Submission Number: BAR-4-5-8

Abstract Number: 99

CRYOPRESERVATION OF CANINE OOCYTES FROM PREANTRAL FOLLICLES.

Mary Ann Olson, Autumn Anderson, Dianne Amodeo and Barbara Durrant*

Center for Reproduction of Endangered Species, Zoological Society of San Diego, San Diego, CA ¹

Abstract:
The reservoir of ovarian preantral follicles represents a largely untapped source of germplasm of great importance in the conservation of endangered species. Previous studies using the domestic dog as a model for exotic carnivores demonstrated the potential of ovarian oocytes for in vitro maturation and fertilization. Cryopreservation of ovarian follicles will contribute to the long-term preservation of genetic diversity; however, long-term storage of mammalian oocytes has not been perfected. This study was the first in a series of experiments to develop freezing techniques for carnivore ovarian oocytes. Healthy Labrador retrievers were 18-20 months of age and had experienced at least one estrous cycle at the time of ovary removal. Each ovary was halved, with one half assigned to each of two follicle preparation protocols: 1) TIS, tissue was minced into 1 mm cubes and slow-frozen at 0.3C/min to 40C in BWW with 1.5 M DMSO before storage in liquid nitrogen; 2) ISO, tissue was minced, then digested in collagenase/DNase for 1 hr at 37C, followed by follicle isolation from tissue and freezing as described above. Oocytes from control follicles were examined without freezing. Tissue and isolated oocytes were thawed for 1.5 and 2 min, respectively, in a 37C water bath and DMSO was removed stepwise over 30 min. Oocytes in the TIS group were isolated by digestion. All oocytes with normal morphology (dense, black, uniform cytoplasm and round shape) were removed from follicles, stripped of attendant granulosa cells and fixed in acetic acid:ethanol (1:3) for 24 hr before staining with aceto-orcein and examination at 400X with Nomarski optics. Integrity of the germinal vesicle was confirmed by an intact nuclear membrane containing dictyate chromatin. Retention of normal morphology and an intact germinal vesicle identified oocytes potentially competent to mature in vitro. Thawed ISO oocytes had 58% of the intact germinal vesicles exhibited by controls, whereas thawed TIS oocytes retained a mere 18% of control values. Clearly, removal of canine preantral follicles from ovarian tissue prior to cryopreservation, rather than after, results in a higher proportion of oocytes with the potential for in vitro maturation and fertilization.

Keywords: preantral follicles, oocytes, canine, germinal vesicle

This abstract is being presented at: 8:00 AM in session:
Cryopreservation