Identification and expression analysis of the interferon-induced protein with tetratricopeptide repeats 5 (IFIT5) gene in duck (Anas platyrhynchos domesticus).

The interferon-induced proteins with tetratricopeptide repeats (IFITs) protein family mediates antiviral effects by inhibiting translation initiation, cell proliferation, and migration in the interferon (IFN) dependent innate immune system. Several members of this family, including IFIT1, IFIT2, IFIT3 and IFIT5, have been heavily studied in mammals. Avian species contain only one family member, IFIT5, and little is known about the role of this protein in birds. In this study, duck IFIT5 (duIFIT5) full-length mRNA was cloned by reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of the cDNA ends (RACE). Based on the sequence obtained, we performed a series of bioinformatics analyses, and found that duIFIT5 was most similar to homologs in other avian species. Also, duIFIT5 contained eight conserved TPR motifs and two conserved multi-domains (TPR_11 and TPR_12). Finally, we used duck hepatitis virus type 1 (DHV-1) and polyriboinosinicpolyribocytidylic acid (poly (I:C)) as a pathogen or a pathogen-associated molecular pattern induction to infect three-day-old domestic ducklings. The liver and spleen were collected to detect the change in duIFIT5 transcript level upon infection by quantitative real-time PCR (qRT-PCR). DuIFIT5 expression rapidly increased after DHV-1 infection and maintained a high level, while the transcripts of duIFIT5 peaked at 8h after poly (I:C) infection and then returned to normal. Taken together, these results provide a greater understanding of avian IFIT5.

Molecular cloning and functional analysis of the duck TLR4 gene.

Toll-like receptor 4 (TLR4) recognizes pathogen-associated molecular patterns in some animals and has been shown to be closely associated with several diseases such as tumors, atherosclerosis, and asthma. However, its function in ducks is not clear. Alternative splicing of the TLR4 gene has been identified in pigs, sheep, mice, and other species, but has not yet been reported in the duck. In this study, alternative splicing of the duck TLR4 gene was investigated using reverse transcription-polymerase chain reaction (RT-PCR). Duck TLR4 gene (duTLR4, accession number: KF278109) was found to consist of 3367 nucleotides of coding sequence. An alternative splice form, TLR4-b, was identified and shown by alignment to retain the intron between exons 1 and 2. Real-time quantitative polymerase chain reaction (qPCR) analyses suggested that duTLR4-a (wild-type) mRNA is widely expressed in various healthy tissues, whereas TLR4-b is expressed at only low levels. Following stimulation of normal duck embryo fibroblasts with lipopolysaccharide, the expression of both isoforms initially increased and then decreased. Expression of the wild-type isoform subsequently increased again, while that of the variant remained low. The expression levels of wild-type TLR4 were further analyzed by transient transfection of a pcDNA3.1(+)-TLR4-a overexpression vector into duck embryo fibroblasts. qRT-PCR analyses showed that after stimulation with LPS and poly(I:C) the expression levels of IL-1ß, IL6, and MHC II increased with a response-efficacy relationship. Our experimental results indicate that TLR4 plays an important role in resistance to both bacterial and viral infections in the duck.

Effects of iron and copper in culture medium on bovine oocyte maturation, preimplantation embryo development, and apoptosis of blastocysts in vitro.

The aim of this study was to investigate the effects of iron and copper on bovine oocyte maturation, preimplantation embryo development and apoptosis of blastocysts. The concentrations of iron in the culture media were 0 (control), 0.45, 0.81, 1.96 and 3.26 mg/l, and the concentrations of copper were 0 (control), 0.093, 0.27, 0.46 and 0.68 mg/l. The changes in the iron (1.96 mg/l) and copper concentrations (0.46 mg/l) in the culture media were measured after oocyte maturation for 22 h and after zygote culture for 48, 96, 144 and 192 h. The results showed that there were no significant differences in oocyte maturation and cleavage between media containing iron and the control, but the media containing iron had higher (P>0.05) rates of 8-cell embryos, morulae, and blastocysts than the control, and addition of 1.96 mg/l of iron increased the blastocyst rate (P>0.05). The effects of copper on oocyte maturation and cleavage were similar to iron, and addition of 0.46 and 0.68 mg/l of copper increased the rates of morulae and blastocysts (P>0.05). Addition of iron or copper significantly decreased the number of apoptotic blastomeres compared with the control (P>0.05). After oocyte maturation for 22 h and zygote culture for 48 h, the iron concentrations decreased by 3.6 and 9.2%, respectively, and the copper concentrations decreased by 6.5 and 10.9%, respectively. After zygote culture for 96, 144 and 192 h, the iron concentrations decreased by 21.4, 25.5 and 27.0%, respectively, the copper concentrations decreased by 23.9, 28.3 and 30.4%, respectively. In conclusion, iron and copper played an important role in the success of culture of 8-cell embryos, morulae, and blastocysts, and long-term lack of iron or copper increased the number of apoptotic blastomeres. Furthermore, transition of primary demand for trace amounts of iron or copper from the cytoplast to culture medium for utilization by zygotes may occur after in vitro zygote culture for 48 h.