In Vitro Maturation Of Oocytes And Culture Of Embryos In Pig

In this study, some key points affecting in vitro production of pig embryos were studied, emphasizing on how to enhance the in vitro maturation (IVM) quality of pig embryos and how to optimize the in vitro culture conditions for pig embryos, in an attempt to establish a more stable, effective and economic in vitro production program for pig embryos, thus provide evidences for modification of pig cloning in the future.Influences of different parameters of electric stimuli, addition of essential amino acid (EAA) and non-essential amino acid (NEAA) into culture media on the IVM and subsequent development of activated oocytes were studied, and effect of Leptin supplement on the IVM and the following development of oocytes after activation was also explored, and finally, the influence of an epigenetic modifier: valproic acid treatment in the process of in vitro culture of pig cloned embryos was evaluated.(1) The matured oocytes were randomly subjected to three electro-activation parameters 1.56 kv/cm, 100μs, 2DC; 1.56 kv/cm, 100μs, 1DC; and 1.00 kv/cm, 80μs, 2DC. Both cleaved rate and blastocyst rate of parthenotes derived from activation by 1.00 kv/cm, 80μs,2 DC were lower than the other groups (Cleaved rate: 65.77±0.31% vs. 78.90±0.19% , 77.78±0.18%; P < 0.05. Blastocyst rate: 27.71±0.39% vs. 39.98±0.38% , 36.55±0.25%; P<0.05).(2) In order to investigate the exact effect of leptin addition into chemically defined maturation medium on the IVM of porcine oocytes and their subsequent development after parthenogenetical activation. Earle's salt buffered TCM199 and 10 IU/mL PMSG, 10 IU/mL hCG, 10 ng/mL EGF, 100 IU/mL pennicilin G and 100 IU/mL streptomycin sulphate served as basic maturation medium, in which Leptin supplemented were test groups, and such basic medium without leptin addition was control group 1, and basic medium added with 5%FBS and 10% porcine follicle fluid served as control group 2. Nuclear maturation rate and subsequent development such as rates of cleavage and blastocyst formation were compared among groups. We failed to observed any significant differences among groups in terms of nuclear maturation rate, and subsequent in vitro developmental ability, specifically the cleavage rate and blastocyst formation rate after parthenogenetic activation.(3) When different concentrations of EAA and NEAA was added (1% EAA+1% NEAA,2% EAA+1% NEAA), the maturation rate and the cleaved rate of oocytes were not significant different between the test group and the control group (P>0.05). But the blastocyst rate of the test groups was notably lower than the control group 1 and the control group 2 (19.07±3.15% , 16.89±2.41% vs. 26.53±2.00% , 27.55±1.64%; P<0.05).(4) When leptin at different concentrations were put into embryo culture media PZM-3 and PZM-4, the rates of blastocyst formation of parthenotes were significant improved when compared to the control groups (P<0.05). In comparison to the control groups, t addition of 50ng/mL leptin resulted in the highest blastocyst rate (PZM-3 ,51.23±3.48% vs. 34.37±4.03%,P<0.05;PZM-4,31.76±5.07% vs. 12.14±1.89%,P<0.05),. But compared to the control groups there were no significant differences between the cleavage rate and the number of total cell in blastocyst.(5) When the cloned embryos were cultured into PZM-3 in which different concentrations of VPA were supplemented all seven day, there were no significant differences observed with regards to the cleavage rate (P>0.05), and there were no blastocyst formation in the experiment groups.Our results showed that:①The best electro-activation parameters is 1.56 kv/cm, 100μs, 2DC.②The supplementation of leptin to chemically defined maturation medium is of no help to porcine oocytes.③The supplementation of EAA and NEAA to IVM medium is of no help to porcine oocytes, and harmful for the in vitro culture, and reduce the rates of blastocyst formation of parthenotes significantly.④Supplementation of leptin is beneficial to increasing the blastocyst formation of pig parthenotes. Supplementation of 50ng/mL leptin is beneficial to get the highest rate of blastocyst.⑤There are harmful for the clone embrys which cultured VPA supplemented all seven day.Taken together, by using electro-activation parameter 1.56 kv/cm, 100μs, 2DC and using PZM-3 which 50ng/mL leptin supplemented, the activated embryos of pig can develop be better.