetrated sperm and their MPN formation percentage both in the sperm lot of high polyspermy (R = -0.560, p < 0.05) and in the sperm lot of moderate polyspermy (R = -0.405, p < 0.05) which suggests that penetration of excessive spermatozoa disables the oocyte cytoplasm to promote MPN formation.

Reproduction, Fertility and Development is an international journal publishing original research , review and comment in the fields of reproduction and developmental biology in humans, domestic animals and wildlife

Previous research revealed that vitrification at the immature (the germinal vesicle, GV) stage triggers premature meiotic resumption in cumulus-enclosed porcine oocytes and causes a damage in gap junctions (Appeltant et al. 2017 Reprod. Fertil. Dev. 29, 2419-2429). However, the correlation between the two phenomena was not investigated yet. The present research was conducted to clarify whether premature meiotic resumption is caused by gap junction disruption and to assess the importance of cumulus cells for the survival of porcine oocytes vitrified at the GV stage. Cumulus–oocyte complexes (COCs) were collected from 3- to 6-mm antral follicles of slaughtered gilts. Immediately after collection, approximately half of them were denuded mechanically (DOs). In each replicate, groups of COCs and DOs were processed without vitrification (control groups). Treatment groups of COCs and DOs were vitrified on Cryotop sheets in a combination of 17.5% propylene glycol and 17.5% ethylene glycol and warmed in 0.4M sucrose. The oocytes were then cultured for 22h in a chemically defined porcine oocyte medium (POM) supplemented with 10ngmL−1 epidermal growth factor, 10IUmL−1 equine chorionic gonadotrophin, 10IUmL−1 human chorionic gonadotrophin, and 1mM dibutyryl cAMP. After culture, COCs were denuded and oocyte survival was assessed by morphological evaluation of membrane integrity under a stereo microscope. Then, live oocytes were fixed and stained with 1% orcein and nuclear status was evaluated under a phase-contrast microscope. The experiment was replicated 5 times. Data were analysed by ANOVA followed by Tukey’s multiple comparisons test. After vitrification and culture, the survival rate in the COC group was higher (P<0.05) than that of the DO group (160/191=84.7±3.4% vs. 153/237=65.0±6.2%, respectively) but reduced (P<0.05) compared with those in the control COC and DO groups (138/143=96.6±1.0% and 152/153=99.3±0.6%, respectively). The majority of the control COCs and DOs were at the GV stage with similar percentages (95.6±2.2% and 94.0±2.2%, respectively). In contrast, the percentages of oocytes at the GV stage in the vitrified COC and DO groups were reduced (71.6±9.4% and 45.7±10.5%, respectively; P<0.05) compared with the control groups, which were associated with increased frequencies of diakinesis and MI stages. Percentages of oocytes at the GV stage in the vitrified COC and DO groups were not significantly different (P=0.23). In conclusion, cumulus cells can prevent vitrification-related membrane damage of oocytes. Furthermore, vitrification induced premature meiosis both in the cumulus-enclosed and denuded oocytes even in the presence of the meiotic inhibitor, dibutyryl cAMP. Nevertheless, cumulus removal without vitrification did not induce premature meiosis in the oocytes. Therefore, disruption in communication with cumulus cells might not be the primary reason for premature meiosis in vitrified oocytes.

Background Male infertility may be due in part to autoimmune orchitis. Experimental models of autoimmune orchitis have yielded valuable information for understanding the underlying pathogenesis. However, previous rodent models show progressive inflammatory cell infiltration, thus differing from human cases. Objective We have established an immunization procedure that induces defective spermatogenesis in rats. Here, we examined the affected testes to clarify the pathogenetic mechanism responsible. Materials and methods Male rats received two subcutaneous injections of spermatozoa in LPS-supplemented oil-based adjuvant. The testes were collected when atrophy was detected by palpation in order to explore the acute phase of the defect. The blood-testis barrier (BTB) permeability was examined in a biotin tracer experiment. Testes were examined immunohistochemically using antibodies against TNF alpha(+) and ED1 (a macrophage marker) to detect inflammatory cells. Antibodies against proteins related to tight and gap junctions, desmosomes, and the Sertoli cell cytoskeleton were also employed. The expression of junction-related molecules and vimentin was also examined by Western blotting and RT-PCR. Results Immunohistochemistry revealed TNF alpha(+) and ED1(+) cells scattered in the interstitium. TNF alpha(+) cells, but not ED1(+) macrophages, were associated with seminiferous tubules in the affected testis. The tracer experiment demonstrated dysfunction of the BTB. Immunohistochemistry revealed delocalization of claudin 11 and tight junction protein 1. Positive immunoreactivity for connexin 43 (a major gap junction protein) and desmoglein 2 (a major desmosome protein) was reduced, probably resulting in germ cell exfoliation. F-actin and vimentin showed aberrant distribution in the seminiferous epithelium, and expression of vimentin was upregulated in the affected testis. Conclusion The observed pathological characteristics demonstrate how inflammatory cells influence Sertoli cells, leading to impaired spermatogenesis, and suggest that this sperm immunization method provides a new rat model of autoimmune orchitis.

