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Whole ovaries, and the follicles and blood vessels they contain, are able to survive cryopreservation. In addition, MRI has shown that blood vessels were intact and that normal blood flow had resumed to the transplant. We conclude that immediate and long-term hormonal restoration and normal ovulation is possible after cryopreservation and transplantation of whole ovaries in sheep.

Zygotes show a higher resistance to chilling injury compared to oocytes at different developmental stages; this might explain the relatively poor survival rates of cryopreserved human oocytes and indicates the necessity to adjust the cryopreservation protocols in order to minimize cryoinjury.

We examined the association between season and expression of genes involved in early embryonic development with an emphasis on cleavage rate and timing of the first embryonic cleavage. In Exp. 1, oocytes were aspirated during the cold (Dec-Apr) and hot (May-Nov) seasons. Matured oocytes were chemically activated and cultured in vitro. The developmental peak to the two-and four-cell stages occurred earlier, with a higher proportion of first-cleaved embryos, during the cold season relative to the hot season (P!0.01). In Exp. 2, a time-lapse system was employed to characterize the delayed cleavage noted for the hot season. Cleavage to the two-cell stage occurred in two distinct waves: early cleavage occurred between 18 and 25 h post activation, and late cleavage occurred between 27 and 40 h post activation. In Exp. 3, oocytes were aspirated during the cold and hot seasons, matured in vitro, fertilized, and cultured for 8 days. In each season, early-and late-cleaved two-cell stage embryos were collected. Total RNA was isolated, and semi-quantitative and real-time PCRs were carried out with primers for GDF9, POU5F1, and GAPDH using 18S rRNA as the reference gene. In both seasons, the expression of all examined genes was higher (P!0.05) in early-versus late-cleaved embryos. POU5F1 expression was higher (P!0.05) in early-cleaved embryos developed in the cold season versus the hot season counterparts. The findings suggest a deleterious seasonal effect on oocyte developmental competence with delayed cleavage and variation in gene expression.

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This method enables maintenance of high cooling rates as well as reduction of cryoprotectant concentration, despite the use of a sealed container that helps to reduce the potential risk of contamination.