Introduction

Milk production (lactogenesis) in a lactating cow is influenced by the interaction of various hormones of which Prolactin (PRL) hormone plays an important role. Prolactin hormone is mainly secreted by the anterior pituitary gland and exerts multiple effects on the mammary gland like initiation and maintenance of lactation (Bole – Feysot et al., 1998). It acts on mammary alveoli to promote the synthesis and secretion of milk proteins, lactose, lipids and other major components of milk (Le provost et al., 1994).

The Prolactin (PRL) gene has been mapped to Chromosome 23 (Hallerman et al., 1988). The PRL gene is 10 Kilo bases in size, consisting of 5 Exons (Camper et al., 1984). Extensive genetic polymorphisms have been detected in PRL exon 3 and 4 by PCR-RFLP analysis utilizing RsaI Restriction endonuclease (Mitra et al., 1995; Chrenek et al., 1998; Udina et al., 2001; Wojdak et al., 2008; Sacravarty et al., 2008). Genetic association studies by various researchers (Brym et al., 2005. Allipanah et al 2007) have shown a significant effect of PRL Exon 3 and Exon 4 polymorphisms with milk production traits. Hence the present study was undertaken to determine the genetic polymorphism of PRL Exon 3 and Exon 4 loci in native Deoni cattle breed and to associate the observed genetic polymorphisms with milk production traits.

Materials and Methods

Sampling and DNA Extraction

Blood samples were collected from 72 lactating Deoni cows maintained at NDRI Southern Campus, Bangalore. About 10 ml of whole blood was collected from each animal in EDTA coated vacutainer tube and stored at -20°C until DNA isolation. The genomic DNA was isolated from white blood cells utilizing High salt method (Miller et al., 1988). The quality and quantity of DNA were checked by using UV – spectrophotometer. The DNA samples were diluted to 100 ng / µl for utilizing as DNA template in PCR amplification reaction.

PCR Amplification of PRL Exon 3 and Exon 4 loci

Allele specific PCR amplification of PRL gene exon 3 and 4 regions were carried out utilizing specific primer sets as described by Lewin et al., 1992 for PRL Exon 3 and Brym et al., 2005 for PRL Exon 4 respectively (Table 1).

The PCR reaction for PRL Exon 3 was performed in a final volume of 25 µl containing 1x PCR buffer (10 mM Tris – HCl, 1.5 mM Mgcl₂, 50 mM KCl, pH-8.3), 100 µm each dNTPs, 5 pm each of forward and reverse primers, 100ng / µl DNA template and 1.5 U Taq DNA polymerase. The thermal cycling conditions was 94⁰C for 5 min followed by 30 cycles at 94°C for 1 min, 59⁰C for 40 sec (Annealing temperature), 72°C for 1 min followed by a final Extension  at 72 ⁰C for 5 min.

The PRL Exon 4 PCR reaction mixture was analogous to the above given composition with thermal cycling condition of 94°C for 5 min, followed by 30 Cycles of 94°C for 30 sec, 58.5°C for 1 min (Annealing temperature), 72°C for 1 min followed by a final Extension of 72°C for 5 min.

Restriction Enzyme Digestion

The PCR – RFLP analysis of PRL Exon 3 and Exon 4 was performed by utilizing the RsaI restriction enzymes.  Approximately 10µl each PCR products of PRL Exon 3 and Exon 4 were separately digested with 1 unit of RsaI restriction enzyme in a final volume of 15 µl as per manufactures instructions. The restriction digested DNA fragments were resolved using 10 % native PAGE (Polyacrylamide Gel Electrophoresis) and electrophoresed at 200 volts in 1X TBE for 4 – 5 hrs and the gels were resolved and stained using Ethidium Bromide. The gels were visualized and documented using BIO-RAD Gel documentation system and band scoring was done manually.

