Buffalo milk is the most preferred commodity in Pakistan due to high fat contents. Hydrolysis of circulating triglycerides and uptake of fatty acids in the mammary gland is regulated by lipoprotein lipase (LPL). This study is designed to identify single nucleotide polymorphisms (SNPs) in LPL gene among high and low milk producing buffalo breeds of Pakistan. We selected samples (n=50) of each Nili-Ravi a high milk producing and Azakheli a low milk producing buffalo breeds. Blood samples were collected for DNA extraction. LPL region of exon 2 region along with exon/intron boundaries were sequenced and data was analyzed for variation detection. Allele frequency was calculated using Hardy-Weinberg equation and in-silico analysis was performed for functional prediction and genetic diversity assessment. We found one single nucleotide polymorphism c.258+43C GT T in the intronic region of LPL gene. This polymorphism followed the Hardy-Weinberg equilibrium in both Nili-Ravi (P-value = 0.538) and Azakheli (P-value = 0.077). Association of T-allele analysis predicted it significantly higher (P-value = 0.009) in Nili-Ravi. We found one novel SNP (c.258+43C GT T) in the LPL gene in both high and low milk producing buffalo breeds of Pakistan but high in Nili-Ravi. Lower splice site effect suggests its less strength of association with milk producing trait.

Mannose Binding Lectins (MBL) gene possibly contributes to bacterial infection resistance and was proposed as a molecular marker for milk production traits to control mastitis A total of 60 Murrah buffaloes were selected to characterize exon 2 of MBL1 gene to identify polymorphism and its association with mastitis. A 401 bp PCR fragment of MBL1 gene targeting g.2686T GT C was amplified and digested with HaeIII restriction enzyme. Genotype analysis using PCR-RFLP revealed a monomorphic CC banding pattern. Sequencing was also carried out to explore other SNPs. The result indicates highly conserved DNA sequence in Murrah buffalo. This study provides preliminary information that the targeted region of MBL1 gene in water buffalo has no significant association with mastitis resistance which maybe a breed specific characteristic. Since present study has formulated the results based on a relatively small sample, further studies are required to validate these results in large samples.

Outcome of bovine tuberculosis (bTB), an infectious disease caused by Mycobacterium bovis, is influenced by host genetic background. For this reason, polymorphism discovery association studies are a powerful tool for selective breeding helping disease control. Interleukin-10 (IL-10) is a regulatory cytokine produced by different cells and fine-tune immune response to bTB. Thus, in this research, we evaluated the role of the single nucleotide polymorphism g.4002C GT T in IL-10 gene for susceptibility to bTB in Mediterranean water buffalo. We characterized 184 animals grouped in cases and controls and demonstrated that homozygous subjects TT are about 3 times more susceptible to bTB compared to CC homozygous. Indeed this polymorphism is responsible of amino acid substitution p. (Thr 152 Met) in the primary protein sequence which affects protein secondary structure. This polymorphism might represent a valid tool for marker assisted selection against bovine tuberculosis in water buffalo.

Systematic studies on expression and characterization of toll like receptors (TLRs) in buffalo and cattle will help to understand the innate immune response specific to each species of animals. TLR4 gene is one of the most studied innate immune receptors which is mainly triggered by lipopolysaccharide. Therefore, the present study was carried out to investigate the mRNA expression and sequence analysis of TLR4 in buffalo in comparison to that of cattle. In vitro expression of TLR4 was assessed by challenging the Peripheral Blood Mononuclear Cells (PBMC) with bacterial LPS which showed significant increase of TLR4 gene expression (P LT= 0.01) in buffaloes when compared to cattle. Further the comparison of mRNA sequence of TLR4 of buffalo and cattle revealed 96 percent similarity with the cattle sequence. The primary structure of protein showed the highest percent of leucine amino acid for both the species of animals. At the same time, the number of Leucine Rich repeats (LRR) of buffalo is varied from those present in cattle TLR4. Alpha helix is the prominent secondary structure, however, there were few amino acid variations between buffalo and cattle in certain regions of TLR4 which altered the three dimensional conformation of TLR4 proteins, this in-turn possibly can influence the binding affinity and interaction with pathogens which may influence the difference in immune response in buffalo when compared to that of cattle.