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Abstract

Lactoferrin (LF), an iron-chelating 80-kDa glycoprotein closely related in structure to iron transport protein transferrin, known as red protein. LF gene is highly conserved among various species and considered as one of the candidate genes for endometritis and mastitis resistance. This study aimed to detect single-nucleotide polymorphisms (SNPs) of lactoferrin gene and if there is any correlation between lactoferrin gene diversity and reproductive status of Egyptian buffaloes. A total of 150 buffaloes were subjected to ultrasonographic examination for diagnosis of fertility and reproductive disorders. Blood samples were collected from 88 fertile, 24 repeat breeder and 38 aneustrum buffaloes. Lactoferrin gene fragment of 301 bp was amplified from isolated DNA samples and PCR-RFLP was performed on the amplified fragment using EcoRI restriction enzymes. Purified PCR amplicons of different electrophoresis patterns were sequenced in both directions using forward and reverse primers. The sequences results were analyzed using NCBI BLASTn and BioEdit Sequence Alignment software. Phylogenetic analysis using UPGMA tree construction for each gene was constructed with consensus sequences. Two alleles of lactoferrin gene; A and B were detected in examined buffaloes. Genotype AA represented with 61.9% and 38.1% in fertile and infertile buffaloes respectively, while the AB genotype was detected with 54.54% and 45.5% in fertile and infertile one. Lactoferrin genotype (AA) showed significant increase in normal fertile animals (P=0.006) in comparison to anestrum and repeat breeder animals. Sequence analysis of both normal and polymorphic one shows point mutations (substitution A/C) in position 54 and 132. Statistically, our results showed significant correlation between the two genotypes of lactoferrin gene (AA and AB) and buffalo reproductive status (fertile and infertile). UPGMA analysis, revealed that the Egyptian buffalo sequence (KX228134.1) was closer to Bubalus bubalis (EU192148.1) and (HM125146.1) sequences compared to those of hybrid cattle. The polymorphisms in lactoferrin gene detected in this study are selectively positive in relation to the improvement of fertility of Egyptian buffaloes. The recorded SNPs may be used as genetic marker for improving buffalo breeding based on marker assisted selection (MAS) programs. This study considered the 1st study on genetic polymorphism of lactoferrin gene in relation to fertility of Egyptian buffaloes. The SNPs in lactoferrin gene may considered as a useful genetic marker for buffalo breeding in Egypt.