94 EFFECT OF OVARIAN SIZE ON THE VIABILITY OF CRYOPRESERVED SYRIAN HAMSTER OVARIES

Abstract

Ovaries can be used in place of gametes as a resource for cryopreservation. Thus far, we have shown that in the Syrian hamster, pups can be successfully produced through the transplantation of fresh and cryopreserved ovaries. In this study, we attempted to simplify the ovarian cryopreservation procedures to facilitate the easy application of these techniques. In particular, the procedure would be easier and quicker if the ovaries did not have to be cut into small pieces before ovarian cryopreservation. Therefore, we examined the effects of ovarian fragment size on subsequent offspring production. Ovaries were cryopreserved by vitrification according to methods described for mouse ovaries (Migishima et al. 2003 Biol. Reprod. 68, 881–887). In brief, ovaries were collected from 3-week-old J2N-n strain (white coat) females. Because we had previously confirmed that quarter-sized ovaries could be used for cryopreservation, intact (∼3.5 × 2 mm) and half-sliced ovaries (∼2 × 1.5 mm) were cryopreserved by vitrification using DAP213 solution (2 M dimethyl sulfoxide, 1 M acetamide, and 3 M propylene glycol; Nakagata 1989 J. Reprod. Fertil. 87, 479–483). Thawed ovaries were transferred into the ovarian bursae (single intact ovary or two half-sliced ovarian pieces per ovarian bursa) of 3-week-old recipient females with agouti-coat (Slc:Syrian) under isoflurane anaesthesia. Not all of the recipients’ ovaries were removed immediately before transfer; both the donor’s and a small portion of the recipient’s ovaries were present in each ovarian bursa. Successful ovarian transplantation was verified by examining the coat colour (non-agouti colour) of offspring produced in test matings between mature recipients and J2N-n males. Of the 3 and 6 recipients that received intact and sliced ovaries, respectively, 2 and 5 recipients delivered pups. Based on the coat colour of the pups, 1 of the 5 recipients delivered a pup produced from transplanted half-sliced ovaries. Our results indicate that half-sliced-size ovaries (smaller than a 2-mm cube) can be successfully used for cryopreservation in the Syrian hamster. Specifically, it is not required to slice the ovary into smaller pieces.