Cryopreservation and in vitro culture of white-tailed deer ovarian tissue

Abstract

The aims of this study were to evaluate (1) the survivability of white-tailed deer ovarian tissue after cryopreservation by slow-freezing (SF) and vitrification (VIT) techniques and in vitro culture (IVC) for up to 7 days, and (2) the effects of cryopreservation techniques on protein expression of proliferative and apoptotic markers of ovarian tissue pre- and post-in vitro culture. Ovaries (n = 14) of seven white-tailed deer fawns (<1.5 years old) were used. Ovarian cortexes were cut into fragments (2 × 2 × 0.5 mm) and split into nine treatment groups: (1) fresh noncultured control, (2) fresh-IVC 1 day, (3) fresh-IVC 7 days, (4) SF noncultured, (5) SF-IVC 1 day, (6) SF-IVC 7 days, (7) VIT noncultured, (8) VIT-IVC 1 day, and (9) VIT-IVC 7 days. Preantral follicle morphology, class distribution, and density; stromal cell density; EGFR, Ki-67, Bax, and Bcl-2 protein expression; and DNA fragmentation were assessed. Results showed that: (i) white-tailed deer fresh ovarian tissue can be cultured for up to 7 days, preserving the tissue integrity and 50% of morphologically normal preantral follicles; (ii) cryopreservation of white-tailed deer ovarian tissue by either slow-freezing or vitrification does not disrupt markers of proliferation and apoptosis after thawing; (iii) ovarian fragments cryopreserved by the vitrification method had greater follicle viability during in vitro culture than the slow-freezing method; and (iv) fragments cryopreserved by slow-freezing suffered apoptosis earlier than those preserved by vitrification. The findings herein reported advance knowledge towards development of adequate cryopreservation protocols for long-term banking programs for Cervidae species.

Keywords: Cervidae; Cryopreservation; In vitro culture; Ovarian tissue; Preantral follicle.