Regeneration and preservation of poultry breeds through transplantation procedures on gonadal tissues

Live poultry flocks are costly to maintain, especially for rare breeds in small populations or non-commercial circumstances. One strategy to reduce these costs could be cryopreservation of the chick gonadal tissues and subsequent transplantation into recipients, which would help to maintain the genetic resources of some heritage chicken breeds that are rare and threatened with extinction. Thus, the overall goal of the study was to produce offspring derived from donors of different chicken breeds, including heritage chicken breeds. In our studies, a vitrification-warming procedure was employed to preserve chick gonadal tissues; and a modified transplantation technique was used to regenerate chicken breeds by the production of donor-derived offspring. An assessment of the vitrification-warming procedure was also conducted by using a morphological scoring system and detecting apoptotic cells via caspase-3 immunofluorescence. Additionally, cryoprotective agent (CPA) diffusivity measurements were made to facilitate mathematical prediction of minimally damaging CPA treatments. One significant aspect of this study focused on post-cryopreservation chick ovarian tissue transplantation to address the failures of this procedure in previous studies. The goal of this experiment was to demonstrate that vitrified/warmed one-day-old chick ovarian tissues could be transplanted and developed generally in the different recipient chicken breeds and can produce the donor-derived offspring; in addition, to evaluate the potential of different chicken breeds to receive a donor graft. In particular, two main experiments were conducted on different chicken lines. The first experiment was transplantation between chickens of similar genetic background of Lohmann White (Oldenhof et al.) and Barred Rock (BR). The second experiment was transplantation between chickens of different genetic background Brown Leghorn (BL), BR and BL/BR. The results differed depending on chicken lines. In the first trial, 50% of ovarian tissues were taken from adult recipients derived from donors of fresh and vitrified-warmed BR ovaries. Moreover, one hen appeared to have a donor-derived graft, as well as two of the embryos from this bird showed alleles of the donor bird. In the second trial, BL and BL/BR background could sustain the development of a graft regardless of the genetic background of the donor. However, all these grafts were enclosed by a membrane and had a variety of growth, suggesting some level of rejection by the recipients. The assessment of the vitrification-warming procedure was conducted on one to three-day-old chick testicular and ovarian tissues. Two methods were used to evaluate the damages of the tissues: Periodic Acid Schiff-Hematoxylin (PAS-H) and an immunohistochemistry technique via caspase-3 immunofluorescence. From the first method, the morphological damages were evaluated, based on the morphological grading system. The second method showed the detection of apoptotic cell execution on the gonadal tissues. Overall, the results show that the vitrification-warming procedures affect the cellular integrity of one to three-day-old chicken gonadal tissues through morphological alterations. While the process does not cause more apoptotic cells on testicular tissues, there are some challenges in ovarian tissues for detecting apoptosis. Taken together, these results suggest the need to optimize this preservation technique for long-term storage and the surgery transplantation, especially in chicken ovarian tissues. Finally, to explore possible relationships between CPA (cryoprotectant agent) equilibration and tissue type, experiments were performed to determine the rate of diffusion of three CPAs into chick testes and ovaries. In the future, this diffusion rate can be used with a mathematical model to create an optimal protocol minimizing the toxicity of CPAs and intracellular ice formation, including diminishing gonadal tissue damages. In summary, the studies of this thesis confirmed the potential of the cryopreservation of chicken genetic material through vitrification and the successful subsequent transplantation of the vitrified-warmed gonadal tissues into the appropriate recipients. Additionally, limitations have been described, such as the effects of the vitrification-warming procedure, the rejections from immune responses of recipients, etc. Some solutions were provided, which could increase the efficiency of the strategy to be practical for poultry studies in diverse fields