Spermatogenesis regeneration by transfected spermatogonial stem cells in infertile roosters through testicular transplantation.

Investigations pertaining to spermatogonial stem cells (SSCs) have led to the use of these cells in a variety of fields including infertility treatments, production of transgenic animals, and genome editing. The aim of the present study was to investigate the plausibility of regenerating spermatogenesis in infertile roosters by transplanting transfected SSCs into testes. Spermatogonial stem cells were isolated and cultured for seven days. Afterward, pDB2, a plasmid vector carrying a reporter gene, GFP, was transfected into the SSCs. Transfected SSCs were transplanted into the left testis of infertile roosters. Tissue samples from the recipients' testes were obtained six weeks after the transplantation and transplanted SSCs were observed in the basement membrane. After eight weeks, GFP-positive spermatozoa were observed in collected semen from the recipient roosters and GFP gene in spermatozoa was confirmed using PCR. The recipient roosters were mated with hens. Hatchlings were visually checked and their tissue samples were tested by PCR to identify transgenesis but both of them were negative. Overall, it seems that regeneration of spermatogenesis in roosters via transfected SSCs is possible but more studies are need to produce recombinant proteins by this way.

Different approaches to establish infertile rooster.

Several methods have been developed to suppress spermatogenesis in recipient males before spermatogonial stem cells (SSCs) transplantation. The aim of this study was to compare two different methods of depleting endogenous spermatogenesis in recipient ROSS 308 strain adult roosters. Gamma-radiation and alkylating agent busulfan were utilized to infertilize adult roosters (ROSS 308 strain). Two radiation therapy regimes (based on 60co isotope) were conducted locally to testes using 40Gy (5×8Gy with three-day intervals) and 30Gy (3×10Gy with three-day intervals). And two different levels of busulfan 60mg(40+20) and 50mg(30+20) with 10-day intervals were injected intraperitoneally. The results showed that both radiation therapy regimes and both busulfan levels reduced sperm motility and sperm concentration significantly compared with control group. Moreover, there were no significant differences between gamma radiation and busulfan treatments in progressive and total motility of sperm reduction. Sperm concentration reached to zero at the end of the 4th week of experiment in all treatment groups. Also histological examinations revealed that both treatments could significantly reduce the diameter of seminiferous tubules and thickness of epithelium. None of the treatments had significant effect on body weight in comparison with control group and the health status of experimental roosters remained good throughout the study. Given that, the risk probability of high doses of radiation exposure and busulfan, it can be concluded that the 30Gy (3×10Gy) and 50mg (30+20) are appropriate for suppression of endogenous spermatogenesis in mature roosters.