Association of genetic variants of bovine prolactin with milk production traits of Black-and-White and Jersey cattle

Associations between polymorphism localised in the third exon of the prolactin gene (PRL-RsaI) and milk production traits of Black-and-White and Jersey cattle were analysed. A total of 427 cows were included in the study. PCR-RFLP method was used. The frequencies of genotypes and alleles were as follows: 0.7107 AA, 0.2851 AB, 0.0042 BB; 0.8533 PRL and 0.1467 PRL for Black-and-White cattle and 0.0919 AA, 0.4324 AB, 0.4757 BB; 0.3081 PRL and 0.6919 PRL for Jersey cattle. Statistically significant differences between the breeds were observed in the frequencies of genotypes and alleles. Associations between PRL-RsaI polymorphism and milk production traits of Jersey cows and lack of associations with these traits in Black-andWhite cows were observed.


Introduction
Prolactin (PRL) is one of the most versatile hormones of the pituitary gland in terms of biological actions.More than 100 different and distinct effects of the hormone have been documented.In most mammalian species, the hormone consists of 197-199 amino acids (SINHA, 1995).Bovine PRL consists 199 amino acids (WALLIS, 1974).Prolactin is essential for the initiation and maintenance of lactation.It acts on mammary alveoli to promote the synthesis and secretion of milk protein.This hormone is, primarily responsible for the synthesis of milk proteins, lactose and lipids, all major components of milk (LE PROVOST et al., 1994).PRL secretion is maintained during lactation by suckling, the most powerful natural stimulus for PRL release (MURAI and BEN-JONATHAN, 1987).PRL regulating reproductive and immunological functions, fluid balance, cellular growth and differentiation (NICOLL, 1980;LORETZ and BERN, 1982;RUSSELL, 1989;KELLY et al., 1991).In addition, members of PRL family have been implicated recently as potential regulators of angiogenesis (CLAPP et al., 1998).Prolactin (PRL) gene is expressed in the pituitary gland, and at several other sites including the central nervous system, the immune system and the mammary gland (SINHA, 1995;BEN-JONATHAN et al., 1996;LE PROVOST et al., 1994).Bovine prolactin (PRL) gene is localised in chromosome 23 (BARENDSE et al., 1997), and consists of five exons separated by interval introns (CAMPER et al., 1984).COWAN et al., (1990) detected a polymorphic site for AvaII restriction endonuclease.Digestion of genomic DNA with the restriction endonuclease AvaII disclosed a probable insertion/deletion of approximately 200 base pairs near the PRL gene.In the studies carried out by HART et al., (1993) showed a four-allele SSCP in the 5'flanking region of the PRL gene.KLAUZIŃSKA et al., (2001) reported the single nucleotide substitutions in the 5' region of the PRL gene.One of these variable sites are also potential binding site for C/EBP transcription factor.A silent A-G transition mutation at the codon for amino acid 103 in exon 3 of bovine PRL gene gives rise to a polymorphic RsaI site (LEWIN et al., 1992).CHUNG et al. (1996) showed that PRL-RsaI locus had a significant effect on milk yield and fat percentage in dairy cattle.In the study carried out by DYBUS (2002) showed that cows with the AA genotypes of the PRL gene had higher milk protein content than AB individuals.The aim of this study was to estimate the allelic frequencies at the PRL-RsaI locus and to investigate the relationship of this polymorphism and milk production traits of Black-and-White and Jersey cows.
Materials and methods A total of 242 Black&White cows (with different proportional share of HF genes) and 185 pure-bred Jersey cows were genotyped.The B&W cows were kept in two herds in West Pomerania region of Poland; Jersey cows were kept in the one herd in the Central region of Poland (Table 1).The PRL-RsaI genotypes were analysed using the PCR-RFLP method (MITRA et al., 1995).Crude DNA was isolated from whoole blood samples using MasterPure TM kit (Epicentre Technologies).A 156-base pair (bp) fragment of the PRL gene was amplified by polymerase chain reaction (PCR) using forward (5'-CGAGTCCTTATGAGCTTGATTCTT-3') and reverse (5'-GCCTTCCAGAAGTCGT TTGTTTTC-3') primers.The PCR reaction contained 80 ng of genomic DNA, 10 pmol of each primer, 1,5 µl 10 x PCR buffer (MBI Fermentas), 1.5 mM MgCl 2 , 200 µM dNTP and 0.4 units Taq-polymerase in a total volume of 15 µl.The following cycles were applied: denaturation -94 °C/5 min, followed by 30 cycles -94 °C/30 sec, primer anneling -59 °C/40 sec, PCR products synthesis -72 °C/20 sec, and final synthesis -72 °C/3 min using a DNA thermal cycler (Perkin Elmer 2400).Amplified DNA was digested with 5 units of RsaI enzyme (MBI Fermentas).The digestion products were separated by horizontal electrophoresis (90 volts, 50 minutes) through 4% agarose gels (Gibco BRL) in 1 x TBE and 1.0 µM ethidium bromide.Data for 305-day milk production in the first, second and third lactation, including production of milk (kg), milk fat and milk protein (kg) and proportions of milk fat, milk protein and sum of milk fat and protein (%), were obtained from the farm documentation.Statistical calculations were performed using procedures of Statistica (StatSoft Inc 2001).Distribution frequencies of alleles and genotypes were compared using chi-square test.The effect of PRL-RsaI genotypes on the milk production traits of dairy cows were analysed using General Linear Model (GLM) procedure.Type III ANOVA was used to determine the differences between parameters.Differences of means were testes with the multiple Duncan test.The models used were as follows: 1. Black-and-White cows:

Jersey cows:
Y ijklmn -analysed trait, µ -overall mean, G i -genotype (i = 1,...3, fixed effect), S j -sire (random effect), b 1 -regression coefficient of calving age, A -mean of calving age, x k -calving age in month of cow, b 2 -regression coefficient of days in milk, DD -mean days in milk, x l -days in milk of cow YS m -year-season (l = 1, ...39, fixed effect), x -trait mean, e ijklmn -random error.

