Transgenic Sheep Reproductive Safety Evaluation

TLR4transgenic sheep were as research model for systematic safety assessment from reproductive point of view in this study. The purpose was to provide some theoretical basis for explaining whether transgene was biosafety, which will help to improve the transgenic animal and technology public awareness in the future. In this study, FO and F1TLR4transgenic sheep as experimental animals were monitored or detected from the aspects of hematology, hisopathology, reproduction and epigenetics.Five FO and seven F1generations transgenic ewes were obtained by Southern blot identification. Transgenic ewes can quickly start the inflammatory response. Immunofluorescence staining results showed TLR4expreesion in the oocyte plasma membrane, and the expression qunatility was obvious increasing in transgenic oocyte. The values of the physiological and biochemical parameters were determined from the blood samples did not differ significantly between the transgenic and wild-type ewes. Moreover, the transgenic ewes showed reproductive traits similar to the wild-type ewes. These traits included characteristics of puberty, the estrus cycle, estrus duration, gestation, the pregnancy rate and the superovulation response. There were slight pathological changes in liver, kidney and small intestine between transgenic and non-transgenic ewes.On this basis, the present study further examined the effect of transgene on oocyte DNA methylation. The experiment was divided into two parts:(1) injection pTLR4-3s and Elution-buffer groups were significantly higher than control group in1,2,4-cell stage embryos methylation level (P<0.05), but there were no significant differences in the8-cell and blastocyst stage (P>0.05);(2) no significant differences were found in the DNA methylation level of the oocytes at various stages and somatic cell between transgenic and wild-type ewes (P>0.05)The results of Flow cytometry analysis showed that sperm motility(85.63%vs.85.90%and85.61%vs.84.38%), proportion of membrance integrity (85.63%vs.85.90%and85.61%vs.84.38%) and high mitochondrial membrane potential (85.58%vs.83.27%and86.61%vs.83.53%) sperms were not significantly different between transgenic and non-transgenic rams in the FO and Fl generation rams (P>0.05). Computer-aided detection analyzer showed that transgene had no significant effect on sperm motility (P>0.05). Transgenic semen artificial insemination pregnancy rate was also similar to control (P>0.05).In conclusion, TLR4trangene had no significant effect on blood physiological and biochemical parameters, major organs pathological conditions and reproductive traits. In addition, TLR4trangene had also no significant effect on various stages oocytes and ear fibroblast DNA methylation levels. In ram, TLR4transgene did not affect sperm quality.