The discovery of how to utilize CRISPR (clustered, regularly interspaced, short, palindromic repeats)-Cas (CRISPR-associated) systems for genome modification has accelerated development of the field of genome editing, especially in large animals such as pigs. The low efficiency of somatic cell nuclear transfer (SCNT) is now becoming a major obstacle in the production of genome-edited animals via cell-mediated approaches and improving efficacy of this technique is crucial. In this study, we propose a few simple modifications to a zona-free SCNT protocol that are effective to produce numerous high-quality blastocysts. To refine the SCNT protocol we modified the following steps/factors: 1) culture medium for SCNT embryos, 2) chemical treatment to prevent precocious activation of the manipulated/reconstructed oocytes and 3) donor cell serum starvation treatment. Although changes in each of these steps only resulted in small improvements, the combination of all modifications altogether significantly enhanced developmental competence of SCNT embryos. Our modified method yielded approximately three times greater blastocyst formation rates. Moreover, resulting blastocysts had roughly twice as many cells as compared to blastocysts produced by the conventional SCNT method. With these significant in vitro improvements, our refined SCNT method is potentially suited for use in the production of genome edited pigs.

In the present study using pig cells, we examined the effect of the cryoprotectant trehalose on the DNA integrity of freeze-dried cells. We then investigated whether donor cell types and storage duration had impact on DNA integrity in freeze-dried cells or developmental competence of oocytes injected with freeze-dried somatic cells. We also examined whether double cytoplasm nuclear transfer (DCNT) would improve developmental competence of such oocytes. Furthermore, using a PCR-based method for sex identification, we determined whether the blastocysts obtained had actually been generated from the freeze-dried cells. It was found that, for a short storage duration at low temperature, trehalose had no beneficial effect on protection from DNA damage, and that donor cell type had no effect on the DNA integrity of freeze-dried somatic cells or the developmental competence of oocytes injected with them. We also confirmed that all of the blastocysts obtained following nuclear transfer were of freeze-dried somatic cell origin. Storage of freeze-dried somatic cells for up to 1 year at low temperature did not degrade DNA integrity in comparison with storage for 1 month, 1 week or 1 day. Following injection of freeze-dried cells, the proportion of oocytes that developed to blastocysts after storage for up to 1 year was similar to that after storage for 1 month, 1 week or 1 day. Moreover, DCNT significantly improved the developmental competence of oocytes treated in this way. In summary, using DCNT, we have demonstrated that freeze-dried porcine somatic cells subjected to long-term storage at 4 °C have nearly the same potential to develop to blastocysts as non-freeze-dried cells.

We examined the allelic expression and positioning of two pluripotency-associated genes, OCT4 and SOX2, and two housekeeping genes, ACTB and TUBA, in 4- and 8-cell porcine embryos utilizing RNA and DNA fluorescence in situ hybridization (FISH) in single blastomeres. The proportion of blastomeres expressing SOX2 bi-allelically increased from 45% at the 4-cell stage to 60% at the 8-cell stage. Moreover, in 8-cell embryos, SOX2 was expressed bi-allelically in significantly more blastomeres than was the case for OCT4, and this was associated with a tendency for SOX2 alleles to move toward the nuclear interior during 4- to 8-cell transition. However, the radial location of OCT4 alleles did not change significantly during this transition. The locations of active and inactive alleles based on DNA and RNA FISH signals were also calculated. Inactive OCT4 alleles were located in very close proximity to the nuclear membrane, whereas active OCT4 alleles were more centrally disposed in the nucleus. Nevertheless, the nuclear location of active and inactive SOX2 alleles did not change in either 4- or 8-cell blastomeres. Our RNA and DNA FISH data provide novel information on the allelic expression patterns and positioning of pluripotency-associated genes, OCT4 and SOX2, during embryonic genome activation in pigs.

