Molecular Characterization of Corticotrophin Releasing Hormone Gene and its Expression in Deoni Cows
Abstract
Improving the production performance in dairy cattle is the main concern of breeding policies, as the production traits are low to medium heritable and it is difficult to select best animals based only on phenotype. Deoni is hardy indigenous cattle with moderate milk production potential. Identification of gene polymorphism and its association with production performance of animals can be very useful for selection of superior animals. The aims of study were: (i) Molecular characterization and expression of CRH gene in Deoni cattle, (ii) Association of CRH gene polymorphism with milk yield and fat content and (iii) Breeding value estimation based on lactation performance. Genomic DNA was isolated from 152 Deoni cattle maintained at SRS of ICAR-NDRI, Bengaluru to evaluate genetic variability through PCR-RFLP method. PCR-RFLP analysis revealed monomorphism in CRH gene and was found to be highly conserved with a single nucleotide change at 19th position in all the Deoni animals compared to Bos taurus sequence. Association studies could not be carried out further because of lack of polymorphism. Mean phenotypic performances of AFC, FLMY, and Fat content were 44.35 ± 0.48 months, 577.64 ± 32.54 kg, and 4.61 ± 0.06 per cent, respectively. Heritability of FLMY of Deoni cattle was found to be 0.24 ± 0.20. MPPA and breeding value for FLMY were estimated by LSA method using GLM procedure of SAS 9.3 based only on phenotypic data without incorporating the genotype information. MPPA was estimated to be 350.77 to 947.25 kg. PBV obtained through LSA method without taking genotype information were in the range from 139.32 to 1520.15 kg. Expression profiling of CRH gene was carried out in PBMC in eight Deoni cows indifferent lactations from the day of parturition to 60th day postpartum. Expression profiling of CRH mRNA was carried out using quantitative RT-PCR with SYBR green as fluorescent dye. Present study revealed a significant decrease of CRH mRNA expression from day 10 of calving today 60 post calving, indicating that levels of stress is higher during parturition than during lactation. This is the first report on characterization and expression of CRH mRNA in Deoni cows.
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Introduction
In dairy enterprise the principal goal of selection is to increase the milk yield and improve its composition. Milk production genes involved in controlling milk fat, protein content and the hormones are excellent candidate genes for linkage analysis with Quantitative Trait Loci (QTL) because of their biological significance on quantitative traits of interest. It is noteworthy that QTLs for milk performance traits has been mapped in all the bovine autosomes, mostly in 6, 14 and 20 [1] Corticotrophin-releasing hormone (CRH), also called as corticotrophin-releasing factor (CRF), is a 41 amino acid peptide deriving from a 191 amino acid precursor which is mainly synthesized in the hypothalamus, and also in other parts of brain. CRH functions as a neuropeptide hormone participating mainly in the stress response in vertebrates. CRH is involved in controlling the energy balance of an organism, which affects body weight and it also participates in modulating immune and reproductive systems [2]. CRH gene expression was found highest in hypothalamus, but the gene is also expressed in many other places, such as ovaries, testes, placenta, uterus, liver, stomach, skin, and immune system. Gene encoding corticotrophin releasing hormone with many QTLs has been mapped in bovine chromosome 14 [3], which govern postnatal growth and it has been considered as a candidate gene for growth traits in cattle [4]. Several single nucleotide polymorphic (SNP) sites have been identified within CRH gene of Bos taurus cattle breeds viz. C22G which causes substitution of amino acids in signal sequence, A145G and C240G, leading to changes of amino acids in a propeptide [4] as well as two SNPs in exon 2 [5]. The present study was undertaken to determine the frequencies of alleles and genotypes with regard to gene polymorphism encoding corticotrophin releasing hormone (CRH-A145G) in Deoni cows and to study the association between genotypes and milk production traits of Deoni cows along with expression levels of CRH mRNA in postpartum Deoni cows during different days of lactation as well as its effect on milk yield and also to estimate the heritability and breeding value of Deoni cows based on First Lactation Milk Yield (FLMY).
Conclusion
CRH gene primarily being a stress regulator was found to be highly conserved in the Deoni breed of Bos indicus cattle. The lack of polymorphism in CRH gene made it difficult to flag it as a candidate gene for production traits in indigenous dairy cattle breeds. Further studies on same gene in other zebu breeds of cattle are to be carried out to ascertain the polymorphic nature of gene. Expression studies of CRH mRNA revealed higher level of stress in Deoni animals during the parturition than the lactation stress. A positive correlation was found among the expression Relative Quantitation (RQ) values and the daily milk yield of Deoni animals, in which both of them revealed a declining trend from the day of calving today 60 post calving. Heritability of the FLMY was estimated to be 0.24 ± 0.20 and bulls used in the herd were ranked based on the Breeding values of FLMY calculated only on adjusted phenotype records excluding the genotypes. The study suggests scope for further evaluation of same gene indifferent Bos indicus breeds of cattle yielding higher quantum of milk to undertake appropriate breeding strategies to decrease the stress levels in lactating animals.
