Molecular Characterization And Biosafety Assessment Of BAC-mediated FSHα/β Transgenic Boars

Follicle-stimulating hormone(FSH) is a critical hormone regulating reproduction in mammals. Transgenic mice show that overexpression of FSH can improve the ovulation rate and litter size of female mice. Previously, 67 Large White transgenic boars harboring FSHα/β genes from Chinese Erhualian pigs were generated using a bacterial artificial chromosome(BAC) system and somatic cell nuclear transfer. Two F0 transgenic boars from the same donor cell were selected to mate with American Large White sows to produce F1 pigs. Here, we conducted the molecular characterization and biosafety assessment for these F1 boars.Litter size data showed that the American Large White sows mating with F0 transgenic boars had normal reproductive performance. F1 pigs were identified by PCR and then conformed by copy number assay. Of these F1 pigs, 50.31% were transgenic. The inheritance of exogenous genes conformed to the Mendel’s law of segregation. Eight to nine copies of exogenous FSHα gene and five to six copies of exogenous FSHβ gene were integrated into the genome of transgenic pigs in a tandem repeat way. Quantitative real-time PCR analysis indicated the FSHα m RNA expression levels in heart, liver, spleen,kidney, brain, testis and epididymis was significantly higher in F1 transgenic boars than in non-transgenic male half sibs. The FSHβ m RNA expression was not detected in these tissues of F1 boars. We found a significant higher level of FSH? protein in the pituitaries of F1 transgenic boars compared with non-transgenic male half sibs by Western blot analysis.RIA data demonstrated that the serum FSH level was significantly higher in F1 transgenic boars than in non-transgenic male half sibs at the age of 90 and 300 days. HE stained sections of testis showed that F1 transgenic boars had significantly higher gonocytes per unit area at the age of 15 days and germ cells per seminiferous tubule in adulthood compared with their non-transgenic male half sibs. F1 transgenic boars had normal sperm quality without disruption of the endogenous reproduction-related genes expression.Analysis of growth curve, hematological and biochemical parameters and histopathology illustrated that there was no significant difference between F1 transgenic boars and non-transgenic male half sibs except the weight of seminal vesicles. By applying 16 S r RNA gene sequencing, we demonstrated that exogenous genes had no impact on the bacterial community structures of F1 transgenic pig guts. Moreover, foreign gene drift did not occur as verified by the horizontal gene transfer detection in American Large White sows mating with F0 transgenic boars, gut microorganism of F1 transgenic boars, soil microorganism and earthworms.In summary, F1 transgenic boars have normal sperm quality and improved spermatogenesis ability without any growth and reproductive defects. These boars grow healthily. Exogenous FSHα/β genes have no impact on the environment, conforming to the requirements of transgenic animal biosafety in China. The findings pave a road to successful breeding of a transgenic Large White pig line with desirable reproductive performance.