The purpose of this study was to examine whether freeze-dried germinal vesicles (GV) can be matured in vitro after being injected into enucleated fresh oocytes in pigs as an alternative method for conservation of genetic resources. Although no reduction of the size of GV (p = .094), resveratrol treatment significantly enhanced the survival rates following GV transfer (GVT) (p < .001). Supplementation with 100 or 200 mmol/L trehalose in freeze-drying medium significantly increased the proportions of GVs with intact nuclear membrane and DNA integrity compared with the control group. Following transfer of freeze-dried GVs into enucleated fresh oocytes, the proportion of reconstructed oocytes reached the metaphase-II stage (2.4% ± 1.4%) was significantly lower (p < .05) than that of the in vitro matured control group (83.2% ± 2.5%), it was comparable with the GVT control group (7.4% ± 2.7%). The rates of freeze-dried GVs with intact nuclear membrane and DNA stored at -20°C for 5 days were significantly higher (p < .05) than those at 4°C and room temperature. The rates of intact nuclear membrane and DNA in the freeze-dried GV stored for 15 or 30 days at -20, 4°C and RT were not significantly different. In conclusion, matured oocytes were produced derived from freeze-dried GVs.

28The aim of the present study was to clarify whether or not our vitrification procedure at the 29 germinal vesicle (GV)-stage triggers the apoptotic cascade in oocytes and subsequent embryos. 30 Immature porcine cumulus-oocyte complexes were either vitrified and warmed (vitrified group) 31 or subjected to cryoprotectant agents (CPA group) or cultured without any treatment (control). 32 Oocytes of all treatment groups were subjected to in vitro maturation (IVM), fertilization, and 33 embryo culture. Apoptosis was assayed in live oocytes at the end of IVM culture and in 34 cleavage-stage embryos after in vitro fertilization (IVF). We detected similar frequencies of 35 DNA fragmentation, levels of caspase activity, phosphatidylserine externalization, and mRNA 36 levels for pro-apoptotic Bax and CASP3 genes in oocytes at the end of IVM and in early 37 embryos among all groups. However, in the vitrified group, the anti-apoptotic Bcl-XL gene was 38 upregulated in 4-8 cell embryos, which caused an 8-fold significant increase in the Bcl-XL/Bax 39 mRNA ratio compared with the control and CPA groups (P < 0.05). In conclusion, vitrification 40 of porcine oocytes at the GV stage by our method did not trigger the apoptotic cascade in 41 oocytes and subsequent embryos but triggered the upregulation of the anti-apoptotic Bcl-XL 42 gene in embryos. 43 44 Key words: apoptosis, immature oocyte, porcine, vitrification 45 46 (IVEP) or cloning. Although porcine oocytes are extremely sensitive to low temperatures and 52 cryopreservation procedures [2], they can be preserved by vitrification; however, the production 53 of offspring by this approach was reported only recently [3, 4]. Despite relatively high survival 54rates, the competence of porcine oocytes to develop to blastocyst stage embryos is greatly 55 compromised by the vitrification process applied either at the mature metaphase -II (MII) stage 56[5] or at the immature germinal vesicle (GV) stage [3]. 57In pigs, perhaps uniquely in farm animals, vitrification at the GV stage seems to be 58 more advantageous than that at the MII stage [6]. Therefore, in a series of studies, we developed 59 a vitrification protocol for immature porcine oocytes [3,[7][8][9][10]. Porcine oocytes survive this 60 procedure at high rates without major reduction in their ability to resume and complete the 61 meiotic process during in vitro maturation (IVM) [11]. However, although live offspring could 62 be obtained by in vitro fertilization (IVF) of such oocytes, embryonic developmental 63 competence of vitrified oocytes remained lower than that of non-vitrified ones [3, 10]. The most 64 notorious manifestation of detrimental effects of oocyte vitrification/warming at the GV stage 65 were reduced cleavage rates and compromised ability of cleaved embryos to reach the 66 blastocyst stage [3, 10]. The exact reason for this phenomenon has not been clarified thus far. 67In previous studies, vitrification at the MII stage reportedly triggered the apoptotic 68 cascade in porcine oocytes, which is...

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&lt;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&lt;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).