Preliminary Study On Preparation Of Boars With Surrogate Sperm

Spermatogonial stem cells(SSCs)are the basis of spermatogenesis and fertility in male mammals.The spermatogonial stem cell transplantation technology has broad application prospects in the preparation of transgenic animals,expansion of excellent male gamete supply,treatment of infertility,and protection of endangered species.However,the development of spermatogonial stem cell transplantation technology in livestock is relatively slow.The key factor for the success of spermatogonial stem cell transplantation is that culture of SSCs in vitro to provide sufficient cell number and preparation of recipients without endogenous SSCs.In this study,pig SSCs were obtained by differential adherence and cultured in vitro,in order to establish the system of culture pig SSCs in vitro.We tried gene editing technology and direct injection of testes with busulfan to prepare recipients for germ cell removal,and transplanted exogenous SSCs to produce donor sperm.The aim of this study was to produce sperm generator replacement by the establishment of a porcine spermatogonial stem cell transplantation technique,using SSCs transplanted to replacers to produce large quantities of sperm,replacing the current boar cloning technology.The major results were showed as follow,We obtained the highest proportion(18.59±0.94)of UCHL-1 positive germ cells from the testis of 3-7 days old Large White piglets by differential adherence method.After collecting the upper layer cells,the proportion of UCHL-1 positive cells was higher than 50% by flow cytometry analysis.The addition of different growth factors to the culture of SSCs showed that the addition of GDNF 20 ng/ml,IGF 10 ng/ml and b FGF 20 ng/ml had the best proliferation effect on SSCs of Large White piglets.SSCs were cultured in vitro with supporting cells as feeder layer,1% serum,GDNF,LIF and b FGF.After 15 days of culture,After 15 days of culture,a large number of SSCswere obtained.Using CRSPR/Cas9 system and Somatic cell nuclear transfer(SCNT)technology,7ETV5 gene homozygous knockout pigs were obtained,the positive rate was100%.The major organ tissues of the knockout pigs were normal,indicating that the ETV5 gene knockout did not pose a fatal risk to the pig.ETV5 knockout pigs had normal testicular development after birth.At 8 months of age,testosterone levels were normal in the blood.SSCs in the seminiferous tubules showed no obvious apoptosis and the structure of the seminiferous tubules was normal and could produce a large number of sperm.Different doses(3,5 mg/kg)of busulfan directly injected into the Duroc boar testicles had no significant effect on their health and growth.The doses of 3 mg/kg and 5 mg/kg could effectively remove endogenous SSCs from Duroc pigs and the maintenance time was long.The number of germ cells was significantly reduced,and the diameter of seminiferous tubules was significantly smaller than control.The early stage of busulfan injection has a certain effect on the hematopoietic function of pigs.With the growth and development of pigs,the effect of 3mg/kg injection on the hematopoietic function of pigs gradually disappeared.Immunohistochemistry and gene expression assay confirmed that the endogenous SSCs were removed and the Sertoli cells remained stable.The SSCs of 3-7 day old from Large White piglets were transplanted into the testes of Duroc receptors.After 80 days of transplantation,colonization of donor SSCs was detected and sperm were generated.In vitro culture and recipient preparation are essential for the transplantation of SSCs.In this study,we established an in vitro culture system for pig SSCs,and tried genetic editing techniques and direct injection of testis with busulfan to prepare recipients.ETV5 knockout pigs failed to show apoptosis of SSCs at 8 months of age,and the direct injection of testes by busulfan obtained receptors for germ cell removal,which did not cause obvious health problems in pigs,while busulfan directly injected testes to obtain receptors for germ cell elimination,and did not cause obvious health problems in pigs.It provides a suitable internal environment for the transplantation of exogenous spermatogonial stem cells,indicating that direct injection of testes bybusulfan is an efficient and safe method.Through the transplantation of SSCs,the colonization and sperm production of donor SSCs were achieved,which laid a good foundation for the establishment of porcine spermatogonial stem cell transplantation technology.