The Agu is the only indigenous pig breed in Japan but its population is very small. In order to estimate the efficacy of testicular xenografting for the conservation of Agu pigs, we investigated whether neonatal testicular fragments would acquire the capacity to produce sperm after they had been cryopreserved and grafted into nude mice. Although on day 180 (day 0 = xenografting), grafts showed a low proportion of seminiferous tubule cross-sections containing sperm (0.1 ± 0.1%, mean ±  SEM for four mice), the proportion reached 36.9 ± 16.7% ( n  = 4 mice) by day 240. When single sperm obtained on day 240 was injected into individual porcine oocytes, 28.2% of the oocytes were found to contain one male and one female pronuclei with the second polar body. Moreover, the blastocyst formation rate after injection of the xenogeneic sperm was 28.4%, whereas that in the absence of sperm injection (attributable to parthenogenesis) was 13.3%. These findings suggest that more than half of the blastocysts resulted from fertilization. Thus, testicular xenografting could assist the conservation of Agu pigs by salvaging germ cells present in neonatal testes even after cryopreservation.

Testicular xenografting, combined with cryopreservation can assist conservation of the genetic diversity of indigenous pigs by salvaging germ cells from their neonatal testes. Using Meishan male piglets as an example, we examined whether testicular tissue would acquire the ability to produce sperm after cryopreservation and grafting into nude mice (MS group). For comparison, testicular tissue from neonatal Western crossbreed male piglets was used (WC group). Sixty days after xenografting (day 0 = grafting), MS grafts had already developed seminiferous tubules containing sperm, whereas in the WC grafts, sperm first appeared on day 120. The proportion of tubules containing spermatids and sperm was higher in the MS group than in the WC group between days 90 and 120. Moreover, in vitro-matured porcine oocytes injected with a single sperm obtained from the MS group on day 180 developed to the blastocyst stage. The blastocyst formation rate after injection of the xenogeneic sperm was 14.6%, whereas the ratio in the absence of such injection (attributable to parthenogenesis) was 6.7%. Thus, cryopreserved Meishan testicular tissue acquired spermatogenic activity in host mice 60 days earlier than Western crossbreed tissue. Such xenogeneic sperm are likely capable of generating blastocysts in vitro.

The aim of the present study was to clarify whether or not our vitrification procedure at the germinal vesicle (GV)-stage triggers the apoptotic cascade in oocytes and subsequent embryos. Immature porcine cumulus-oocyte complexes were either vitrified and warmed (vitrified group) or subjected to cryoprotectant agents (CPA group) or cultured without any treatment (control). Oocytes of all treatment groups were subjected to in vitro maturation (IVM), fertilization, and embryo culture. Apoptosis was assayed in live oocytes at the end of IVM culture and in cleavage-stage embryos after in vitro fertilization (IVF). We detected similar frequencies of DNA fragmentation, levels of caspase activity, phosphatidylserine externalization, and mRNA levels for pro-apoptotic Bax and CASP3 genes in oocytes at the end of IVM and in early embryos among all groups. However, in the vitrified group, the anti-apoptotic Bcl-XL gene was upregulated in 4–8 cell embryos, which caused an 8-fold significant increase in the Bcl-XL/Bax mRNA ratio compared with the control and CPA groups (P < 0.05). In conclusion, vitrification of porcine oocytes at the GV stage by our method did not trigger the apoptotic cascade in oocytes and subsequent embryos but triggered the upregulation of the anti-apoptotic Bcl-XL gene in embryos.

The aim of this study was to examine whether a morphological approach is efficient for selecting high-quality porcine embryos produced by in vitro fertilization (IVF) under high polyspermy conditions. Frozen-thawed Meishan epididymal spermatozoa showing moderate and high polyspermy were subjected to IVF (1 × 105  sperms/ml). Under conditions of moderate polyspermy, 4-cell embryos selected at 48 hr after IVF (single selection) and 8-cell embryos selected at 79 hr after IVF from the collected 4-cell embryos (double selection) showed high developmental competence. Likewise, 4- and 8-cell embryos produced by IVF under high polyspermy conditions also showed high competence for development to blastocysts. However, blastocysts derived from high polyspermy conditions had significantly fewer cells than those produced under moderate polyspermy conditions. Furthermore, the frequency of nuclear and chromosomal abnormalities in 4- and 8-cell embryos produced under conditions of high polyspermy was significantly (p < .05) higher in comparison to moderate polyspermy conditions. These findings suggest that although high polyspermy affects the frequency of nuclear and chromosomal anomalies in porcine IVF embryos, subsequent selection based on morphological features of 4- and 8-cell embryos even under high polyspermy conditions, could be an alternative option for selecting porcine IVF embryos with high development ability.

