Introduction
Prolactin (PRL) is the best known hormone for the multiple effects it exerts on the mammary gland. The varied effects of PRL on the mammary gland include growth and development of the mammary gland (mammogenesis), synthesis of milk (lactogenesis) and maintenance of milk secretion (galactopoiesis). Hence, PRL gene is a potential quantitative trait locus and genetic marker of production traits in dairy cattle (Brym et al, 2005). Bovine PRL gene has been located on chromosome 23 and is composed of five exons and four introns with an overall length of approximately 10 kb encoding the 199 amino-acid mature protein (Camper et al, 1984). A silent A→G transition mutation at the codon for amino acid 103 in exon-3 of size 156 bp bovine PRL gene gives rise to a polymorphic Rsa I (Rhodopseudomonas sphaeroides). This site has become a popular genetic marker used for genetic characterization of cattle populations by means of PCR-RFLP analysis (Chrenek et al, 1998; Dybus et al, 2005). Many workers have reported that PRL gene is highly polymorphic and has been found associated with milk production (Alipanah et al, 2008; Maksymiec et al, 2008; Ghasemi et al, 2009). The present study was conducted to study polymorphism in the PRL gene and its association with milk production in Frieswal cattle.

1 Statistical Analysis
The frequency of PRL gene allele A and B and genotype in each breed were estimated using the POPGENE Popgene software package (Yeh et al, 1999). Frequencies of distribution of alleles within the herds were compared using the Chi-square test. Milk productivity traits like lactation length, lactation yield and service period were analysed using the simple analysis of variance model as given below:

Y_ij = µ + G_i + e_ij

Where, Y_ij is the milk productivity trait of the jth cattle for ith genotype, µ is overall mean, G_i is the effect of ith genotypes (i= 1, 2, 3) and e_ij is random error.

2 Results and Discussion
Present study revealed amplified PCR product on 2% agarose gel as a single compact band of 156 bp size in Frieswal breeds under UV transilluminator and documented by gel documentation system. The PCR product of the same bp size was also reported by Kumari et al (2008) in exotic and Zebu cattle, Sacravarty et al (2008) in Kankrej cattle, and Ghasemi et al (2009) in Montebeliard cows.

Restriction digestion with Rsa I restriction enzyme revealed restriction fragments of 15 682 bp and 74 bp sizes. The different restriction fragments generated three different RFLP patterns. The DNA restriction fragments were obtained for the PRL-Rsa I polymorphism were: for the AA genotype 156 bp (no digestion), for the AB genotype 156 bp, 82 bp and 74 bp and for the BB genotype 82 bp and 74 bp. These three different RFLP patterns were obtained at two restriction sites corresponding to AA, AB and BB genotypes. Similar RFLP patterns using Rsa I restriction enzyme were reported by Kumari et al (2008) in exotic and Zebu cattle, Sacravarty et al (2008) in Kankrej cattle and Ghasemi et al (2009) in Montebeliard cows.

The genotypic frequencies for AA genotype 0.315 for AB genotype 0.629 and for BB genotype 0.056 whereas, allelic frequencies for A allele 0.630 and for B allele 0.370 in Frieswal cattle breeds. The significant (PË‚0.05) Chi-square value for all the genotypes in Frieswal cattle showed that the population was not in Hardy-Weinberg equilibrium. Similar results were reported by Klauzinska et al (2004) in Polish Red and Polish Black and White cattle, Brym et al (2005) in Jersey and Black and White cattle and Skinkyte et al (2005) in Lithuanian Black and White and Lithuanian Red cattle. The results obtained in the present study are not in agreement with those reported by Alipanah et al (2007a) in Red Pied cattle, Alipanah et al (2007b) in Russian Black Pied and Red Pied cattle and Alipanah et al (2008) in Russian Black Pied and Red Pied cattle.

The significant (P<0.05) Chi-square value indicates that the animals differ in their genotypic distribution in respect to gene frequency. The deviation in observed and expected genotypic frequencies among all these breeds may also be due to small population size and selective breeding. The result of analysis of variance showed non-significant effect of genotypes on lactation length, lactation yield and service period in Frieswal cattle breeds may be due to small sample size resulting in higher error variance. The result of analysis of variance showed non-significant effect of genotypes on lactation length, lactation yield and service period in Frieswal cattle breeds may be due to small sample size resulting in higher error variance. Non-significant differences were observed between mean lactation length and lactation yield among various genotypes. However, the significant (P<0.05) differences between mean service period of various genotypes was observed in Frieswal cattle breed. Less number of days for service period was observed for AA genotype in Frieswal cattle, which indicates superiority of AA genotype as compared to AB and BB genotypes.

