Nucleotide variability of protamine genes influencing bull sperm motility variables.

Protamines (PRMs), important proteins of chromatin condensation in spermiogenesis, are promising candidate genes to explore markers of sperm motility. The coding and in-silico predicted promoter regions of these genes were investigated in 102 crossbred and 32 purebred cattle. Also, mRNA quantification was done to explore its possibility as diagnostic tool of infertility. The PCR-SSCP analysis indicated there were two band patterns only in fragment I of the PRM1 and fragment II of the PRM2 gene. The sequence analysis revealed A152G and G179A transitions in the PRM1 gene. Similarly, G35A, A49G and A64G transitions were identified in the PRM2 gene which resulted in altered amino acid sequences from arginine (R) to glutamine (Q), from arginine (R) to glycine (G) and from arginine (R) to glycine (G), respectively. This caused the reduction in molecular weight of PRM2 from 2157.66 to 1931.33 Da due to reduction in the number of basic amino acids. These altered properties of the PRM2 protein led to the reduction in Mass Motility (MM: P < 0.01), Initial Progressive Motility (IPM; P < 0.05) and Post Thaw Motility (PTM; P < 0.05) in crossbred bulls. The least squares analysis of variance indicated there was an effect of PRM2 haplotypes on MM (P = 0.0069), IPM (P = 0.0306) and PTM (P = 0.0500) in crossbred cattle and on PTM (P = 0.0408) in the overall cattle population. Based on the RT-qPCR analysis, however, there was not any significant variation of PRM1 and PRM2 gene expression among sperm of Vrindavani bulls with relatively lesser and greater sperm motility.

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.