It remained unknown whether HDAC6 affected the histone deacetylation of in vitro maturation oocytes and the reprogramming of nuclear transplantation in pig. Our results indicated that HDAC6 specific inhibition did not affect overall HDAC activity and meiosis process, which increased histone H3K9/K14 and H4K8 acetylation of porcine in vitro maturation oocytes and pseudopronucleus embryos. HDAC6 inhibition also significantly enhanced the cleavage and blastocyst of nuclear transfer embryos (0.81 ± 0.12 vs. 0.68 ± 0.12 and 0.46 ± 0.19; 0.73 ± 0.13 vs. 0.63 ± 0.18 and 0.40 ± 0.16, P<0.05). The inhibition of HDAC6 significantly enhanced histone H3K9/K14 and H4K8 acetylation, and upregulated the OCT4 and CDX2 expressions (1.83 ± 0.16 vs. 1.00 ± 0.00 %; 2.07 ± 0.09 vs. 1.00 ± 0.00; P<0.05) in porcine SCNT blastocysts. Interestingly, HDAC6 inhibition significantly increased the pseudo-pronucleus volume during somatic cell reprogramming. Thus, HDAC6 was required for porcine histone deacetylation during the in vitro maturation and pseudo-pronucleus stages. HDAC6 inhibition improved the in vitro development of nuclear transfer embryos. HDAC6 may restrict the reprogramming of somatic nuclear transfer by regulating pseudo-pronucleus expansion. We need further research to confirm this in the future.

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Cytoplasmic microinjection (CI) of foreign gene into in vivo fertilized zygotes has emerged as a useful tool for transgenic pig production. In the current study, we investigated factors affecting transgenic efficiency and developmental potential of parthenogenetic (PA) and in vitro-fertilized (IVF) porcine embryos produced by CI. These factors included adding of RNase inhibitor, DNA or RNA concentration, injection time, and different structures of plasmids. Our results showed that adding of 1-4 U/μL of RNase inhibitor did not have negative effect on development potential of CI-PA embryos, and RNase inhibitor injection significantly increased EGFP expressing rate of CI-PA embryos. High injection DNA concentration and long injection interval after PA significantly reduced blastocyst formation. Different molecular structures such as DNA or RNA affected CI-PA embryos development, and RNA had little harmful effect on pig's early embryonic development. EGFP expression rate of CI-IVF embryos was improved following the increase of foreign DNA concentration, but blastocyst formation rate was decreased. Injection time after IVF did not show any significant difference on embryonic development, but longer interval resulted in a significantly lower EGFP expressing rate. Cas9 mRNA and myostatin (GDF-8) sgRNA co-injection indicated that the mutation rate of CI-IVF group was significantly higher than that of CI-PA. The CI-IVF-generated embryos were then transferred to six recipient pigs, but no live piglets were obtained. The following pronuclear formation assessment showed more than 76.1% IVF zygotes were polyspermy. These results demonstrate that CI-PA and CI-IVF were effective methods for production of transgenic pig embryos. However, polyspermic fertilization and poor quality of porcine IVF blastocysts are still the main problem of resulting in pregnancy failure.