The aim of this study was to develop a method for spermatogonial stem cell transplantation into the bovine testis. Five-month-old Holstein-Friesian calves were used and half of the calves were hemicastrated to allow autologous transplantation and the other half were used for homologous transplantation. Approximately 20 g of each testis was used for cell isolation. On average 106 cells per gram of testis containing about 70% type A spermatogonia were isolated. The cells were frozen in liquid nitrogen until transplantation. Testes were irradiated locally with 10-14 Gy of X-rays to deplete endogenous spermatogenesis. At 2 months after irradiation, cells (approximately 10 x 10(6) were injected into the rete testis through a long injection needle (18 gauge), using ultrasonography and an ultrasound contrast solution. At 2.5 months after transplantation, calves were castrated and samples of testes were taken for histological examination. After 2.5 months in the irradiated non-transplanted control testes, only 45% of the tubules contained type A spermatogonia. However, after autologous spermatogonial transplantation, >80% of the tubule cross-sections contained type A spermatogonia. In addition, only 20% of the tubules of the control testes contained spermatocytes and, except for a few tubules (5%) with round spermatids, no more advanced germ cells were found. After autologous spermatogonial transplantation, about 60% of the tubules contained spermatocytes; 30% contained spermatids and in about 15% of tubules spermatozoa were found. No improvement in spermatogonial repopulation was found after homologous transplantation. The results of this study demonstrate, for the first time, successful autologous transplantation of bovine spermatogonial stem cells resulting in a complete regeneration of spermatogenesis.
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