| Poorer development of in vitro derived porcine embryos indicates that either in vitro maturation of oocytes or in vitro culture conditions of embryos is still suboptimal compare with their in vivo counterparts.In order to establish a system for reliably providing good oocytes and steadily producing cloned embryos by somatic cell nuclear transfer in pigs,we performed the following experiments.Effects of different basic maturation media,supplementation of Ghrelin or Insulin to a selected maturation media on the nuclear maturation of oocytes and their subsequent developmental potential were examined.To our knowledge,this is the first time that Ghrelin was added into maturation media.Afterwards,we evaluated the effects of treatment durations,such as 0h,12h,36h and 48h,of 50nM trichostatin(TSA) on the developmental competence in vitro of pig somatic cloned embryos.Finally,we investigated if supplementation of Insulin could promote the in vitro development of porcine parthenotes.The results were:(1) The maturation rate of oocytes cultured in media when NCSU23 as basic medium was significantly higher than when DMEM as basic (69.54±2.74%vs52.82±5.72%,P＜0.05);the in vitro development of embryos activated parthenogenetically from oocytes matured in NCSU23 was obviously superior to those matured in TCM199 and DMEM(cleavage rate:74.25±8.39%vs 60.25±1.42% vs43.75±1.55%,P＜0.01);(2) When the Ghrelin was added,the maturation rate of oocytes was slightly lower than control group though no significant differences exist.After activation of these oocytes,although there was a trend that cleavage rate increased in 50ng/mL group and blastocyst rate improved in 100ng/mL group,no statistical differences were observed among groups;(3) When Insulin of various concentrations was supplemented in the maturation medium,the rate of maturation in the test groups was all remarkably higher than that in the control group;after parthenogentic activation,the cleavage rate for 5μg/mL group was significantly higher than that for the control group;for the blastocyst rate,significant difference was observed between the 5μg/mL group and the control,and no differences were found among the other groups;(4) When the cloned embryos were treated by TSA at 50nM of various durations,no differences were observed with regards to the cleavage rate;the rate of blastocyst formation was significantly higher in 36 h group that that in 12 h group and the control;(5) When Insulin of different concentrations were put into culture medium,the rates of cleavage and blastocyst formation of parthenotes were both drastically improved regardless of how many Insulin was added when compared to the control groups;Our results showed that:①NCSU23 was an ideal basic medium for in vitro maturation of porcine oocytes;②Addition of Ghrelin into the maturation medium is not necessary;③Addition of Insulin at the concentration of 5μg/mL to the IVM medium could not only improve the nuclear maturation of oocytes but also could enhance the preimplantation development of embryos activated from such oocytes;④Treatment by TSA with the duration of 36 h could promote in vitro developmental ability of cloned embryos in pigs;⑤Supplementation of 2.5μg/mL,5μg/mL or 10μg/mL Insulin is beneficial to increasing the rates of cleavage and blastocyst formation of pig parthenotes.Taken together,by using NCSU23 as basic medium and supplementation of 5μg/mL Insulin for IVM,and addition of 2.5μg/mL Insulin in embryo culture media and treatment of cloned embryos with TSA for 36 h,we successfully obtained a system for IVM of pig oocytes and IVC of pig parthenotes and cloned embryos.
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