Ovarian xenografting makes it possible to obtain oocytes with fertilization ability from immature pigs of Western breeds. In this study, we applied these methods to the Meishan, an indigenous Chinese pig breed, and investigated the developmental competence of oocytes grown in their neonatal tissue after grafting into nude mice. First, mice harboring neonatal ovarian tissue were infused with follicle stimulating hormone (FSH) (62.5 U/ml) for 13 days starting at 10, 30, and 60 days after vaginal opening (D10-, D30-, and D60-FSH groups, respectively). Development of antral follicles and their oocytes was most enhanced in the D60-FSH group. For the next step, we examined the in vitro maturation ability of the oocytes recovered from host mice after infusion with FSH at a dose of 62.5 U/ml or 125 U/ml (FSH-62.5 or -125 group) for 13 days starting at 60 days after vaginal opening. Many more oocytes with maturation ability were obtained from the FSH-125 group. The FSH-125 mature oocytes were fertilized in vitro, as shown by formation of male and female pronuclei, but did not reach the blastocyst stage. These results indicate that Meishan neonatal ovaries are able to produce oocytes with fertilization ability after being grafted into nude mice.

Previous studies reported the activation of the apoptotic cascade by vitrification in mature porcine oocytes (Vallorani et al. 2012 Anim. Reprod. Sci. 135, 68-74) and that the cathepsin B inhibitor E-64 improved developmental competence of bovine oocytes via an antiapoptotic effect (Balboula et al. 2013 Reproduction 146, 407-417). The present study was carried out to test whether E-64 affected the developmental competency of porcine oocytes vitrified at the germinal vesicle stage. Cumulus-enclosed porcine oocytes were vitrified in microdrops and warmed by our method (Somfai et al. 2015 J. Reprod. Dev. 61, 571-579). Then, the oocytes were subjected to in vitro maturation (IVM) for 46h in a chemically defined porcine oocyte medium supplemented with 10ng mL−1 of epidermal growth factor, 10IU mL−1 of eCG, and 10IU mL−1 of hCG and during the first 22h of IVM with 1mM dibutyryl cyclic adenosine monophosphate. Then, cumulus-oocyte complexes were fertilized in vitro and presumptive zygotes were cultured in 50-µL drops of porcine zygote medium-3 for 7 days in 6-well dishes covered by paraffin oil in an atmosphere of 5% CO2, 5% O2, and 90% N2 at 39°C. On Day 5 (Day 0=IVF), the porcine zygote medium-3 was supplemented with 10% (vol/vol) FCS. The effects of 1.0μM of E-64 supplementation during IVM of non-vitrified and vitrified cumulus-oocyte complexes were investigated in a 2×2 factorial design. Survival rates after IVM, cleavage rates on Day 2, blastocyst rates, and total cell numbers in blastocysts on Day 7 were compared among groups. The experiment was replicated 5 times. Results were analysed by ANOVA and Tukey’s multiple comparison test. The percentages of live oocytes were statistically similar when oocytes were matured in the absence or presence of E-64 both in non-vitrified (99.2% v. 99.6%, respectively) and vitrified (94.3% v. 90.8%, respectively) groups. Similarly, IVM without or with E-64 supplementation had no effect on subsequent cleavage and blastocyst development rates in non-vitrified (67.4% v. 71.2% and 38.7% v. 43.2%, respectively) and vitrified (46.8% v. 48.8% and 14.6% v. 22.8%, respectively) oocytes. Irrespective of E-64 treatment, all survival and developmental rates in the vitrified groups were significantly lower (P<0.05) compared with those of their non-vitrified counterparts except for the blastocyst development rate in the E-64-treated vitrified group, which did not differ significantly from those of the non-vitrified groups with or without E-64 treatment. There was no statistical difference in mean blastocyst cell numbers among the groups, ranging between 86.5±15.8 and 118±10.6. In conclusion, E-64 treatment had no effect on embryo production rates, which suggests that in our system, cathepsin-mediated apoptosis during IVM might not be the factor to limit embryo production using either fresh oocytes or those vitrified at the immature stage. This work was supported by JST/JICA SATREPS.

