Bioinformatics analysis of candidate genes for milk production traits in water buffalo (Bubalus bubalis).

Water buffalo (Bubalus bubalis) is an important livestock species in developing countries due to its contribution to meat, milk production, and a certain form of labor. However, the genetic potential of buffalo milk production traits has not been fully exploited. To date, 516 candidate genes associated with milk production traits of buffalo have been identified. The present study aimed to explore the possible molecular mechanisms underlying milk production traits of this species through functional genomics analysis of these candidate genes by using different bioinformatics tools. Gene ontology (GO) analysis indicated that these candidate genes were associated with complex biological processes, such as cell proliferation and mitotic nuclear division. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis demonstrated that these candidate genes were enriched in multiple signaling pathways, such as AMPK, ErbB, Toll-like receptor, and Jak-STAT. In addition, one function module consisting of 57 nodes and 139 edges were identified from the protein-protein interaction (PPI) network. GO analysis showed that the 57 candidate genes in this function module were enriched in three main biological processes, including homeostasis, metabolism, and cell response. These three distinct biological processes are well known for regulating mammary gland activities, which explained clearly the mechanism underlying milk production traits. This study provides a novel perspective for better understanding of the biological processes linked with milk production traits. This knowledge is conducive to the improvement of milk yield and composition of this species.

Polymorphisms in the bone morphogenetic protein 15 gene and their effect on sperm quality traits in Chinese Holstein bulls.

Bone morphogenetic protein 15 (BMP-15) expression has been detected in the testis, but its roles in this organ has not been well elucidated. We evaluated polymorphisms of the BMP-15 gene by PCR-SSCP and PCR-RFLP in 212 Chinese Holstein bulls, and investigated possible associations with sperm quality traits, including semen volume per ejaculate, sperm density, fresh sperm motility, thawed sperm motility, acrosome integrity rate, and abnormal sperm rate. A single nucleotide polymorphism (C5697T) in intron 1 of the BMP-15 gene was identified in these bulls. Age was found to have significant effects on both fresh sperm motility and abnormal sperm rate. A significant effect of genotype on fresh sperm motility was also observed. Least square analysis showed that CT genotype bulls had significantly lower fresh sperm motility than CC or TT genotype bulls. In conclusion, BMP-15 should be considered as a potential genetic marker for sperm quality, based on its association with fresh sperm motility.

Effects of the polymorphisms of Mx1, BAT2 and CXCL12 genes on immunological traits in pigs.

It is necessary that genetic markers or biomarkers can be used to predict resistance towards a wide range of infectious diseases. In the present study, we estimated the potential markers and measured their relationship with heritabilities of a wide range of immune traits. Polymorphisms in exon 13 of Mx1, intron 25 of BAT2 and intron 3 of CXCL12 were identified by sequencing, and the genotypes were analyzed by PCR-RFLP in a resource population composed of 352 pure breed Landrace piglets at days 0, 17 and 32 after birth. Associations of single-nucleotide polymorphisms (SNPs) in these genes with a variety of immunological traits and antibody levels for pig reproduction and porcine respiratory syndrome virus (PRRSV), pseudorabies virus (PRV) and classical swine fever virus (CSFV) were performed. The performance of GG genotype of BAT2 on hemoglobin concentration (HBG) and hematocrit (HCT) of piglets at day 0 was significantly higher than that of the AA and AG individuals. For Mx1, compared with CT genotype, the pigs with TT or CC generated more PRRS antibody at day 0. The piglets with CT genotype had highly significant difference of PRV antibody from those with CC and TT genotypes at day 0. And the piglets with CC genotype had higher level red blood cell count (RBC), hemoglobin concentration (HBG) and hematocrit (HCT) than those with CT and TT genotypes at day 17. For the C7462G SNP in the intron 3 of CXCL12, the PRV antibody level of piglets with the CG genotype were higher than that of piglets with CC and GG genotypes at day 17, and the mean corpuscular volume (MCV) of GG piglets were larger than that of CC and CG individuals at day 0. At the locus 7331 bp in the intron 3 of CXCL12, there were significantly differences of mean corpuscular hemoglobin concentrations (MCHC) at day 0 and white blood cell count (WBC) at day 32, which showed the trend GG or AG>AA, AA>AG or GG, respectively. The pigs with AA or GG genotype had more platelet distribution width (PDW), mean platelet volume (MPV) and platelet-large cell ratio (PLR) at day 17 than those with AG. The results of this study indicated that polymorphisms in Mx1, BAT2 and CXCL12 genes were significantly associated with the immunological traits in Landrace piglets and had potential application value for marker-assisted selection of pig breeding with disease resistance.

An immunological method to screen sex-specific proteins of bovine sperm.

This study was designed to identify sex-specific antibodies (SSAb) in rabbit antisera against bovine sex-sorted sperm, and capture sex-specific proteins of bovine X- or Y- proteins by SSAb. The rabbit antisera against bovine X- or Y-sperm were first produced by a series of immunological approaches, and further purified through immuno-neutralization with excess sex-sorted Y- or X-sperm, respectively, to remove non-sex specific antibodies and enrich sex-specific antibodies. After removal of non-sex specific antibodies, the purified rabbit sera with enriched sex-specific antibodies were screened for sex-specific antibodies by immunofluorescence staining and flow cytometry. The results showed that 3.0, 2.2, and 4.2% of unsorted sperm, sex-sorted X-sperm, and sex-sorted Y-sperm were recognized by the purified rabbit antisera against Y-sperm, respectively, whereas 29.2, 19.7, and 3.9% of unsorted sperm, sex-sorted X-sperm, and sex-sorted Y-sperm were recognized by the purified rabbit antisera against X-sperm. These results suggested that the purified rabbit antisera against X-sperm contained SSAb that preferentially bound to sex-sorted X-sperm. Subsequently, the purified rabbit antisera against X- or Y-sperm were used to immunoprecipitate sex-specific proteins in bovine sperm proteins, and a 30-kDa protein was specifically captured by the rabbit antisera against X-sperm. In conclusion, our results implied that this 30-kDa protein might be a sex-specific protein in bovine X-sperm, which has the potential to be used in immunological procedures for sexing sperm.

