Successful cryopreservation of buffalo ovaries using in situ oocyte cryopreservation

To improve the efficiency and efficacy of cryopreservation of ovaries, we developed a new method termed in situ oocyte (ISO) cryopreservation. ISO cryopreservation is a multistep procedure that involves aspiration of follicular fluid and then perfusion of antral follicles and diffusion of whole buffalo ovaries with cryoprotectant agent (CPA), rapid cooling, storage, thawing and, finally, dilution and removal of the CPA with return to physiological environment. Our study compared ISO cryo ovaries with cryo-diffused ovaries. We systematically examined the effects of ISO cryo and diffuse cryo on ovaries by morphological examination and with viability tests. The percentages of morphologically normal and viable follicular oocytes from ISO cryo were significantly higher than those that resulted from the cryo-diffused method (p<0.01). The quality of follicular oocytes from 150 cryo ovaries appeared better than that achieved from cryo-diffused ovaries. In conclusion, this study shows that ISO cryo is highly efficient for cryopreservation of oocytes and ovarian tissue.