Polymorphisms of leptin, β-lactoglobulin and pituitary transcription factor have no effect on milk characteristics in crossbred cows

Milk production systems installed in the Brazilian semiarid region are used on a large scale, in which the European breeds specialized in milk production are hybridized with zebu breeds more suited to a climate with semiarid conditions (Façanha et al., 2017). The result of this crossbreeding produces animals that are less productive, although better adapted to the tropics, which has increased the productivity and viability of dairy farming in the semiarid northeast. Allelic identification of the different genotypes of dairy cows, using molecular biology techniques, is used to make adjustments in genetic improvement programs, directing crosses of different genetic groups for more efficient gains in animal production systems. 

Milk production systems installed in the Brazilian semiarid region are used on a large scale, in which the European breeds specialized in milk production are hybridized with zebu breeds more suited to a climate with semiarid conditions (Façanha et al., 2017). The result of this crossbreeding produces animals that are less productive, although better adapted to the tropics, which has increased the productivity and viability of dairy farming in the semiarid northeast. Allelic identification of the different genotypes of dairy cows, using molecular biology techniques, is used to make adjustments in genetic improvement programs, directing crosses of different genetic groups for more efficient gains in animal production systems.

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The strategy of detecting genetic polymorphisms associated with genes which are important for a studied trait has been successfully employed (Hax et al., 2017). For milk production, polymorphisms of the κ-casein and βlactoglobulin genes stand out. According to Otaviano et al. (2008) and Freitas et al. (2012), casein (S1α, S2α and β) and serum proteins (βlactoglobulin and α-lactalbumin) are associated with increased protein levels in milk. Similarly, other polymorphisms have been investigated, for example, some single nucleotide polymorphisms (SNPs) in leptin genes (Madeja et al., 2004) and pituitary transcription factor or PIT1 (Renaville et al., 1997), because they have significant effects on milk production, feed conversion, weight gain, meat tenderness and subcutaneous fat deposition. In many countries, these studies are well advanced in the Holsten breed. On the other hand, for dairy zebu, there is still a gap. Today, only Australia and the United States are researching the Bos indicus group, giving greater importance to these breeds, especially the Brahman breed. Currently, little is known about the effects of crossbreeding between Holstein and Guzerá cows, kept in a hot climate and a semiarid region.
The aim of the present work was to identify genetic polymorphisms of leptin, β-lactoglobulin and pituitary transcription factor (PIT1) and to evaluate their effects on the chemical composition and somatic cell count of milk from crossbred dairy cows. All procedures performed with the animals of the present experiment were in accordance with the ethical standards stablished be the committee of the Universidade Federal Rural do Semiárido, UFERSA (nº. 23091.003895/2014-71).
We obtained 291 blood samples for DNA extraction and milk samples from crossbred cows. The samples were of three genetic groups: , genomic DNA extraction, we used the protocol according to Montgomery and Sise (1990). The amplification reactions of the leptin, βlactoglobulin and PIT1 genes were conducted in a thermocycler. Then, 5µl aliquots of the amplified product were digested with restriction enzymes for restriction sites found in each marker (Table 1). The resulting fragments were separated by electrophoresis in tris-borate-EDTA (TBE) in 10% polyacrylamide gel and stained with silver nitrate. The animal genotype was determined by the analysis of fragment size reported as base pairs. Based on the results of the PCR/RFLP analyses, the genotypes of each animal were identified. The estimates of allele and genotype frequencies were calculated using the GENEPOP software version 1.2. The genotypic and allelic frequencies of the population sample for the gene fragment of leptin, β-lactoglobulin and PIT1 were established by the following equations: and , where n ii and n ij correspond to the number of homozygotes and heterozygotes observed in gene i, respectively; n corresponds to the total number of individuals (Otaviano et al., 2008). The Hardy-Weinberg equilibrium was tested for each gene to according Falconer and Mackay (1996).
