Effects Of L-arginine Supplementation On Development Of Porcine Parthenogenetic Activated Or Somatic Cloned Embryos

Pig somatic cell cloning technology plays an important role on breeding and biomedical disease models.But the somatic cell nuclear transfer embryos still exist many problems,such as low efficiency,fetal placental abnormalities and the presence of physiological defects.Therefore,to achieve somatic cell cloning technology out of the lab and in the service of animal production and widely used in the medical field,we should improve the cloned embryo production system and give full play to its development potential.L-arginine(L-Arg)as a condition essential amino acid,supplemented in the diet can promote the reproductive performance of normal pregnancy sow,supplemented in vitro also increases porcine embryo development.However,whether L-Arg is beneficial to porcine somatic cloned embryos remains unknown.The aim of this paper was to investigate the effects of L-Arg supplementation during in vitro culture on the development competent of porcine parthenogenogenetic activation embryos(PAEs)and porcine somatic cell nuclear transfer embryos(NTEs),and the impacts of dietary supplementation with L-Arg on fetal development of cloned embryos surrogate sows,detailed test and the results are as follows:Experiment l was designed to reveal the effects of different developmental competence of parthenogentic activated embryos from oocytes in vitro matured with L-Arg supplementation.The test groups were supplemented 0.12 mM,0.36 mM,0.72 mM,1.08 mM L-Arg during chemically defined oocyte maturation medium,the control group used the same medium but without supplementing L-Arg.We found that the maturation rate,cleavage rate,blastocyst rate and total cell number per blastocyst in test groups were all higher than the control group,but the difference was not significant(P > 0.05).The results showed that simply supplemented L-Arg during oocyte maturation medium cannot improve maturation of oocyte and the quality of preimplantation embryo.Experiment 2 aimed to estimate the influence of L-Arg on early embryogenesis of porcine PAEs.The test groups were supplemented 0.12 mM,0.36 mM,0.72 mM,1.08 mM L-Arg during PZM-3 medium,the control group used the same medium but without supplementing L-Arg.We found that the blastocyst rate and blastocyst cell number was significantly increased in the 0.12 mM L-Arg-treated group compared with the control group compared with the control group(P<0.05);Other test groups were also improved blastocyst rate and the total cell number of blastocyst,but there was no significant difference(P>0.05).The results show that L-Arg supplementation in PZM-3 medium Enhances PAEs development.Experiment 3 was further to reveal the effects of L-Arg supplementation during in vitro oocyte maturation and in vitro embryo culture on the development competent of embryos.The test groups were supplemented 0.12 mM,0.36 mM and 0.72 mM L-Arg during both chemically defined oocyte maturation medium and PZM-3 medium,the control group used the same medium but without supplementing L-Arg.These results showed that all the experimental groups blastocyst rate have a tendency to develop into more blastocysts compared with the control group,but the difference was not significant(P> 0.05);Only the group treated with 0.72 mM L-Arg showed a significant increase of the blastocyst rate of PAEs(P<0.05).The results suggest that L-Arg supplementation during in vitro oocyte maturation and in vitro embryo culture promoted the development competent of porcine PAEs;It may require a higher concentration of L-Arg to play a role compared with only supplemented L-Arg during PZM-3 medium.Experiment 4 was to investigate Effects of L-Arg supplementation during the different initial time of embryo culture on Development competent of porcine PAEs.The test group supplemented 0.12 mM L-Arg on 0-168 h,0-72 h and 72-168 h during PZM-3 medium,the control group used the same medium but without supplementing L-Arg.The cleavage rate,blastocyst rate and total cell number per blastocyst tended to be slightly higher in the all test groups,although these values did not significantly differ from those of the controls(P> 0.05).The trend of 0-72 h group improved most obvious,and the trend of 72-168 h group improved most significantly.The results suggest that the impact L-Arg on the development of embryo development may occur in the later.Experiment 5 was designed to reveal the influence of L-Arg on early embryogenesis of porcine NTEs.The test groups were supplemented 0.12 mM,0.36 mM,L-Arg during PZM-3 medium,the control group used the same medium but without supplementing L-Arg.L-Arg supplementation of the PZM-3 medium increased the cleavage rate,blastocyst rate,and the blastocyst cell number of NTEs compared with the controls.Which the blastocyst rate and the total cell number of blastocyst was significantly higher than control group(P<0.05).The results showed that supplemented of L-Arg to NTEs in vitro significantly increased the development efficiency and quality of NTEs.The goal of experiment 6 was to investigate the effects of maternal dietary L-Arg supplementation during pregnancy on NTEs development in vivo.In the case of the strict implementation of the standard dietary feeding,respectively,supplemented 0.8% L-Arg in the test group of 22 cloned embryos surrogate sows,supplemented 1.64% L-Ala in the control group of 25 cloned embryos surrogate sows(equal nitrogen content).We found that dietary supplementation with L-Arg to cloned embryos surrogate sows during gestation increased average litter weight of live-born piglets,average placental weight live-born piglets,average weight at ablactating,although the values did not differ from those of the controls(P>0.05).Our results suggest that maternal dietary L-Arg Supplementation Enhanced the development of NTEs in vivo,and it was conducive to health of cloned piglets before weaning.In summary,Supplementation with 0.12 mM L-Arg during PZM-3 medium could obviously promote the blastocyst formation rate and the total cell number of blastocysts of NTEs and PAEs.The blastocyst rate but not the blastocyst quality of PAEs increased when we supplemented 0.72 mM L-Arg both in vitro oocyte maturation medium and in vitro embryo culture medium.The PAEs were treated by 0.12 mM L-Arg could slightly improve cleavage rate and blastocyst rate of PAEs,and embryonic development ability improvement trend is more obvious at 72-168h(P> 0.05).It is the first to reveal 0.8% L-Arg supplementation enhances the reproductive performance of cloned embryos surrogate sows via improving the quality of placenta.It was also beneficial for health of the cloned piglets.In conclusion,the present study suggest exerts its positive effects of development of porcine somatic cell nuclear transfer embryos in vivo and in vitro with L-Arg.