Identification of sperm-specific proteins associated with high fertility of Punjab Red and RIR x Local cross roosters with SDS-PAGE and Immunoblotting.

The present study aimed at characterization of fertility-associated proteins in PR and RIR x Local roosters and was conducted on two generations of birds. Roosters were divided into high- (>50%) and low-fertility groups (<50%) based on sperm function tests and fertility rate in both the generations. Polyclonal antibodies were raised in rabbits against sperm proteins of first generation highly fertile roosters and tested for characterization of fertility-associated sperm proteins in the second generation of same roosters. IgG fraction against proteins (anti-SP IgG) was reacted with sperm proteins of both high and low fertile roosters of second generation on immunoblots. SDS-PAGE of sperm extracts of PR and RIR x Local cross breeds resulted in resolution of 12 and 23 proteins on 12% acrylamide gels and anti-SP IgG reacted only with 8 and 9 sperm proteins of PR and RIR x Local cross roosters on immunoblots. The SDS-PAGE and immunoblotting analysis also indicated a variation in sperm proteins among two breeds and high/low fertile roosters. It can be concluded that the selection of roosters on the based on proteins of 65/ 25; 70/ 46/ 30 kDa may be specifically associated with high fertility of PR and RIR x Local cross, respectively. The proteins 62 kDa (PR) and 40kDa (RIR x Local cross) may be specifically responsible for low fertility.

Profiling of sperm gene transcripts in crossbred (Bos taurus x Bos indicus) bulls.

Crossbred cattle in some sectors of the world have a significant role in enhancing milk production thereby enhancing the per capita milk availability as a human food source. However, there are certain constraints associated with crossbred animals, such as disease susceptibility, increased reproductive problems, repeat breeding and poor seminal quality. The semen of crossbred bulls has a poor freezing capacity, increased cryo-damage, poor mass cell motility, greater percentages of dead/abnormal sperm and poor initial and post-freeze cell motility. The rejection rate of crossbred bulls for cryostorage of semen has been reported to be as great as 50% as a result of unacceptable semen quality. The identification of superior bulls using molecular technologies is needed which necessitates identification of the genes having a role in sperm function. The present study was, therefore, conducted to gain information on identification and expression of genes having a role in sperm motility in crossbred bulls. The gene transcripts in bulls with sperm of superior and inferior quality were profiled in Vrindavani crossbred cattle by microarray analyses and the results were verified by real time-quantitative PCR. Microarray analyses revealed 19,454 genes which were differentially expressed. At a two-fold cut off, 305 genes were differentially (P<0.01) expressed with 160 genes upregulated and 145 genes down regulated. Some of the upregulated candidate genes were further validated by RT-qPCR. These genes had a four to 16 fold upregulation in sperm with inferior motility as compared to sperm of crossbred bulls with superior motility.

In Silico Characterization of Functional Divergence of Two Cathelicidin Variants in Indian Sheep.

The present work focuses on the in silico characterization of functional divergence of two ovine cathelicidin coding sequence (cds) variants (ie, Cath1 and Cath2) of Indian sheep. Overlapping partial cds of both the cathelicidin variants were cloned in pJet1.2/blunt vector and sequenced. Evolutionary analysis of the Cath2 and Cath1 indicated that the mammalian cathelicidins clustered separately from avian fowlicidins. The avian fowlicidins, which are very different from mammalian cathelicidins (Caths), clearly displayed signatures of purifying selection. The pairwise sequence alignments of translated amino acid sequences of these two sheep cathelicidins showed gaps in the antimicrobial domain of Cath1 variant; however, the amino terminal cathelin regions of both the Caths were conserved. Amino acid sequence analysis of full-length cathelicidins available at public database revealed that Cath1, Cath2, and Cath7 of different ruminant species (including our Cath1 and Cath2 variants) formed individual clads, suggesting that these types have evolved to target specific types of microbes. In silico analysis of Cath1 and Cath2 peptide sequences indicated that the C-terminal antimicrobial peptide domain of Cath2 is more immunogenic than that of the ovine Cath1 due to its higher positive antigenic index, making Cath1 a promising antigen for production of monoclonal antibodies.

Sex determination in 6 bovid species by duplex PCR.

Sex determination in domestic animals is of potential value to livestock breeding programs. The aim of this study was to develop a simple and accurate PCR-based sex determination protocol, which can be applicable to 6 major domesticated species of the family Bovidae, viz. Bos frontalis, B. grunniens, B. indicus, Bubalus bubalis, Capra hircus, and Ovis aries. In silico analysis was done to identify conserved DNA sequence in the HMG box region of the sex-determining region of the Y-chromosome (SRY gene) across the bovids. Duplex PCR assay, including the SRY gene and the GAPDH housekeeping gene, was optimized by using genomic DNA extracted from blood samples of known sex. It was possible to identify the sex of animals by amplifying both gender-specific (SRY) and autosomal (GAPDH) genes simultaneously in the duplex reaction, with the male yielding two bands and the female one band. The protocol was subjected to a blind test that showed a 100 percent specificity and accuracy, thus it can be used in sex determination in livestock breeding programs.