Optimization Of Production And Immunofluorescence Staining Against Histone Acetylation Of Cloned Embryo In Pigs

Porcine somatic cell nuclear technology (SCNT) has wide applications in animal husbandry, human medicine and biology basic research. However, up to date, SCNT in pig is still not expanded outside the lab due to its chanllanging, complex procedures, low efficiency and high cost. The aim of the current study was to establish a cost-effective, efficient, convenient and stable platform for prodcing porcine cloned embryos, and lay the solid foundation for deciphering epigenetic regulation mechanisms underlying the fate of porcine cloned embryos. It consists of the following 6 independent experiments.Experiment 1. Effects of lung air, the atmosphere exhaled after deep inhalation, on nuclear maturation of gilt oocytes and subsequent development of parthenogenetic activated (PA) and cloned embryos were explored. It showed that no significant difference among lung air, low oxygen (5% O2), high oxygen (20% O2) atmosphere environments as for nuclear maturation rate (66.5% vs. 60.2%, 63.2%), cleavage rate (94.8% vs. 94.2%, 85.2%), blastocyst formation rate (39.5% vs. 40.3%, 32.5%) and total cell number per blastocyst (37 vs. 38, 32). Moreover, as for porcine cloned embryo, no significant difference between lung air and high oxygen (20% O2) group was observed in the cleavage rate (88.3% vs. 80.3%), blastocyst formation rate (7.3% vs. 10.7%) and total cell number (34 vs.36). The above results indicated that porcine oocytes can be matured in vitro safely and efficiently using the lung air atmosphere.Experiment 2. In the current experiment, in vitro developmental competence of porcine zona-free PA embryos cultured in lung air, low oxygen (5% O2), high oxygen (20% O2) atmosphere environment were studied. No obvious difference among three groups as for the rates of cleavage, blastocyt formation and total cell number was observed, but blastocyt formation rate and total cell number in lung air, low oxygen (5% O2) were higher than those in the rest group (P<0.05). These results show that lung air may be not suitable for porcine zona free PA embryos.Experiment 3. The present experiment was to investigate the feasibility of culturing porcine cloned embryos in chemically defined medium in vtro. As for PA embryos, the cleavage rate was not significantly different among three medium groups (PZM-3, PZM-4 and PZM-5), while the blastocyst rate in PZM-3 was superior to PZM-4 and PZM-5(P<0.05). Moreover, total cell number per blastocyst in PZM-3 was higher than that in PZM-5, but similar to that in PZM-4. As for SCNT embryos, no significant difference was found for the cleavage rate or the blastocyst rate among three groups. However, total cell number of blastocyst in PZM-3 was notably higher than PZM-5 (P<0.05), but was similar to that in PZM-4. The results suggest that chemically semi-defined medium PZM-3 and total chemically defined medium PZM-4 can be used to culture efficiently the porcine PA and SCNT embryos in vitro.Experiment 4. The current experiment was carried out to compare activation effect of fusion simultaneous activation (AFS) and fusion before activation ( delayed activation) on porcine cloned embryos. It showed that no significant difference existed between two activation methods as for the rate of cleavage, blastocyst formation and total cell number; but blastocyst formation rate using the delayed activation may become higher compared to AFS group. The above outcomes reveal that two activation methods can be used to activate porcine cloned embryos efficiently, but delayed activation may be more efficient.Experiment 5. The present study was to investigate effect of adding enucleated cytoplasmic quantity or volumn on developmental potential of porcine zona free cloned embryos. The outcomes showed that significant higher cleavage rate, blastocyste formation rate and total cell number in cloned embryos derived from two enucleated cytoblasts were observed compared to cloned embryos derived from one enucleated cytoblast only (P<0.05). The results demonstrate that increasing recepient cytoplasmic quantity or volumn can improve the developmental competence and quality of porcine cloned embryos in vitro.Experiment 6. The present experiment was designed to optimize the procedure of the indirect immunofluorecence staining of H3K9 acetylation in porcine PA embryos and fetal fibroblast cell. The outcomes indicated staining effect using the following protocols, namely imported secondary antibody, fixing time for 15min, permeabilizing time for 20-30min, zona-intact embryo, will be significantly improved.Taken together, to our knowledge, the present study demonstrated for the first time that porcine oocytes can be cultured in lung air. And in vitro developmental competence of porcine cloned embryos was elevated by optimizing the key procedures. Moreover, protocol of immunofluorescence staining against H3K9 was also optimized.