The mouse sperm genome is resistant to in vitro heat treatment, and embryos derived from heated sperm can support full-term embryonic development, but the blastocyst rate and implantation rate are lower compared to embryos derived from fresh sperm. In the present study, the patterns of DNA methylation, histone H4K12 (ACH4K12) acetylation, H3K9 trimethylation (H3K9-TriM), and H3K27 trimethylation (H3K27-TriM) in preimplantation embryos derived from 65°C-heated sperm were investigated. Although no evident changes in global DNA methyaltion, histone H4K12 (ACH4K12) acetylation, and H3K9 trimethylation (H3K9-TriM) were found, significantly lower levels of H3K27-TriM, which was thought to be one of the reasons for low efficiency of mouse cloning, were found in the inner cell mass of heated-sperm derived blastocysts. Thus, defective modification of H3K27-TriM might contribute to compromised development of embryos derived from heated sperm.

To evaluate the effects of H5 subtype avian influenza DNA vaccine with HA gene and expressive vector optimization, groups of 3-week-old specific pathogen free (SPF) chickens were inoculated intramuscularly with a single dose of 100 μg or 10 μg of pCIHA5, pCAGGHA5, pCIoptiHA5 or pCAGGoptiHA5 in 200 μl volume. Another group of chickens was injected with 200 μl phosphate-buffered saline (PBS) as controls. Sera were collected every week after vaccination to detect the HI and agar-gel precipitin (AGP) antibodies. Four weeks after the single vaccination, all chickens were challenged with 100LD50 of the highly pathogenic A/Goose/GuangDong/1/96(H5N1) [Gs/GD/1/96(H5N1)]. Oropharyngeal and cloacal swab specimens were collected from all chickens 3, 5 and 7 days after inoculation for titration of virus in eggs. Chickens were observed daily for 2 weeks for signs of disease and death. Results showed that in the groups vaccinated with 100 μg pCAGGoptiHA5 and pCAGGHA5 chickens were completely protected from virus challenge (no disease signs, no virus shedding and no deaths), while only partial protection occurred in chickens vaccinated with 100 μg pCIoptiHA5 (75%) and pCIHA5 (50%). In groups vaccinated with 10 μg pCAGGoptiHA5 and pCAGGHA5 chickens were protected from virus challenge (no disease signs and no deaths). In the group vaccinated with 10 μg pCIoptiHA5 the protection ratio was 75%, while chickens vaccinated with 10 μg pCIHA5 all died after challenge. Results indicated that codon usage optimization of the HA gene and β-actin promoter enhanced the protection efficiency of H5 subtype avian influenza DNA vaccine and the construction pCAGGoptiHA5 could protect chickens from lethal H5N1 virus challenge even at the low dose of 10 μg, implying the possibility for the commercialization of an avian influenza DNA vaccine in the near future.