Cryopreservation of female germplasm has basic importance in preservation and distribution of genetic lines in farm animals. Although vitrification technology has been applied for the cryopreservation of porcine oocytes and ovarian tissues, reduced developmental competence of preserved oocytes and the lack of offspring produced from them underlines the importance for further developments in cryopreservation protocols for this purpose. This review discusses the problems of female germplasm cryopreservation in pigs and the possible strategies to overcome them and gives an update on the present status of cryopreservation of porcine oocytes and ovarian tissues.

Porcine immature oocytes can survive vitrification at high rates and retain their ability to undergo maturation and fertilization; however, the procedure reduces their competence for subsequent embryo development via unknown mechanisms (Somfai et al. 2014 Plos One 9, e97731). The aim of the present study was to clarify whether our vitrification procedure at the germinal vesicle stage triggers apoptosis in oocytes and subsequent developing embryos. Immature porcine cumulus-oocyte complexes obtained from slaughterhouse-derived ovaries were vitrified and warmed by our method (Appeltant et al. 2018 Cryobiology 85, 87-94) immediately after collection (vitrified group). The oocytes were equilibrated in 2% (vol/vol) ethylene glycol and 2% (vol/vol) propylene glycol for 13-15min. Then, they were vitrified by dropping them into liquid nitrogen in 2-μL microdrops of a medium composed of 17.5% ethylene glycol, 17.5% propylene glycol, 0.3M sucrose, and 50mgmL−1 polyvinylpyrrolidone. After warming, they were subjected to IVM, fertilization (IVF), and embryo culture using chemically defined media (Yoshioka et al. 2008 J. Reprod. Dev. 54, 208-213). From each collected batch, a group of oocytes was processed without vitrification (control group). Apoptosis was assayed in membrane-intact oocytes at the end of IVM and in cleavage-stage embryos on Day 2 after IVF (Day 0) by the CaspACE FITC-VAD-FMK In Situ Caspase Marker (Promega; Experiment 1), deoxynucleotidyl transferase (TdT) dUTP nick-end labelling (TUNEL; Experiment 2), and analysis of mRNA levels by RT-qPCR for the pro-apoptotic Bax and CASP3 genes (Experiment 3). Each experiment was replicated three times. Data were analysed by Kruskal-Wallis test followed by Dunn's multiple comparisons test. The mean survival rate of vitrified oocytes was 89.2%. There was no significant difference between the control and vitrified groups in relative caspase levels in IVM oocytes and in 2- to 4-cell embryos after IVF; however, significantly increased caspase activity (P<0.05) was detected in oocytes and embryos after treatment with 10 μM staurosporine (positive control). There was no significant difference between the control and vitrified groups in the proportion of TUNEL-positive oocytes (4.1 and 0.8%, respectively) and embryos (0 and 0%, respectively), whereas 96.6% of oocytes and 100% of cleavage stage embryos treated with 1000IUmL−1 deoxyribonuclease I (positive control) were proven to be TUNEL positive (P<0.05). Similar mRNA levels for Bax and CASP3 genes were detected in oocytes at the end of IVM and subsequent developing 4- to 8-cell embryos between the control and vitrified groups. In conclusion, vitrification of porcine oocytes at the germinal vesicle stage by our method did not trigger apoptosis in oocytes and subsequent developing embryos. This work was supported by the Japan Science and Technology Agency (JST)/Japan International Cooperation Agency (JICA) Science and Technology Research Partnership for Sustainable Development (SATREPS).

We tested the effects of resveratrol both as a pre-treatment and as a recovery treatment after warming during in vitro maturation (IVM) on the viability and developmental competence of porcine oocytes vitrified at the germinal vesicle stage. Pre-treatment before vitrification of oocytes for 3 hr with 2 μM resveratrol did not affect survival, oocyte maturation and embryo developmental competence to the blastocyst stage after parthenogenetic activation. However, supplementation of the medium with resveratrol during subsequent IVM after vitrification and warming significantly improved the ability of surviving oocytes to develop to the blastocyst stage, and this effect was observed only on vitrified, but not on non-vitrified oocytes. The intracellular levels of glutathione and hydrogen peroxide in oocytes were not affected by vitrification and resveratrol treatment. Also, there was no significant difference in the occurrence of apoptosis measured by annexin V binding between vitrified and non-vitrified oocytes, regardless of the resveratrol treatment. In conclusion, resveratrol did not prevent the cellular damages in immature porcine oocytes during vitrification; however, when added to the IVM medium, it specifically improved the developmental competence of vitrified oocytes. Further research will be necessary to clarify the mechanisms of action of resveratrol on the recovery of vitrified oocytes from vitrification-related damages.