CRYOPRESERVATION, CULTURE AND XENOTRANSPLANTATION OF DOMESTIC CAT OVARIAN TISSUES IN NUDE MICE

Abstract

Fertility preservation of endangered wild animals that decease unexpectedly is concerned as a challenge for preserving the high valuable genetic materials within the gonads to be future restore into offspring using ARTs. Ovarian tissue cryopreservation is the option for fertility preservation in prepubertal animals or animals that decease unexpectedly. The present thesis aims to investigate the potential of ovarian tissue cryopreservation, in vitro culture and xenotransplantation of domestic cat as a model for other wild felid species. Firstly, different sucrose supplementations (0 M, 0.1 M and 0.3 M) in the standard freezing medium were investigated for the effects on the follicular viability, follicle morphology, DNA integrity and gap-junction protein (Cx43) expression. Ovarian tissue slow frozen using 0.1 or 0.3 M sucrose showed better follicular viability, morphology, and fewer DNA damaged follicles than the control group (P < 0.05). Next, the cryopreservation methods were compared between slow-freezing and vitrification protocols. Slow-freezing groups showed a better quality of follicular viability, morphology, and less DNA damage follicles than vitrification group (P < 0.05). To achieved a better quality of ovarian tissue obtained from the fields, FCCP (carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone), a mitochondrial uncoupling agent, have been studied for pre-exposing of this substance before in vitro culture of ovarian tissue in order to lower the mitochondrial activity. Ovarian tissues incubated with 200 nM FCCP for 120 min showed a protective effect on the follicular viability, morphology, and proliferation index (Ki-67) after in vitro tissue culture up to 7 days. However, this beneficial effect of FCCP pre-exposure, was absence after tissue freezing and thawing. Xenotransplantation of the cryopreserved ovarian tissue into the immunodeficient animal (nude mouse) was studied for histological changes within the grafted ovarian tissue after 15 days of transplantation. Percentage of morphologically normal primordial follicle of the cryopreserved graft was lower (1.8 ± 2.6%; P < 0.05) than the cryopreserved tissue before transplantation (57.9 ± 11.8%). Additionally, the normal primary follicle in the cryopreserved graft was also lower (2.8 ± 3.4; P < 0.05) than the cryopreserved tissue before transplantation (54.1 ± 11.7). Xenotransplantation of ovarian tissue still has potential to increase percentage of growing preantral follicles within the fresh tissue but not in cryopreserved tissue. Ovarian tissue cryopreservation and further follicle development i.e, in vitro ovarian tissue culture and xenotransplantation of ovarian tissue into nude mouse suggest the possibility to systematically preserve female gamete and develop the preantral follicle in feline species.