Estimation of Milk Fat and Protein Percentage

Milk samples (10 ml from each cow) were collected from the same 72 Deoni lactating cows from which the blood was collected. The Fat percent of milk samples was estimated using the standard Gerber method (Gerber, 1935) and the protein percent was estimated using Kjeldahl method.

Statistical Analysis

Gene and genotypic frequencies of PRL Exon 3 and 4 alleles which were observed in Deoni cattle breed population were determined by direct counting and individual allele frequencies were calculated as follows:

H_i = ∑n_i / N

Where H_i is the frequency of allele i, n_i is the number of allele i in a population and N is the total number of alleles in the population.

Hardy – Weinberg (HW) equilibrium was tested for PRL exon 3 and PRL exon 4 genotypic frequencies and the possible deviations from HW equilibrium probability values was estimated using Chi – Square (χ²) test. The observed heterozygosity and gene diversity (H_E) of PRL Exon 3 and PRL Exon 4 alleles was estimated for Deoni population. All the above analysis was performed using POPGENE Version 1.32 Software (Table 3).

Genetic association of PRL Exon 3 and PRL Exon 4 genotypes with production performance traits was analyzed using General Linear Model (GLM) of SPSS Version 17 statistical software. The effect of PRL genotypes on milk production traits (Lactation Milk Yield, Lactation Length, Fat Percent and Protein percent) were estimated using the formula:                                        `           Y_i = µ + G_j + e_ij

Where,

Y_i = i^th production trait (LMY, LL, FP and PP) of animal in j^th genotype.

µ = overall mean

G_j = effect of j^th genotype (where j =1, 2 and 3 genotypes of Exon 3 and 4)

E_ij   = Random error associated with Y_ijk observation, and assumed to be NID (0, s²e).

Results

The allele specific PCR amplification of PRL Exon 3 and Exon 4 loci resulted in clear DNA bands of expected base pair sizes of 156bp for PRL exon 3 and 294bp for PRL exon 4 respectively.

Table 1: Primers utilized for gene specific PCR amplification of PRL gene Exon 3 and Exon 4 fragments

Table 2: Genotypic and allelic frequencies observed for PRL gene Exon 3 and Exon 4 in Deoni cows

Table 3: The Expected and observed heterozygosity, homozygosity and Hardy Weinberg Equilibrium analysis of PRL Exons 3 and 4 in Deoni cattle using POPGENE

Table 4: Genetic association of PRL Exon 3 genotypes with Least Square Means ± S.E of milk production traits in Deoni cows

Means followed by different superscript letters within columns differ significantly (P ≤ 0.05)

Table 5: Genetic association of PRL Exon 4 genotypes with Least Square Means ± S.E of milk production traits in Deoni cows

Means followed by different superscript letters within columns differ significantly (P ≤ 0.05)

            Fig.1: PRL Exon 3 Gel                                                                       Fig.2: PRL Exon 4 Gel

PCR RFLP Analysis of PRL Exon 3 and PRL Exon 4 loci

The PCR RFLP analysis of PRL Exon 3 in Deoni population revealed three genotypes viz., AA, AB and BB. The genotypic frequencies of AA, AB and BB genotypes were 0.097, 0.58 and 0.32 respectively. Thus the allele A was observed in the frequency of 0.39 and B allele in the frequency of 0.61.

Similarly PCR RFLP analysis of PRL Exon 4 revealed three genotypes viz., AA, AG and GG. The genotypic frequency of AA was 0.097, GG was 0.18 and AG was 0.72. Thus the A allele observed in the frequency of 0.488 and the G allele in the frequency of 0.542.

Population Genetic Analysis of PRL Exon 3 and Exon 4 Genotypes

The PRL exon 3 in Deoni population showed the observed heterozygosity value of 0.583 which was comparatively higher than the expected heterozygosity value of 0.478. Similarly the PRL exon 4 in Deoni population showed the observed heterozygosity value of 0.722 which was higher than the expected heterozygosity value of 0.500. The above heterozygosity values observed reveals a high degree of heterozygosity maintained in both the PRL Exon 3 and PRL Exon 4 genotypes of Deoni population.

