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

Authors: GASTAL, G - Southern Illinois University; AGUIAR, F - Southern Illinois University; RODRIGUES, A - Southern Illinois University; SCIMECA, J - Southern Illinois University; APGAR, G - Southern Illinois University; BANZ, W - Southern Illinois University; FEUGANG, J - Mississippi State University; GASTAL, G - Southern Illinois University

Submitted to: Theriogenology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/5/2018
Publication Date: 3/8/2018
Citation: Gastal, G., Aguiar, F., Rodrigues, A., Scimeca, J., Apgar, G., Banz, W., Feugang, J., Gastal, G. 2018. Cryopreservation and in vitro culture of white-tailed deer ovarian tissue. Theriogenology. 113:253-260.

Interpretive Summary: The mammalian ovary contains a finite number of follicles that establish female fertility. Unfortunately, various endogenous and exogenous factors to the female can affect this number, jeopardizing fertility in clinical, livestock, and wildlife settings. Despite the knowledge of the ovarian function in the mammal, the development of novel technical approaches to maintaining the viability of ovarian tissues and identify the critical markers for further real-time and functional investigations are imperative. Here we tested the white-tailed deer ovarian tissue for cryopreservation and in vitro culture. This study will permit the continuous development of conservation biology and further real-time investigations of molecular function.

Technical 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.