Comparative analysis of ovarian transcriptomes between prolific and non-prolific goat breeds via high-throughput sequencing.

To increase the current understanding of the gene expression in the pre-ovulatory ovary and identify the key genes involved in the regulation of ovulation rate, we compared the transcriptomes of ovaries from the prolific Jintang black goat (JTG) and the non-prolific Tibetan goat (TBG) during the follicular phase using the Illumina RNA-Seq method. Three ovarian libraries were constructed for each breed. On average, we obtained approximately 49.2 and 45.9 million reads for each individual ovary of TBGs and JTGs, respectively, of which 79.76% and 78.67% reads were covered in the genome database. A total of 407 differentially expressed genes (DEG) were detected between these two breeds, in which 316 were upregulated, and 91 were downregulated in the ovaries of JTGs versus TBGs. Based on the results of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment, some of these DEGs potentially play an important role in controlling the development of ovarian follicles. SRD5A2, MSMB, STAR and 3BHSD, etc. were the most significantly differentially expressed between these two distinct breeds. In addition, each ovary expressed 1,066 versus 989 novel transcripts, and 171,829 versus 140,529 putative SNPs in TBGs versus JTBs, respectively. All data sets (GEO and dbSNP) were available via public repositories. Our study provides insight into the transcriptional regulation of the ovaries of two distinct breeds of goats that might serve as a key resource for understanding goat fecundity. SRD5A2, MSMB, STAR and 3BHSD may be associated with the high fecundity of JTGs.

Identification of candidate miRNAs and expression profile of yak oocytes before and after in vitro maturation by high-throughput sequencing.

Small RNA represents several unique non-coding RNA classes that have important function in a wide range of biological processes including development of germ cells and early embryonic, cell differentiation, cell proliferation and apoptosis in diverse organisms. However, little is known about their expression profiles and effects in yak oocytes maturation and early development. To investigate the function of small RNAs in the maturation process of yak oocyte and early development, two small RNA libraries of oocytes were constructed from germinal vesicle stage (GV) and maturation in vitro to metaphase II-arrested stage (M II) and then sequenced using small RNA high-throughput sequencing technology. A total of 9,742,592 and 12,168,523 clean reads were obtained from GV and M II oocytes, respectively. In total, 801 and 1,018 known miRNAs were acquired from GV and M II oocytes, and 75 miRNAs were found to be significantly differentially expressed: 47 miRNAs were upregulated and 28 miRNAs were downregulated in the M II oocytes compared to the GV stage. Among the upregulated miRNAs, miR-342 has the largest fold change (9.25-fold). Six highly expressed miRNAs (let-7i, miR-10b, miR-10c, miR-143, miR-146b and miR-148) were validated by real-time quantitative PCR (RT-qPCR) and consistent with the sequencing results. Furthermore, the expression patterns of two miRNAs and their potential targets were analysed in different developmental stages of oocytes and early embryos. This study provides the first miRNA profile in the mature process of yak oocyte. Seventy-five miRNAs are expressed differentially in GV and M II oocytes as well as among different development stages of early embryos, suggesting miRNAs involved in regulating oocyte maturation and early development of yak. These results showed specific miRNAs in yak oocytes had dynamic changes during meiosis. Further functional and mechanistic studies on the miRNAs during meiosis may beneficial to understanding the role of miRNAs on meiotic division.

Molecular characterization and expression analysis of the NLR family CARD containing five transcripts in the pig.

The NOD-like receptor (NLR) family caspase recruitment domain-containing 5 (NLRC5) is one of the newly discovered and largest NLR family members. The NLRC5 has recently received extensive attention because of its important role in regulating innate and adaptive immune responses. The NLRC5 in many vertebrates, such as humans, mice, cattle, and horses, has already been proven and studied. However, the NLRC5 gene characteristics of pigs remain unclear. Thus, we completely cloned the NLRC5 cDNA sequence of the pig using the rapid amplification of cDNA ends(RACE) technology. A characteristic and tissue expression analysis was also conducted on the pig sequence. The sequence analysis showed that the complete cDNA sequence of the NLRC5 of the pig is 6638 bp, and the open reading frame is 5538 bp which encoded 1846 amino acids. The protein prediction analysis indicates that the overall performance of the NLRC5 protein of the pig is hydrophilic and possesses a typical nucleotide binding and oligomerization domain(NBD) and 20 leucine-rich repeats(LRRs). The homology analysis result indicates that the NLRC5 transcript in pigs is highly homologous to cattle, sheep, macaques, and humans, and accounts for around 80%. The genetic evolutionary tree analysis shows that the NLRC5 transcript in pigs has the closest evolutionary relationship with cattle and sheep. Further tissue expression analysis shows that immune organ systems (e.g., lymph node and spleen) and mucosa organs (e.g., intestinal lymph node, stomach, and lungs) possess high expressions with NLRC5 mRNA. The result of this study indicates that the NLRC5 transcript in pigs is relatively conservative among mammals and may play a vital role in immune reaction, which provides a basis for further studies on the NLRC5 function in the pig immune system and the role in comparative immunity.

Serological investigations on West Nile virus in birds and horses in Shanghai, China.

West Nile virus (WNV) infection is an emerging zoonosis that threatens global public health. In this study, a total of 95 bird serum samples from 14 species and 341 horse serum samples were collected from 2008 to 2010 in Shanghai, China. All serum samples were screened initially for WNV-reactive antibodies using a competitive ELISA. The positive samples detected by ELISA were further confirmed using a plaque-reduction neutralization test (PRNT) for WNV and its most closely related flaviviruses in the area to avoid false positives due to cross-reactivity. Five (5·3%) of the bird serum samples and none (0·0%) of the horse serum samples tested positive for WNV antibodies. The findings strongly suggest that some of the birds, specifically the resident birds in China, had been exposed to WNV.

