Effect of cysteamine supplementation of in vitro matured bovine oocytes on chilling sensitivity and development of embryos

In vitro techniques for production of bovine embryos including in vitro oocyte maturation (IVM), fertilization (IVF) and culture (IVC) are becoming increasingly employed for a variety of research purposes. However, decreased viability following cryopreservation by conventional methods has limited commercial applications of these technologies. A practical alternative to facilitate transport would be to arrest development by chilling without freezing. The present research was undertaken to evaluate chilling sensitivity of IVM-IVF embryos at different stages of development, and to determine possible beneficial effects of cysteamine treatment during IVM, previously shown to enhance embryo development in culture, on survival following chilling at different stages. Embryos produced by standard IVM-IVF-IVC methods were chilled to 0 degrees C for 30 min at 2-cell (30-34 h post-insemination, hpi), 8-cell (48-52 hpi) or blastocyst (166-170 hpi) stages. Viability after chilling was assessed by IVC with development to expanded blastocyst stage determined on days 7 and 8 post-insemination (pi) and hatching blastocyst stage determined on days 9 and 10 pi. Control embryos at the same stages were handled similarly, but without chilling, and development during culture similarly assessed. The effect of cysteamine supplementation (100 microM) of the IVM medium was determined for both chilled and non-chilled (control) embryos. Cysteamine supplementation during IVM had no significant effect on oocyte maturation or fertilization, but increased the proportions of oocytes developing to blastocyst stage by day 7 (13.7+/-0.9% versus 7.2+/-0.9%; P<0.05), total blastocysts (20.5+/-0.9% versus 15.3+/-1.3%; P<0.05), and hatching blastocysts (16.8+/-1.6% versus 12.0+/-1.5%; P<0.05). The greater survival in terms of hatching (78.6+/-7.0) following chilling of blastocysts produced by IVM-IVF of oocytes matured in media supplemented with cysteamine offers promise for applications requiring short-term storage to facilitate transport of in vitro produced bovine embryos.