Successful in-vitro fertilization of frozen—thawed rabbit oocytes

The objective of this study was to evaluate the capacity of frozen-thawed rabbit oocytes to be fertilized in vitro. After superovulation with FSH a total of 1040 oocytes was obtained by puncturing the follicles 6 or 9 h after the injection of LH or by flushing the oviducts 12 h after LH application. 1.5 M dimethylsulphoxide (DMSO) was used as cryoprotective agent and the oocytes were transferred into 0.25 ml French straws and cooled in a methanol bath to -30°C and transferred to liquid nitrogen. After 1–14 days of storage the oocytes were thawed rapidly in a 15°C water bath and DMSO was diluted in a stepwise manner. Subsequently the oocytes were cultured in Ham's F-10 + 10% fetal calf serum at 37°C and 5% CO2 for 14, 7 and 4 h according to the time of oocyte collection. The survival rates of the oocytes based on morphological criteria were 5.4, 20.0 and 28.8%, respectively. For chromosomal analysis, morphologically intact frozen-thawed oocytes were fixed and stained using the technique described by Tarkowski. In 44 successful chromosomal preparations, 2 of 2, 10 of 19 and 22 of 23 preparations of oocytes collected 6, 9 and 12 h after LH application were in metaphase-II, respectively. Furthermore, the viability of the oocytes was also examined by using fluores-cein diacetate. Out of 52 morphologically intact oocytes, 50 showed a positive intracellular fluorescence. Finally 30 intact frozen-thawed oocytes were incubated with in-vivo capacitated spermatozoa for 5 h in Brackett's defined medium, transferred to Ham's F-10 medium and examined 20 h later for cleavage. Eighteen out of 30 oocytes (60%) divided into 2- or 4-cell stages. However, only one embryo (5.5%) was able to reach the blastocyst stage after 5 days in vitro.