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Despite many advances in the field of oocyte cryopreservation, the adverse effects of cryopreservation on oocyte competence are still an open challenge in most mammalian species. Using ovine in vitro-matured oocytes, the differential effects of vitrification steps, embryo production methods, and parental origin of pronuclei were systemically investigated to unravel (1) the most critical stage (if any) of oocyte vitrification, (2) the most suitable method (if any) of embryo production for a vitrified oocyte, and (3) differential contributions of male or female pronuclear formation to the poor quality of vitrified oocyte. Although cryoprotectants used during vitrification had some inevitable adverse effects on oocyte competence, the damages caused by low temperature per se (chilling injury) were the main cause of poor quality of vitrified oocytes. When vitrified oocytes underwent either IVF or intracytoplasmic sperm injection (ICSI), embryo development rates were substantially lower than those of fresh ones. In contrast, when vitrified oocytes underwent either parthenogenetic activation (PA) or SCNT, embryo development rates were very similar to those of fresh ones. Evaluation of nuclear morphology after IVF, ICSI, PA, and SCNT oocytes revealed that vitrification had no apparent effect on the female (IVF, ICSI, and PA) and somatic (SCNT) pronuclear formation rates but significantly reduced male pronuclear formation after either IVF or ICSI compared with fresh counterparts. Quantitative analysis of transcripts revealed comparable mRNA abundances of CNX43, HSP90, GMNN, NPM, and OCT4 between vitrified and fresh oocytes, whereas CCNB, ATP1A1, and PAP transcripts were significantly lower in vitrified versus fresh oocytes. Although underlying mechanisms of poor quality of vitrified oocytes are multifactorial, the ability to obtain equivalent development after PA and SCNT, but not IVF and ICSI, in vitrified versus fresh oocytes may argue that the cytoplasm of vitrified oocyte has the necessary components to support in vitro embryonic development of the maternal, even adult somatic cell, chromosomes but fails to do so with sperm chromosomes., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The present study was carried out to investigate how the interactions between aging, vitrification and post-warming interval affect the credibility of sheep MII-oocytes for in vitro fertilization (IVF), intracytoplasmic injection (ICSI), and parthenogenetic activation (PA). According to our results, aged oocytes had significantly higher rates of chromosome and spindle abnormalities compared to young oocytes. However after vitrification-warming, the total rates of these abnormalities were not significantly different between aged and young oocytes. Unvitrified-aged, and vitrified young and aged oocytes had comparable ultrastructural characteristics, whereas they were completely dissimilar in compared with unvitrified-young oocytes. Although mRNA abundance was reduced during vitrification-warming in both aged and young oocytes, the post-warming interval could improve the relative mRNA abundance. Aged oocytes had lower capacity for IVF and ICSI in compared with young oocytes, but had similar pattern for PA process. The vitrification process decreased developmental competence of both aged and young oocytes in compared with young ones, particularly when warmed oocytes were rested for 2 h before IVF, ICSI and PA. The results of the present study showed that in vitro aged oocytes had higher capacity to be used for parthenogenetic studies rather than IVF and ICSI. Furthermore, it was shown that vitrified oocytes had a time-dependent decline in quality and developmental potential. Notably, the speed of this decline was higher in vitrified-young oocytes, indicating that the vitrified oocytes do not require to be rested post warming. Conclusively, the results of this study can be useful in preserving in vitro aged oocytes to provide a valuable and easy access source of oocytes for research purposed studies., (Copyright © 2012 Elsevier Inc. All rights reserved.)

It is a common practice to rest vitrified-warmed matured oocytes for 1-3 h, as a treatment to recover spindle and cytoskeleton, before commencing a further treatment. Vitrified-warmed matured oocytes, however, are very sensitive and may resume meiosis spontaneously during this recommended rest time. Therefore, the aim of this study was to assess spindle and chromosome status as well as developmental competence of vitrified in vitro matured sheep oocytes activated parthenogenetically, either 0 h (immediately) or 2 h (delayed) after warming. There was no significant effect of post-warming interval on the proportion of degenerated oocytes. Evaluation of chromosomes and meiotic spindle configuration showed that 11.11% of oocytes in the immediate group and 8.82% of oocytes in the delayed group had normal chromosomal alignment on well-structured spindles, compared to non-vitrified group (79.41%). Meanwhile, majority of the chromosomal abnormalities in the immediate and delayed groups were categorized as absent (unobservable) (77.78%) and anaphase II (70.59%), respectively. Oocytes in immediately activated group showed significantly higher blastocyst rate (28.86%) compared to delayed activated group (16.47%). In conclusion, the results suggest that post-warming interval may have important consequence on meiotic progression and parthenogenetic activation of vitrified oocytes. In sheep, it appears that chemical activation without having to await microtubule reorganization improves embryonic development., (Copyright © 2011 Elsevier Inc. All rights reserved.)