Polymorphism of the glutathioneperoxidase-1 gene (GPX-1 g. 189 T/C) and biochemical parameters of the blood serum of Holstein cattle

. The study examined the polymorphism of the glutathione peroxidase-1 gene ( GPX-1 , g. 189 T/C) effect on the GPx enzyme activity and biochemical parameters in the blood serum of Holstein cows of indigenous and foreign selection. The work was carried out in the Lenin Breeding Farm Integrated Agricultural Production Center (IAPC) and the Mukhametshin Z.Z. Peasant Farm Enterprise (PFE) of the Republic of Tatarstan. DNA genotyping by the locus of the Bos taurus GPX-1-Bsc4 I gene was performed by PCR-RFLP. As a result of genetic typing of cattle, 2 alleles (C and T) and 3 genotypes (CC, TC and TT) were identified. The classification carried out by PIC values in two populations of Holstein cattle showed that there is a moderate polymorphism of the GPX-1 gene. A negative inbreeding coefficient (F) indicates excessive outbreeding in both populations. In both populations, the numerical advantage of heterozygous genotype TC cows is 60.1 and 56.7 % of the total livestock. Indigenous breeding cows, compared with imported cattle had higher: GPx activity, AST, ALT, cholesterol level, total protein and albumin fraction. Analysis of the association between the GPX-1 gene polymorphism and the GPx enzyme level indicates that the activity of this enzyme varies depending on the genotype, and there is also a variation in the biochemical parameters of the Holstein cattle blood serum.


Introduction
Currently, it is well known that intensive industrial technologies in animal breeding are stressogenic.The impact of a number of stress factors accompanying this technology leads to an increase in the load on the adaptive abilities of the body, which entails various physiological and biochemical disorders in the cattle's body.At the same time, the metabolic processes violation is accompanied by a change in the homeostasis parameters, the components of which are indicators of the immune and biochemical status, the state of the oxidant-antioxidant system.
The transition period is critical for the cows' health and it is a time when cows seem more sensitive to oxidative stress, which also contributes to impaired immune function and perinatal metabolic diseases.A decrease in antioxidant activity affects the immune system and the phagocytic activity of cells and, thus, increases the incidence of mastitis and postpartum diseases [1].Proinflammatory markers blood level increase at the moment when stress is known to occur may indicate their role in the pathogenesis of dairy cattle diseases that occur during the perinatal period [2].It was previously reported that the peripartal period is characterized by antioxidant status depletion and, as a consequence, oxidative stress [3].Additional research in this area is needed to further clarify the potential protective role of antioxidants in the perinatal period [4].
Earlier studies suggested a possible link between body weight loss and oxidative status [5].It was reported that cows with elevated levels of non-esterified fatty acids (NEFA) in blood plasma also had higher levels of active oxygen metabolites, but lower levels of antioxidants.Low antioxidant potential, as a consequence of increased fatness, may be the result of excessive accumulation of reactive oxygen species (ROS), antioxidant protection depletion, or a combination of both [3].It has been established that cows with a high fatness index are more sensitive to oxidative stress [6].The results obtained by other researchers indicate that the oxidants and antioxidants level in the blood can affect the cows' reproductive qualities [7].
In the current situation, there is a need to search for biomarkers to stratify the risk of dairy cows' oxidative stress.It is known that antioxidant activity is provided by enzymes such as glutathione peroxidase, catalase, superoxide dismutase, paraoxonase, etc., as well as some vitamins [8].
Glutathione peroxidase (GPx) is the common name of a multiple isoenzymes family that catalyze H₂O₂ or organic hydroperoxides reduction to water or corresponding alcohols using reduced glutathione (GSH) as an electron donor [9].It is also able to protect milk lipids from oxidation [10].The basis of the enzyme is protein, but there is also an important non-protein part (cofactor) -this is the selenium trace element, which largely determines its antioxidant properties.With an insufficient amount of selenium, the enzyme activity of glutathione peroxidase decreases.This often causes free radicals excessive accumulation.Studies of scientists indicate a two-fold decrease in the glutathione peroxidase level of cows with mastitis, as opposed to healthy animals [11][12][13][14].There are data on the variability of GPx activity depending on the season -in summer it increases, and in winter it decreases [15,16].
The bovine GPX-1 gene encoding the glutathione peroxidase enzyme is located on chromosome 22, contains 2 exons and 1 intron [17].Replacing proline with leucine (Pro → Leu) at position 189 in a fragment 442 bp long can control the transcriptional gene activity and, as a consequence, the GPx protein's structure and action.
The aim of the work is to research the polymorphism of the GPX-1 gene (transition C → T; C189T) and its association with dairy cows' blood serum biochemical parameters in the populations of Holstein cattle of indigenous and foreign selection.

