Optimizing the place of autologous (heterotopic and orthotopic) ovarian transplantation following vitrification-warming procedure in rabbit

Abstract Background: To maintain survival, notably for endangered species, it is essential to preserve female and male gametes. Ovarian tissue cryopreservation and subsequent re-transplantation can restore both endocrine function and fertility in humans and animals. The aim of this study was to evaluate the optimal transplantation site for ovarian tissue fragments after freezing/thawing for restoring fertility in rabbits. Methods: Rabbits were randomly assigned into 3 experimental groups according to ovarian auto-transplantation sites (n=6), as follows: I) cervical subcutaneous transplanted group (orthotopic transplantation), II) intramuscular transplanted group (orthotopic transplantation) and III) ovarian bursa transplanted group (heterotopic transplantation). After anesthesia, oophorectomy was performed on each rabbit. The ovarian cortex was separated from the medulla and divided into four strips. One strip was saved as a control in 10% formalin, the other three were frozen for two weeks, then one of the frozen ovarian parts, after warming process, was re-implanted into the implantation sites. Biopsies from ovarian strips were taken eight weeks after transplantation. Vaginal cytology and hormonal levels were determined a day before oophorectomy, two weeks after oophorectomy, and 2, 4, 6, and 8 weeks following implantation. Microscopically, the number and area (µm²) of primordial follicles were measured. Results: The number of intact primordial follicles was significantly reduced in the re-implanted ovarian tissues compared with the fresh ovary in all groups. The implanted ovarian tissues in three groups did not show any evident changes in area and number of intact primordial follicle. There was a statistically significant difference in serum hormonal concentrations two weeks after oophorectomy compared to other sampling times in each group but, there were no significant differences among three groups as to the serum hormonal concentrations in implanted ovarian tissues. The present study revealed that the morphological changes of vaginal cells in the vaginal smear after implantation were similar to those before oophorectomy. Conclusions: Cryopreservation and implantation of small pieces of ovarian tissues could restore fertility and the endocrine function of ovarian tissue. Generally, the subcutaneous, ovarian bursa and intramuscular area are all available sites for implantation and have similar rates of acceptance, despite some differences in the details of implantation. This study can be used strategically to preserve the female gametes of endangered species, wild and important domestic animals.