The Chi-square (χ²) test for determining the Hardy-Weinberg equilibrium showed Chi-square (χ²) values of 3.499 and 14.425 for the PRL Exon 3 and PRL Exon 4 genotypes in the studied Deoni population, respectively. The PRL exon 4 genotypic frequencies deviated significantly from HW equilibrium probabilities showing a high probability value of 0.000146 (P ≤ 0.05) which may be due the significant variation observed between the heterozygotic and homozygotic ratios, whereas the PRL exon 3 genotypic frequencies did not deviate significantly from HW equilibrium probabilities.

The PRL Exon 3 and PRL Exon 4 revealed high negative Fis values of -0.227 and -0.454, respectively, the negative Fis value observed clearly indicates a heterozygosity excess in the studied Deoni population.

Genetic Association Studies of PRL Exon 3 and PRL Exon 4 with Milk Production Traits

The polymorphisms in Exon 3 and Exon 4 were associated with milk production traits in order to find possible correlation between specific genotypes and milk production traits using GLM analysis available in SPSS 17 software.

The genetic association between PRL Exon 3 genotypes and lactation milk yield revealed the PRL genotype BB was associated with highest lactation milk yield (1007.354 ± 92.328 kg) followed by AA and AB genotypes, respectively (Table 2). The heterozygotic PRL Exon 3 genotype AB was associated with highest fat percentage (4.780 ± 0.126) and highest protein percent (3.290 ± 0.033) followed by BB and AA genotypes, respectively (Table 2). Genetic association with lactation length showed the highest lactation length (224.750 ± 18.635 days) being associated with PRL Exon 3 genotype BB followed by AB and AA genotype, respectively. Even though some variations in the least square means of milk production traits in relation to specific PRL Exon 3 genotypes were observed in the Deoni population, the General Linear Model of SPSS (version 17) using Multivariate analysis of variance performed to determine any significant variations in the estimated least square means of milk production traits in relation to PRL Exon 3 genotypes revealed no significant probability values in the studied population (Table 2).

Similarly, in PRL Exon 4 of Deoni population, the General Linear Model of SPSS using ANOVA to determine any significant variations in the estimated least square means of milk production traits in relation to PRL Exon 4 genotypes revealed no significant probability values in the studied population (Table 3). Small variations observed in the least square means of milk production traits in relation to specific PRL Exon 4 genotypes are as follows; The PRL Exon 4 genotype GG was associated with highest milk yield (994.638 ± 101.100 kg) followed by AG and AA genotypes, respectively (Table 3). The PRL Exon 4 heterozygotic genotype AG was associated with highest fat percent (4.743 ± 0.121) and highest protein percent (3.287 ± 0.031), respectively followed by AA and GG genotypes (Table 3). And the highest lactation length (220.000 ± 22.308 days) was associated with PRL exon 4 genotype GG followed by AG and AA genotypes (Table 3).

Discussion

The genetic characterization and association studies of important genes of the Quantitative Trait Loci (QTL) influencing specific traits is one of the useful methods to determine the genetic polymorphism and to analyze the influence of observed genetic polymorphisms of the studied candidate genes on the economic traits in dairy animals. In marker-assisted selection of dairy cattle for milk production, some genes are proposed as potential candidates associated with dairy performance traits. Among the various candidates genes studied, the Prolactin gene seems to be promising, because of the crucial role played by Prolactin hormone during mammary gland development, initiation and maintenance of lactation and expression of milk protein genes. Determining allelic variation in the structural and regulatory sequences of the Prolactin gene can provide us with insight into the possible direct or indirect role played by PRL genetic polymorphisms on milk production and composition. In the above consent the PRL gene Exon 3 and Exon 4 were analyzed by PCR-RFLP technique utilizing RsaI restriction enzyme to determine the genetic polymorphisms and to associate the observed genetic polymorphisms with milk production traits in native Deoni cattle breed population.