Materials and methods
The research was carried out in two farms of the Republic of Tatarstan: in the Lenin Breeding Farm Integrated Agricultural Production Center (IAPC) of Atninsky district -148 cows of the Holstein breed of indigenous selection (HIS) with an average milk yield of 32.0 kg / day and in the Mukhametshin Z.Z.Peasant Farm Enterprise (PFE) of Sabinsky district -231 cows of the Holstein breed of foreign selection (HFS) with an average milk yield of 28.0 kg / day.For biochemical and DNA studies on the 60th day of lactation, whole blood samples were taken into K3E (EDTA) and Z (Serum Clot Activator) vacuum tubes.The laboratory part of the experiment was conducted in the Physiology, biochemistry, genetics and animal nutrition department of TSRIA of FRC Kazan Scientific Center of RAS.
Biochemical analysis of blood serum was carried out on a semi-automatic analyzer SINNOWA BS-3000M (China) according to generally accepted methods.The activity of the glutathione peroxidase (GPx) selenoenzyme in blood was measured by spectrophotometric method.Using the ready-made AmpliPrime DNA-sorb-B kit (Next BIO, Russia), DNA was recovered from biological material, which was subsequently tested for the GPX-1-Bsc4 I gene locus.
Polymorphism of the GPX-1 gene in exon 2 (transition C → T; C189T) was detected by PCR-RFLP under optimized temperature-time regimes [18,19].Primers F: 5' -GAA AAG TGC GAG GTG AAT GG -3', R: 5' -GCT GTG GTC TGG GAA AGG -3' (Evrogen, Russia) were used for PCR [20].To determine the polymorphism of the GPX-1 gene, amplified fragments treated with endonuclease Bsc4 I (Bacillus schlegelii) (SibEnzyme, Russia) were separated in an agarose gel of 2.6% with the addition of 5 µl of 10% Ethidium bromide in a 1 x TB buffer in a horizontal electrophoresis chamber (Helicon, Russia) at an electric field strength of 20 V/cm during 20 min.Visualization and fixation were carried out using the Gel&Doc and GelDoc Go documentation system with the Image Lab Touch V. 3.0 (BIO RAD, USA) software.Genotypes were identified by the detected polymorphism of DNA sequences.
The genetic equilibrium in the studied cattle population was tested according to the Hardy-Weinberg law.The variability between the observed and expected genotype distribution was checked by chi-squared (χ 2 ) method.Inbreeding in populations and polymorphism information content (PIC) of molecular marker were evaluated.The assessment of GPX-1 gene polymorphism effect on biochemical status indicators was determined in groups of animals which were formed according to the identified genotype.