Identification of complex vertebral malformation carriers in Holstein cattle in south China.

Complex vertebral malformation (CVM) is a recently described monogenic autosomal recessive hereditary defect of Holstein dairy cattle that causes premature birth, aborted fetuses and stillborn calves. Guanine is substituted by thymine (G>T) in the solute carrier family 35 member A3 gene (SLC35A3). A valine is changed to a phenylalanine at position 180 of uridine 5'-diphosphate-N-acetyl-glucosamine transporter protein. CVM is expected to occur in many countries due to the widespread use of sire semen. We developed a created restriction site PCR (CRS-PCR) method to diagnose CVM in dairy cows. This was tested on 217 cows and 125 bulls selected randomly from a Holstein cattle population in south China. Five Holstein cows and five Holstein bulls were identified to be CVM carriers; the percentages of CVM carriers were estimated to be 2.3, 4.0 and 2.9% in the cows, bulls and entire Holstein cattle sample, respectively.

Effects of parity on uterine involution and resumption of ovarian activities in postpartum Chinese Holstein dairy cows.

Favorable uterine involution and ovarian activity are very important for the next reproductive cycle of postpartum cows. The objective of this study was to evaluate the effect of parity on uterine involution and resumption of ovarian activity in Chinese Holstein dairy cows after calving under similar postpartum nutritional conditions. Traits of the status of uterus and ovaries detected by ultrasonography, dry matter intake (DMI), milk yield, body condition score (BCS), and estradiol concentration in milk samples were analyzed for 46 Chinese Holstein dairy cows in various parities (primiparous=18; biparous=13; multiparous=15). The results showed that there was no significant difference for DMI, BCS, and milk yield among different parities; all cows were considered to be under similar nutritional conditions. Days of the previous gravid uterine horn involution were significantly greater in primiparous dairy cows than in biparous and multiparous dairy cows. Days from calving to ovulation (first and second) and the number of follicular waves to first ovulation were significantly greater in primiparous cows than in multiparous cows. In summary, there was a significant negative relationship between parity and postpartum uterine involution and resumption of ovarian activity in Chinese Holstein cows under similar body conditions.

Construction of a fusion protein expression vector pGS/2SS-M4GFP without antibiotic resistance gene and its subcellular localization in different cell lines.

A novel plasmid pGS/2SS-M4GFP was constructed in the present study by recombination of GS/2SS gene and enhanced green fluorescent protein (M4GFP) sequence. The GS/2SS fusion gene encoding two copies of somatostatin genes was firstly introduced into pVAX-asd vector in which the kanamycin resistance cassette was replaced by the asd cassette. The M4GFP gene was then fused into 3' end of GS/2SS gene in the proper reading frame. After purified, plasmid pGS/2SS-M4GFP was transfected into different cell lines derived from pig kidney and human cancer cells. The transcription process of GS/2SS gene was confirmed by RT-PCR, and the localization as well as expression of GS/2SS-M4GFP fusion protein was observed by confocal microscopy and ELISA. Transfection results revealed that sole M4GFP was localized within the cytosol and the nucleus, while fusion protein GS/2SS-M4GFP was localized only in the cytoplasm. Furthermore, it should be noted that subcellular localization of GS/2SS-M4GFP was not specific to one cell line, but appeared to be common across a variety of cell lines. These results provide for the first time valuable evidence that M4GFP is a versatile tool to trace GS/2SS protein and pave the way for further study on its tissue distribution and immunological mechanism in vivo.

Polymorphism of the growth hormone gene and its association with growth traits in Boer goat bucks.

In the present study, the polymorphism of growth hormone (GH) gene was analyzed as a genetic marker candidate for growth traits in Boer goat bucks. Two single nucleotide polymorphisms (SNPs) - A781G (Ser/Gly35) and A1575G (Leu147), were identified by GH gene sequencing and PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) analysis. AA genotype resulted in a significant decrease in birth chest girth (P=0.03) and weaning weight (P=0.014) comparing to AB genotype, while CC genotype contributed to weaning height (P=0.04) greater than CD genotype. When in combination, AACD genotype was undesired for lower scores in a series of growth traits including body weight, length, height, and chest girth at birth and weaning, as well as the pre-weaning daily gain and body weight at age of 11 months. These results indicate that new molecular markers associated with caprine growth traits can be used in MAS (marker-assisted selection) in Boer goat bucks.

Evaluation of the VP22 gene adjuvant for enhancement of DNA vaccine against somatostatin in mice.

Somatostatin (SS) is a hormone that inhibits the secretion of growth hormone. Immunization against SS can promote the growth of animals. A novel SS-VP22 fused vaccine, pEGS2SS-V, was constructed from pEGS2SS plasmid with a VP22 gene fragment. Two times of immunization with pEGS2SS-V-induced anti-SS antibodies in mice. Compared with mice immunized with pEGS2SS and 0.85% saline, the growth performance of mice immunized with pEGS2SS-V was increased by 14.1% (P < 0.05) and 48.4% (P < 0.01) on the 2nd week after the first vaccination, respectively. The results indicated that the effects of the somatostatin DNA vaccine could be improved effectively by VP22 gene adjuvant.