Mean performance of genotypes showed non-significant (P<0.05) differences among genotypes for lactation length and lactation yield. Though the differences among genotypic means were non-significant but superiority of BB genotype for lactation length (329.14 days) and AB genotype for lactation yield (3161.19 kg) were seen among all the three genotypes.

Non-significant association of lactation length was reported by Aravindakshan et al (2004) in Vechur and Kassargode cattle. The results revealed in the present study are in accordance as reported by Alipanah et al (2007a) in Russian Red Pied cows, Alipanah et al (2007b) in Russian Black Pied and Russian Red Pied cows, Alipanah et al (2008) in Russian Black Pied and Russian Red Pied cows, and Ghasemi et al (2009) in Montebeliard cows. Non-significant association of lactation yield was reported by Aravindakshan et al (2004) in Vechur and Kassargode cattle, Khatami et al (2005) in Russian and German Black and White and Yaroslavl cattle breeds and Sacravarty et al (2008) in Kankrej cattle.

Mean performance of genotypes showed significant (P<0.05) differences for service period. The genotype AA (117.45 days) seems to be superior for service period among the three genotypes. Non-significant association of service period was reported by Sacravarty et al (2008) in Kankrej cattle. Hence, it can be concluded that the animals of BB genotype for higher lactation length and AB genotype for lactation yield and animals of AA genotype for less service period may be used for future breeding.

3 Materials and Methods
The present study comprised of 54 blood samples of Frieswal lactating cows collected from military dairy farm, Jabalpur, along with their lactation length, lactation yield and service period records. Genomic DNA was extracted from venous blood by the method of John et al (1991) with slight modification. Purity and concentration of genomic DNA was determined by using Nano-drop spectrophotometer. Genomic DNA quality was assessed by using 0.8% horizontal submarine agarose electrophoresis. PCR products were amplified using Oligonucleotide primers specific to bovine PRL gene locus were custom synthesized by Ocimum Biosciences, Bangalore in accordance with Lewin et al (1992) primer sequences: forward (5′-CGAGTCCTTATGAGCTTGATTCTT-3′) and reverse (5′-GCCTTCCAGAAGTCGTTTGTTTTC-3′) primers.

Polymerase chain reaction was carried out in a final reaction volume of 25 µL. PCR reaction mixture used for amplification of DNA was 2X PCR master mix 12.5 µL, forward primer (10 pmol/µL) 1.0 µL, reverse primer (10 pmol/µL) 1.0 µL, genomic DNA 3.0 µL and DNAase free water 7.5 µL. Amplification was performed in PCR thermal cycler programmed for 32 cycles with an initial denaturation at 95℃ for 10 min, denaturation at 95℃ for 1 min, annealing at 56℃ for 1 min and extension at 72℃ for 1 min with a final extension at 72℃ for 10 min. Total 5 µL of amplified PCR product of each sample was mixed with 1 µL of 6X gel loading dye buffer from each tube. The samples were loaded on 2% agarose gel containing ethidium bromide (1% solution @ 5µL/100mL) along with 80bp DNA ladder at a constant voltage of 80 V for 30 min. in 0.5X TBE buffer. Amplified DNA was digested with the Rsa I enzyme. Restriction digestion of the PCR product was performed in a total 30 µL. Content having 10X Buffer Tango 2 µL, PCR reaction mixture 10 µL, restriction enzyme (10units/µL) 1 µL and 17 µL nuclease free water. The reaction mixture was spined for few seconds for uniform mixing and then incubated at 37℃ for 4 h and 30 min in the water bath. After restriction digestion, the restriction product mixtures were electrophoresed on 3.5% agarose gel containing 1% ethidium bromide @ 5 µL/100mL at constant voltage of 80 V for 80 min using 0.5 X TBE buffer. Gel loading dye (6X) was used to load the digested PCR samples. DNA ladder (Range 100-1000 bp) was used as a molecular size marker. The DNA PCR-RFLP bands were visualized under UV light and documented by gel documentation system. The band size was judged by comparing with molecular size marker and recorded. Genotyping of PRL gene locus was carried out according to the band pattern of respective genotypes.

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