Сельскохозяйственная биология, 2012, № 2, с. 39-44.

doi: 10.15389/agrobiology.2012.2.39eng

УДК 636.2.034:575.113

POLYMORPHISM OF GROWTH HORMONE AND PROLACTIN GENES IN CONNECTION WITH MILK QUALITY DETERMINANTS IN CATTLE OF THE YAROSLAVSKAYA BREED

I.V. Lazebnaya¹, O.E. Lazebnyi², V.F. Maximenko³, G.E. Sulimova¹

The authors investigated the dependence between milk productivity of cattle of the Yaroslavskaya breed and bGH and bPRL genes of growth hormone and prolactin, respectively, in AluI and RsaI restriction sites. The complex genotypes of these genes were established, which determined more high and low percentage of milk fat and protein. The obtained principles can be used for revelation of animals, having prospects in relation to milk productivity at the early detection of the applicant to pedigree or commercial stock.

Keywords: bPRL and bGH genes, Yaroslavl cattle breed, PCR-RFLP, milk production.

A current state of animal breeding and agriculture requires the use of genetic data based on DNA analysis supplementing conventional time-tested techniques. This task is especially relevant in selection of valuable traits manifested only in mature animals, such as milk production in cattle when cows can be evaluated only after the first calving and sires are estimated by productivity of their daughters. The genes directly or indirectly affecting milk production traits are in focus of many studies. The first group of genes encodes milk proteins such as casein and lactalbumin (1, 2), the second –various products involved in regulatory metabolic processes. In the second group, a special role is performed by the genes for prolactin (bPRL) and growth hormone (bGH) responsible for the appropriate hormones (3, 4). Their receptors activate transcription of target genes including the genes for milk proteins.
A lot of available data describe various SNPs (SNP - single nucleotide polymorphism) localized in these genes and associated with milk performance traits. C-G transversion in the 5th exon of the growth hormone gene bGH (2141st nucleotide position) is associated with milk production traits in cattle. In this case, the restriction site for AluI-endonuclease disappears and the resulting protein product of bGH gene contains at position 127 the amino acid valine instead of a normally present leucine (Leu is substituted by Val). LL genotype of bGH gene is associated with better milk yield compared with LV genotype in Black-and-White and Holstein-Friesian cattle (5, 6). At the same time, in Holstein and Polish Black-and-White breeds, V-allele is positively correlated with milk yield and contents of milk fat and protein (7, 8).
The gene bPRL shows a synonymous A-G transition (in the codon for the 103rd amino acid  in a gene product) located in the 3rd exon of bPRL, which results in appearance of a polymorphic RsaI-site (9). Several authors reported about a positive effect of AA and AB genotypes on milk yield and milk protein content in Polish Black-and-White, Holstein-Friesian and Brown Swiss cows (10-12). However, a distinct correlation was observed in Red Pied cattle (13).
Earlier in cows of Kostroma breed the authors investigated the relationship between fat percentage in milk with SNP-polymorphism of genes for growth hormone and prolactin (14). Genetic structure of three herds was determined along with total variability for each gene-specific marker. Although a correlation with fat content in milk (%) was insignificant, it showed a trend to association with AluI-polymorphism of bGH gene. The same markers were used by S.R. Khatami et al. (15) to study genetic variation and its association with milk production in Black-and-White and Yaroslavl cattle. The effect of bGH genotypes (AluI) on milk fat content (%) was established in Yaroslavl cattle breed. However, the combined influence of genes on milk productivity wasn’t studied in most of such works.
The purpose of this research was evaluation of individual and combined effects of growth hormone bGH (AluI) and prolactin genes bPRL (RsaI) on milk yield, percentage content of milk protein and fat (%) in cows of Yaroslavl breed.
Technique. PCR-RFLP (restriction fragment length polymorphism) was performed to determine the diversity of bPRL (RsaI) and bGH (AluI) genes in 113 purebred Yaroslavl cows bred in cattle pedigree farms “Mikhailovskoye” (n = 59, 2003) and “Gorshikha” (n = 54, 2003) (Yaroslavl province). DNA was extracted from whole blood (200 ml) of the animals using the kit DIAtomTM DNA Prep (“IsoGeneLab”, Moscow). The gene fragments bPRL (156 bp) and bGH (223 bp) were amplified by standard methods (9) with GenePakTM PCRCore (“IsoGeneLab”, Moscow) in a thermocycler Tertsik (“DNA-Technology Corp.”, Russia). RsaI- and AluI-restriction endonucleases were applied according to the manufacturer’s instructions (“MBI Fermentas”, Lithuania).
Pairwise comparison of the observed heterozygosity in studied breeds was conducted using Fisher's exact test (F). Correlations of bGH and bPRL gene polymorphisms with milk production traits (yield, percentage content of milk fat and protein in three primary lactations) was assessed by variance analysis in the program Statistica 6.0.
Results. The comparison of the herds has revealed the absence of any significant difference in distribution of frequencies of genotypes and alleles bPRL and bGH  (respectively, G² = 0,338, d.f. = 2, P = 0,845 and G² = 4,522, d.f. = 2, P = 0,104), so all tested groups were united and further analyzed as one generalized sample.
The presented data (Table 1) indicate that the studied group of cattle included representatives of all possible genotypes of bPRL and bGH in terms of the studied SNPs and, therefore, all combined genotypes. The distribution of frequencies of individual genes was found to be an equilibrium state according to Hardy-Weinberg criteria: bPRL  — χ² = 0,120, P = 0,729; bGH — χ² = 1,982, P = 0,152. Values of expected heterozygosity (H_e) corresponded to observed heterozygosity (H_o) for both genes; for bPRL these values amounted to, respectively, 0,442 ± 0,047 and 0,461 ± 0,066, for bGH - 0,558 ± 0,047 and 0,497 ± 0,028.
Relatively high degree of heterozygosity for bPRL gene in Yaroslavl cattle wasn’t reliably different from the earlier established values of Kostroma and Yakut breeds (P > 0,05) (14, 16), but differed from those of Russian Red Pied and Black-and-White cattle (respectively, P <0,05 and P <0,001) whose values were calculated by the authors upon the literature data about genotype frequency (13, 17). He and Ho of bGH gene in Yaroslavl cattle were comparable with high values peculiar to the previously studied group of Yakut cattle (P> 0,05) (16), and they reliably exceeded those of Polish Black-and-White cattle (P < 0,001, calculated on the basis of genotype frequencies stated by L.V. Zwierzchowski et al.) (8), as well as in Kostroma cattle (P <0,01), which was shown by the authors previously (14).