Detection of astrovirus infection in pigeons (Columbia livia) during an outbreak of diarrhoea.

Avian astrovirus infections are widespread in many countries, and infections have been linked to enteritis and increased mortality in young poultry. Although pigeons are treated as an important poultry product in some countries, their diseases are often poorly understood and astrovirus infection in pigeons has not been reported. In the present study, faecal samples were collected during an outbreak of gastrointestinal illness in a population of Shanghai pigeons. The samples were examined for astroviruses by reverse transcription-polymerase chain reaction. Eighty-nine per cent (40/45) and 4% (2/45) were found to be positive for avian nephritis virus (ANV) and chicken astrovirus, respectively. One positive sample indicated a co-infection with both ANV and chicken astrovirus. Phylogenetic analysis based on the partial polymerase gene sequence and full-length capsid protein from published avian astrovirus sequences in GenBank revealed that the pigeon viruses detected in this study were evolutionarily closely related to chicken ANV. The present study provided evidence for the presence of astrovirus in pigeons and suggests that cross-infection between pigeons and commercial chickens was likely. Whether the astroviruses in pigeons were responsible for the diarrhoea remains to be determined.

Identification of differential abundances of mRNA transcript in cumulus cells and CCND1 associated with yak oocyte developmental competence.

The development of an accurate and noninvasive preselection process for competent oocytes is essential to achieve a highly efficient in vitro production (IVP) of embryos. Cumulus cells (CCs) have important functions in oocyte growth, development, maturation, and fertilization. It, therefore, is important to know if the quality of oocytes can be ascertained by assessment of gene expression of the surrounding CCs or not. The aim of this study was to identify differentially expressed genes in yak CCs from oocytes with varying developmental competences as possible biomarkers for distinguishing oocyte competence. The isolated CCs were pooled into immature and mature groups in accordance with the maturation outcome of oocytes. A total of 9516 genes were differentially expressed in the two CC categories (P <  0.05). With a minimum change of 2.5-fold, 45 up-regulated and 79 down-regulated genes were observed in CCs belonging to the mature group compared with those in the immature group (P <  0.01). These genes were primarily enriched for the cell cycle, meiosis, cell signaling, metabolism, and apoptosis. The selected candidate genes (CCND1, BMP15, GDF9, H19, KLF4, GPC1, SYCP3, and CTSB) were validated using quantitative real-time polymerase chain reaction (RT-qPCR) and there were expression patterns similar to those detected with transcriptome analysis. The CCs from fertilized oocytes arrested at the 2-cell (2-cell group), or 8-cell (8-cell group) stages or that developed into blastocysts (the blastocyst group) had a 1.5-, 1.8-, and 2.3-fold increase, respectively, in mRNA relative abundance of CCND1 compared with CCs from unfertilized oocytes (P <  0.05). The results with the RT-qPCR analysis confirmed that the relative abundance of CCND1 mRNA in CCs was associated with oocyte developmental competence. In conclusion, RNA-Seq is useful in extracting transcriptomes and selecting markers associated with oocyte developmental competence. Furthermore, the expression of the CCND1 gene in yak CCs can be used to preselect oocytes for IVP efficiency.

Segmentation expression of capsid protein as an antigen for the detection of avian nephritis virus infection in chicken flocks.

Subclinical pathological changes in the kidneys of broiler chickens and suppression of growth caused by the avian nephritis virus (ANV) affect poultry flocks worldwide. A test for detection of virus-specific antibodies in serum would be useful for epidemiological investigations, however the poor propagation in cell cultures has restricted the development of serological tests based on the use of ANV particles as antigens. An enzyme-linked immunosorbent assay (ELISA) was developed for detection of ANV-specific antibodies in chicken serum, using a recombinant protein antigen prepared by segmentation expression of the capsid protein antigen epitope of ANV (HM029238) transfected into Escherichia coli. The expressed fusion protein was detected by Western blotting with ANV-positive serum, and the optimal immunoreactive fusion P1 protein was determined. Using the optimized P1-ELISA, ANV-specific antibodies were detected in commercial chicken flocks aged 10-25 days obtained from the Liaoning Province, China. Out of 960 serum samples, 459 (47.8%) were positive for infection with ANV. These results indicate that the P1-ELISA is helpful for preliminary serological diagnosis of ANV infection, and could be used to for screening in ANV infection and for determining antibodies against ANV.

Molecular detection and sequence analysis of feline Torque teno virus (TTV) in China.

In the present study, two isolates (SH-F1 and SH-F2) of Torque teno felis virus (feline TTV) were detected in 16 (12.5%) serum samples collected from cats in China. Their full length genomes were cloned and sequenced. The results showed that they were 2063bp in length and contained three open reading frames (ORFs) (ORF1: nt438-1748, ORF2: nt268-585 and ORF3: nt268-581, 1461-1842). Phylogenetic analysis showed that they were clustered with the strain of Japan (Fc-TTV4, AB076003) and the strain of France (PRA4, EF538878). Sequence analysis indicated that SH-F1 had high (97.5% and 93.3%) identity with the strain of Japan and the strain of France, and SH-F2 shared 94.5% and 92.1% homology with them, respectively. In conclusion, we demonstrated that feline TTV is present in China.