Date of Award


Degree Name

Doctor of Philosophy


Agricultural Sciences

First Advisor

Gastal, Eduardo


Preserving viability of frozen gametes and reproductive tissues is crucial to understand and overcome species-specificities in respect to the diversity in cryobiological properties and requirements among cell types and tissues. The use of different animal models to study ovarian tissue cryopreservation will help to uncover several important factors related to germ cells preservation. Horses (Equus ferus caballus) have been proven to be an excellent model for reproductive biology studies with implications for humans. White-tailed deer (Odocoileus virginianus) are one of the most abundant wild species in the United States, but little information about their reproductive features are known. Therefore, five studies were conducted in this Dissertation with the following general objectives: (i) to develop ovarian tissue cryopreservation techniques for horses and white-tailed deer species; and (ii) to determine the effects of ovarian tissue cryopreservation techniques on morphological and molecular mechanisms related to folliculogenesis in horse and white-tailed deer species. In study one, equine ovarian tissue was used to determine the ideal ovarian fragment size for better cooling resistance under storage at 4°C. In study two, equine ovarian tissues were used to determine the toxicity effect of cryoprotective agents on ovarian tissue pre- and post-cryopreservation. In study three, equine ovarian tissues were used to compare slow-freezing versus vitrification; and to determine the best cryoprotective agents for each cryopreservation method. In study four, white-tailed deer reproductive tracts were used to characterize the age effect on reproductive features. In study five, white-tailed deer ovarian tissue was used to compare slow-freezing versus vitrification methods to preserve preantral follicles under in vitro culture. The main findings of the horse studies were: (i) equine ovarian tissue can be stored at 4°C for up to 24 h when biopsy ovarian fragments are used; (ii) ethylene glycol seems to be a less harmful cryoprotectant agent to equine preantral follicles; and (iii) both slow-freezing and vitrification methods similarly preserved the follicle morphology after time of culture. The main findings of the white-tailed deer studies were: (i) aging caused quantitative and qualitative effects on the ovarian reserve of white-tailed deer; (ii) fresh ovarian tissue can be cultured for up to seven days preserving the tissue integrity; and (iii) fragments cryopreserved by vitrification had higher follicle viability during in vitro culture than by the slow-freezing method. In conclusion, this work demonstrated the viability to cryopreserve equine and white-tailed deer ovarian tissue. Furthermore, the frozen-thawed equine and white-tailed deer ovarian tissue can be cultured for up to seven days.




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