Two milk samples were collected after predipping sterile containers containing preservatives that varied according to the type of analysis. The preservative Bropanol® was used for the somatic cell composition and counting analyses and the preservative Azidiol® as used for total bacterial counts. The analysis of milk composition (fat, protein, lactose and non-fat dry extract), somatic cell count and yields was also performed.
The genetic groups and the genetic polymorphisms for each gene (leptin, βlactoglobulin and PIT1) were used as fixed effects in the analysis of variance (t-test). In addition, the interaction between the two fixed effects was also evaluated.
For the leptin gene, the amplified and digested product with the restriction enzyme Kpn2I generated two 75 and 19bp fragments for the C allele with a restriction site. CT heterozygotes showed fragments of 94, 75 and 19bp and the T allele showed only a fragment of 94bp, because it does not contain a restriction site for Kpn2I.
For the β-lactoglobulin gene, the fragment digestion obtained by the PCR-RFLP technique showed a restriction polymorphism for the HaeIII enzyme. The AA genotype was characterized by the presence of two fragments (153 and 109bp), AB by the presence of four fragments (153, 109, 74 and 79bp) and the BB genotype by the presence of three fragments (109, 79 and 74bp).
In the study of the variability of the PIT1 gene, a fragment of 168bp was analyzed using the Hinf1 enzyme, which allowed for the identification of the (+) and (-) alleles. Only the (+) allele presented a restriction site for Hinf1, characterized by the presence of two fragments with 96 and 76bp. Homozygous (+/+) animals were identified by the two (-) band patterns, corresponding to 96 and 76bp fragments. The (+/-) heterozygotes were indicated by three bands (168, 96 and 76pb) and the (-/-) homozygotes by a single band (168bp).
The allele frequencies were described together with the genotype frequencies according to the three genetic groups studied (Table 2). For the allele frequencies of leptin, the absence of the CC genotype was found in the group of Holstein x Guzerá animals, demonstrating an imbalance in the Hardy-Weinberg equilibrium (P< 0.04). The same observation was true for PIT1, where the TT allele frequency in the group of Holstein x Guzerá animals was equal to zero. For PIT in particular, a Hardy-Weinberg imbalance was shown all three groups of animals (P< 0.03).  Buchanan et al. (2002) reported the frequency of the T allele in cattle of the Angus (0.5), Charolais (0.3), Hereford (0.5) and Simental (0.3) breeds. According to Konfortov et al. (1999), the T allele is probably present in the taurine breed at a frequency close to 0.4 and does not appear in the Indian breed, where the C allele appears to be fixed. The present study suggests the predominance of taurine genes in the investigated crossbred dairy cow population, since the frequency of T was similar to that found in European breeds.
β-lactoglobulin is encoded by a single locus in autosomal genes; twelve allelic variants have been reported for this gene, with A and B being the most frequent variants in domestic cattle. In the present study, the populations were in Hardy-Weinberg equilibrium (P> 0.05) and no change was found in relation to these genes, resembling the results found by Veli and Rivas (2010) in populations of crossbred dairy cows.
The results obtained for genotypes of crossbred dairy cows no showed significant differences (P< 0.05) in milk composition and quality variables Arq. Bras. Med. Vet. Zootec., v.71, n.2, p.715-719, 2019 (Table 3). However, other studies have associated some leptin polymorphisms with milk production characteristics (Madeja et al., 2004) with significant effects (P< 0.01) of the TT genotype (HphI polymorphism) on the genetic value of some characteristics, such as milk, fat and protein. Although genetic variants A and B of βlactoglobulin are most strongly related to milk yield and fat production (Bonfatti et al., 2010 andFreitas et al., 2012), the variation between the means found in this study was minimal and there was no difference between the genotypes.
In the present study, it was expected that most of the composition variables would vary among the genotypes. It is already known that crosses give rise to animals with both phenotypic and genotypic characteristics. However, we found in this study that polymorphisms leptin, β-lactoglobulin and PIT1 do not influence the composition and quality of milk from , and Holstein x Guzerá cows kept in a hot climate.