Individual effects of the studied genes and their combined effects on milk productivity traits of Yaroslavl cattle were assessed by variance analysis (bPRL: RsaI-marker; bGH: AluI-marker), which has revealed reliable correlations between individual combined genotypes for these genes and percentage content of milk protein (F = 2,93, d.f.₁ = 2, d.f.₂ = 104, P = 0,041, 1st lactation) and fat (F = 2,59, d.f.₁ = 2, d.f.₂ = 104, P = 0,041, 1st lactation).

Comparative posteriori (post-hoc) analysis of milk productivity traits in studied cows with different combined genotypes has revealed those providing more high or lower contents of fat or protein in milk (Table 2). Thus, average percentage of fat in milk of cows ABVV [6] and BBVL [8] was reliably different from that in a group of animals with genotypes ABLL [4] and BBVV [9] (probability P6 _{[4, 9]} = 0,008 and P8 _{[4, 9]} = 0,009, resp.), as well as AAVV [3], ABLL [4] and BBLL [7] reliably differed by mean percentage of milk protein from AALL [1], ABVV [6 ] and BBVV [9] (P3 _{[1, 6, 9]} = 0,017, P4 _{[1, 6, 9]} = 0,008, P7 _{[1, 6, 9]} = 0,020, resp.) (Table 2).
According to these data (Table 2), ABVV cows produced milk with high percentage of milk fat and low protein content. An opposite relation was observed in ABLL cows. In a group of BBVL cows fat content in milk was reliably higher than in ABLL and BBVV , but the intermediate value of milk protein content compared with ABVV cows (not shown in Table 2). An intermediate value of fat content in milk was associated with genotypes AAVV and BBLL, which animals though showed high percentage of milk protein.

Differences between mean absolute values of fat percentage in milk from animals with genotypes involved in positive and negative associations amounted to 0,43-0,47% (Table 3). These differences are important in terms of the total volume of milk. However, the available scientific literature has no data about fat content in milk from cows with combined genotypes in respect to the used marker systems.
At the same time, there are the published data on complex genotypes of bGH gene (for AluI and MspI restriction sites) in Polish Black-and-White cattle having the difference of only 0,10-0,22% (8). In Russian Black-and-White cattle, fat content in milk of  cows with bGH AluI-genotypes amounted to 0,16% during the 1st and 0,29% - during the 3rd lactation (17).
In the studied Yaroslavl cows, mean percentage content of protein varied between combined genotypes associated with higher and lower protein content within 0,17-0,29% (Table 3), which is also economically significant (18, 19). In Russian Black-and-White breed, differences between groups of animals with bPRL RsaI-genotypes were less than in Yaroslavl cattle: 0,05% for the 1st and 0,09% - for the 3rd lactation (17, 18).
The performed research has revealed in Yaroslavl cattle the presence of combined effects of genes bPRL and bGH on percentage content of milk protein and fat. Each trait is associated with individual combined genotypes providing reliable positive or negative influence, but there’s no genotype positively associated with both parameters. It has been identified individual genotypes associated with high values of one parameter and low – of the other, as well as genotypes associated with maximum mean for a one trait and good value of the other. The observed dependence is useful for selection of highly productive animals with varying degrees of particular productivity traits. This process should consider the only combined genotype BBVV unfavorable for both parameters, but these animals must not be discarded as they are double homozygotes useful in crosses as a source of genotypes with high contents of  milk fat (ABVV and BBVL) and protein (AAVV and BBLL). It’s noteworthy that the total frequency of promising genotypes in terms of percentage contents of milk fat and protein are, respectively, 12,4 and 23,9%.
Thus, in Yaroslavl cattle it has been established the association of several combined genotypes for growth hormone bGH and prolactin bPRL genes with high and low percentage content of milk fat and protein. The obtained facts about joint action of these genes can help to improve productive properties of cattle. The revealed regularities can be used to detect promising individuals in terms of milk fat and protein content during an early testing of candidates for pedigree livestock or commercial herds.

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