OVARIAN TISSUE BANKING IN ANIMAL MODELS

Ovarian tissue preservation offers limitless opportunity of fertility insurance for all ages and at any state of reproductive cycle in addition to multiple restoration options that can rekindle both reproductive and endocrine functions of the gonad. Vitrification has been argued as a superior cryopreservation technique for ovarian tissue fragments because of its desirable output, simplicity and suitability in the field. However, as with any cryopreservation technique, vitrification is associated with challenges including cryogenic insults and CPA toxicity which could generate supra-physiological levels of ROS that may culminate in apoptosis. In addition to the need for mitigating these challenges, there is a broad necessity of optimizing evaluation technique for preserved ovarian tissue. Consequently, the aim of this PhD thesis was to establish reliable ovarian tissue banking protocols including valid end point evaluation tools for feline and bovine models. In the quest for reliable fixation techniques suitable for both morphological and immunohistochemical assessment of ovarian tissue, this thesis has provided an in-depth perspective on the effect of fixatives on both morphological and immunohistochemical evaluation of feline ovarian tissue and has found that form-acetic maintained ovarian tissue architecture with excellent follicular morphology and maintained good immunohistochemical signals. Furthermore, in order to optimize vitrification and subsequent in vitro culture of bovine ovarian tissue, this thesis demonstrated in vitro culture of bovine vitrified-warmed ovarian tissue on agarose inserts which maintained good follicle morphology, low follicle activation, and low apoptosis of both follicles and stromal cells vis-a-vis culture inserts. Similarly, determining a field suitable protocol for the vitrification of feline ovarian tissue was achieved wherein vitrification protocol with low equilibration time at room temperature was reported. Finally, to further refine the protocol, role of melatonin in vitrification and culture of feline ovarian tissue was assessed and it was found that melatonin supplementation at 10-7 M promoted follicular viability and activation with reduced apoptosis during in vitro culture of vitrified-warmed feline ovarian tissue.