Results
The following DNA restriction fragments were obtained for the PRL-RsaI polymorphism: 82 and 74 bp for the BB genotype, 156, 82 and 74 bp for the AB and 156 bp (no digestion) for the AA (Fig. ).In Black-and-White cattle the AA genotype was the most frequent in the studied herds (0.7222 and 0.6983), followed by the heterozygotic AB (0.2778 and 0.2931), whereas the BB was the least frequent (0 and 0.0086).In contrary, in Jersey cows the BB genotype was the most frequent (Table 2).Table 3 shows the influence of the PRL-RsaI polymorphism on milk production traits in Jersey cows.Table 4 shows the influence of the PRL-RsaI polymorphism on milk production traits in Black-and-White cows  Frequencies in columns with the same letter differ significantly; capitals P ≤ 0.01, small letters P ≤ 0.05; n -number of cows; F&P -sum of fat and protein content, * -excluded from the statistical analysis Discussion In dairy cattle, the primary goal of the selection is the improvement of yield and composition of milk.With the advances in molecular biology, the identification of the genes underlying livestock production traits -called quantitative trait loci (QTL) -is now possible and is likely to lead to more efficient breeding programs (PARMENTIER et al., 1999).Although a number of strategies can be envisaged to identify candidate genes markers, essentially two approaches are presently developed to estimate the favourable QTL implicated in a specific production trait.The first method, the positional cloning, consist of the localization of the genes of interest using marker-QTL associations covering the whole genome.The second strategy uses the candidate gene approach (COPPIETERS et al., 1999;SCHALKWYK et al., 1999;SEYFERT, 1999).GRUPE and SCHWERIN (1998) mapped QTL, affecting milk yield and milk fat content, on the telomeric region of bovine chromosome 23.The genetic equilibrium in the populations under study was not disturbed and the size of certain genotypes of the PRL was not statistically different from the theoretically calculated size.Frequencies of PRL-RsaI alleles obtained in this study were similar to the frequencies obtained earlier for the Black&White cattle.Higher frequency of the PRL A (0.95) in Holstein breed was observed by CHRENEK et al. (1998).Somewhat smaller frequency of the PRL A (0.80) was observed in studies of MITRA et al. (1995).Considerably smaller frequencies of PRL A (0.73) were observed by CHUNG et al. (1996).The frequencies of PRL-RsaI alleles found in this study for Jersey cows considerably deviate from the values found for Holstein-Friesian cows.World literature lacks reports on PRL-RsaI polymorphism in Jersey cattle, which hinders any inference.Table 3 shows the influence of the PRL-RsaI polymorphism on milk production traits in the analysed Jersey cows.The statistically significant differences between individuals of different PRL genotypes were found in milk fat yield, milk fat content and and sum of fat and protein content.In the first 305-day lactation, the cows of the AA genotype produced less milk fat (-15.9 and -14.6 kg) than the AB and BB individuals (P ≤ 0.01).In the case of milk fat content, statistically significant differences between individuals of different PRL genotypes were found in the first lactation (P ≤ 0.01).The cows with the AA genotype produced milk with lower fat content (-0.19 and -0.18%) than AB and BB individuals, respectively.Different results were obtained by CHUNG et al., (1996) who reported that Holstein-Friesian cows with AA genotype produced milk with higher fat content than BB individuals.CHRENEK et al. (1999) examined an influence of PRL-RsaI polymorphism on the milk production traits of Brown Swiss cows, and there were no statistically significant differences between the cows with different PRL genotype.In reference to sum of milk fat and protein (%), statistically significant differences (P ≤ 0.01) between the cows with different PRL-RsaI genotype were observed.In the third lactation cows with AB genotype had higher F&P content (+0.44%) than AA individuals.In our study, no associations between RFLP in PRL gene and milk production traits were found for Black-and-White cattle.Bearing in mind the weak associations in Jersey cattle and no associations in B&W cattle, it should be stressed that the usefulness of PRL-RsaI polymorphism for the improvement of production traits of dairy cattle seems uncertain.

Table 2
Frequencies of genotypes and alleles of the PRL-RsaI (Frequenz von Genotypen und Allelen von PRL-RsaI) Means and standard deviations of milk production traits in Jersey cows carrying different PRL-RsaI genotypes (Mittelwerte und Standardabweichungen der Milchleistungsmerkmale bei Jersey Kühen mit verschiedenen Genotypen PRL-RsaI) Frequencies in columns with the same letter differ significantly; capitals P ≤ 0.01, small letters P ≤ 0.05; n -number of cows; F&P -sum of fat&protein content; L -lactation; Gen -genotype; SD -standard deviation