The genotyping of PRL Exon 3 in Deoni breed revealed allele frequencies of 0.3889 and 0.6111 for A and B alleles, respectively. Allele frequency distribution as shown above was previously reported for Jersey breed (Dybus., et al 2005) which showed a lower frequency of 0.3065 for A allele and higher frequency of 0.695 for allele B. Similarly, in Red Sindhi Bos indicus cattle breed (Kumari. et al 2008) the frequency of A allele was 0.46 and B allele was 0.54 respectively. Whereas, in most of the previously studied cattle breeds viz. Kankrej cattle (Sacravarthy., et al 2008), Korean cattle (chung., et al 1998) Black and white cattle (Dybus., et al 2002), Vechur and kassargode cattle (Arvindakshan., et al 2004) showed contrasting distribution of PRL exon 3 alleles showing predominantly higher frequency of A allele when compared to B allele, respectively.

The genotyping of PRL Exon 4 in Deoni breed revealed allele frequencies of 0.4883 for allele A and 0.5416 for G allele. Similar allele frequency distribution observed for Deoni breed was previously reported for Black and White cattle breed (Brym et al., 2005) which showed allele frequencies of 0.113 and 0.887 for A and G alleles respectively. Whereas in Jersey breed (Brym et al., 2005) the allele A showed higher frequency of 0.706 and allele G showed lower frequency of 0.294 which is contradictory to the allele frequency observed in the studied breed.

The genetic associations of individual PRL Exon 3 and Exon 4 genotypes with least square means values of various milk production traits in Deoni breed, revealed very small differences among the observed milk production traits in relation to individual PRL Exon 3 and Exon 4 genotypes, thus producing non significant p-values as estimated by Duncan’s Multiple Range Test (DMRT). The non-significant genetic association of PRL genotypes with milk production traits observed in Deoni breed may be due to small no of samples utilized for the study. Thus much wider study involving more number of Deoni animal samples must be carried out to determine the potential genetic association of PRL genotypes with milk production traits.

Significant associations between specific PRL Exon 3 genotypes and Milk production traits have been previously reported in various breeds. Similar genetic associations observed for PRL Exon 3 with milk production traits in Deoni population was previously reported in Russian Red Pied cattle where the PRL Exon 3 RsaI genotype BB was associated with higher milk yield and genotype AB was associated with higher milk fat percentage (Alipanah et al., 2007a). Whereas contradictory to the results observed in the studied Deoni breed has been previously reported in various breeds (Alipanah et al., 2007b. Khatami et al., 2005; Brym et al., 2005; Dybus, 2001; Chung et al., 1997).

The results of genetic associations between specific PRL Exon 4 genotypes and milk production traits observed for the studied Deoni population were not in accordance with previously studied breeds. For instance, in Black and White cattle breed PRL Exon 4 genotype AG was associated with higher milk yield and genotype GG was associated with higher fat content (Brym et al., 2005). Jersey cattle breed showed that the PRL Exon 4 genotype AA was significantly associated with higher fat yield (Brym et al., 2005).

The significant variations in the frequency distribution of PRL Exon 3 and Exon 4 genotypes and the genetic association studies performed in Deoni population reveal a unique distribution of PRL genotypes in the studied population along with some differences in the milk production traits with respect to individual PRL genotypes. Thus the above study provides a preliminary insight into considering PRL gene as a potential genetic marker for genetic characterization and genetic association studies with various production and reproduction traits.

Further rigorous genetic analysis of PRL and other PRL regulatory gene like STAT5 and their genetic association studies involving a large population of indigenous Bos indicus and exotic Bos taurus cattle breeds under various geographical locations may provide us with in depth information, optimal genetic characterization and significant genetic association results, which could be utilized for considering PRL gene and its regulatory genes as potential genetic markers for genetic breeding and marker assisted selection programs.

Acknowledgements

The authors wish to express their thanks to the Director, National Dairy Research Institute and ICAR for funding this project and providing the facilities to carry out the research work..

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