| The previous research of this research group found that the addition of 10-7 M melatonin to the in vitro maturation medium for bovine and pig oocytes,the maturation efficiency of bovine and pig oocytes.,and embryonic developmental potential of females is significantly increased.In order to investigate whether melatonin has the same results in sheep oocyte maturation,this study added melatonin at a concentration of 10-7 M to sheep in vitro maturation and embryonic development to calculate the oocytes maturation efficiency,and fertilization and somatic cell nuclear transfer.Ovarian endogenous overexpressing melatonin synthase AANAT/ASMT gene sheep was established by somatic cell nuclear transfer,and animal models were established to investigate the effects of endogenous melatonin on reproduction.The biological safety of sheep with over-expressed melatonin synthase AANAT/ASMT genes was tested to investigate the safety and effectiveness of transgenic animal models.In vitro maturation results show that the addition of 10-7 M melatonin to the in vitro maturation medium can effectively promote the expansion of cumulus granulosa cells and increase the efficiency of oocyte first polar body excretion(69.69±2.27%),forming a significant difference with the control group.The difference was(52.72±0.68%)(P<0.05).The fertilization efficiency of melatonin-treated group(63.2±0.0%)was significantly higher than that of the control group oocytes(50.451±1.21%)(P<0.05).After fertilization of mother cells,melatonin blastocyst rate(26.60±1.12%)was added during early embryo culture,and melatonin-treated melatonin-treated oocyte embryo blastocyst rates(29.60±1.07%))And melatonin mature oocyte control group blastocyst rate(27.28±1.09%)was significantly higher than the blank control group(19.74±1.13%)(P<0.05).The somatic cell nuclear transfer of melatonin-treated oocytes showed that the fusion rate(69.57±2.77%)in the melatonin-treated group was significantly higher than that in the control group(57.46±1.22%).The embryonic cleavage efficiency(57.65±8.23%)was significantly higher than the reconstructed embryo cleavage rate(18.31±0.90%)and the blank control group(8.79±1.46%)in the melatonin-treated control group(P<0.05).Biosafety evaluation of sheep with overexpressing melatonin synthase AANAT and ASMT genes showed that:positive transgenic sheep had four growth indicators of body weight,body length,height,and bust at ages 0,6,and 12 months,and There was no significant difference in ordinary sheep(P>0.05).The total blood protein,albumin,globul in,cholesterol,and various types of cells were within the nonnal range and there was no significant difference with ordinary sheep(P>0.05).The results of urine nitrite,protein,and glucose in normal sheep showed negative results,indicating that the sheep excretory system is free of infectious diseases.Sequencing of rectal fecal flora showed positive sheep and normal sheep intestinal microbial communities.There was no significant difference in composition and dominant flora(P>0.05).The expression level of melatonin in blood of transgenic sheep was not significantly different from that of ordinary sheep,and the level of melatonin and lactose in milk were significantly higher than those of ordinary sheep(P<0.05).The content of somatic cells was significantly lower than that of ordinary sheep(P<0.05).In summary,melatonin was added during in vitro maturation culture,fertilization,early embryo development,and cloning,which significantly improved the quality of oocytes and early embryo development potential,and improved cloning efficiency.Secondly,the safety evaluation research results of transgenic sheep show that transgenic technology will not endanger the growth and development performance of melatonin synthase gene in the breast,and will not change the blood,urine,milk composition and overexpression of melatonin synthase gene in the breast Effectively increase the content of melatonin in transgenic goat milk and reduce the number of somatic cells in milk.Through the establishment of a safe and reliable transgenic animal model,to explore the impact of endogenous and exogenous melatonin on animal reproductive activities,to provide theoretical support for improving production efficiency in production practice,and to provide experience for exploring the biological safety of transgenic animals. |
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