Results and discussion
Identification of polymorphism by the GPX1-Bsc4 I gene locus in two populations of Holstein cattle showed that 2 alleles (C and T) and 3 genotypes (C, TC and TT) are present in both herds.This frequency indicates the genetic biodiversity of the studied populations.Conducted DNA testing of 148 Holstein cows of the Lenin Breeding Farm IAPC of Atninsky district of the Republic of Tatarstan revealed that the studied livestock is represented by all possible genotypes of the GPX-1 gene (Table 1).In the process of genotyping individuals of the Holstein breed of Mukhametshin Z.Z.PFE аббр.оставляем foreign selection, the numerical superiority of cows with heterozygous TC genotype (56.7%) was also established, the second largest group of cows had homozygous CC genotype (23.0%) and the least were individuals with the TT genotype (20.3%).The frequency of C and T alleles was 0.513 and 0.487, respectively, demonstrating a slight dominance of the GPX-1 gene C allele.
The variability between the observed and expected distribution of genotypes in this population is set at the level of χ 2 = 4.21, which is below the critical values.
In the expected distribution of GPX-1 genotypes in both populations, there is a slight shift in the direction of increasing homozygosity.In a cattle herd of indigenous selection, the genetic Hardy-Weinberg Equilibrium is violated (not observed).
The classification carried out by PIC values in two populations of Holstein cattle showed that there is a moderate polymorphism of the GPX-1 gene.A negative inbreeding coefficient (F) indicates excessive outbreeding in both populations.
The authors' researches aimed at studying the polymorphism of the GPX-1 gene in Indian cattle of the Nimari and Malvi breeds report genetic biodiversity and variability in populations [20,21].
In the work of R. Jagtap et al. ( 2012), C and T alleles in the Malvi cattle had a frequency of 0.741 and 0.249, respectively, and CC genotypes -53.8%,TC genotypes -42.3 and TT genotypes -3.9% [21].The experimental population of the same breed in the study by S.Singh et al. ( 2011) is represented by C and T alleles with a distribution of 0.870 and 0.130 [20].And the following values corresponded to the animals of the Nimari cattle: C allele -0.930 and T allele -0.070.The maximum number of animals that were carriers of the homozygous CC genotype was 86.0%, the heterozygous TC group accounted for only 14.0% of the total, and individuals with the TT genotype were not found among this breed.
Evaluation of the data obtained indicates that in our experiment, the "normal" C allele of the GPX-1 gene has an advantage over the "mutant" T allele in populations of different selection, as in cattle in India.
The parameters of the oxidative status and biochemical profile of the Holstein cattle blood serum were analyzed.The results of experimental animals' blood serum biochemical studies, shown in Tables 2 and 3, which can be used to judge the degree of metabolism and oxidative status, indicate that the obtained indicators of cows of various groups were mainly within the physiological norm.However, when comparing the indicators, differences can be traced, both by animal genotypes and by farms.

GPX-1 TC (n = 89) GPX-1 TT (n = 29)
GPx, U/ml 0. The results of the biochemical parameters study indicate the variability of the GPx level depending on the genotype of animals.In two populations of Holstein cattle, the GPx content in the serum of cows with CC genotypes is significantly higher (*p < 0.05), slightly lower in individuals with TC genotypes and cows with TT genotypes of the GPX-1 gene have a minimum amount.Cows of different genotypes had the difference in this indicator: between CC and TT -0.028 U/l (8.7%) between TC and TT -0.019 U/l (6.3%).
According to the data obtained, the level of GPx in the blood serum of indigenous selection dairy cows with high milk productivity is higher than that of imported cows with lower milk yields.In the imported cattle, the GPx content in blood serum was statistically significantly (p < 0.05) reduced among cows with TC and TT genotypes.The difference between individuals with CC and TC genotypes was 0.022 U/l (7.5%), CC and TT -0.034 U/l (11.6%), and TC and TT -0.012 U/l (4.4%).
The study of lipid metabolism products showed that cows of indigenous selection had a statistically significant high level of cholesterol with the TT genotype and amounted to 0.56 and 0.26 mmol/l (p < 0.05), respectively, compared with TC and TT genotypes individuals.In the population of foreign selection cows, cholesterol was 0.29 and 0.25 mmol/l (p < 0.05), which is higher among CC and TC genotypes cows than among homozygous TT animals.When comparing the two populations, there is a suspended content of cholesterol in the blood of indigenous selection cows.
Regarding the content of triglycerides in the blood, a significant high value was found in two populations of the TT genotype.However, in the population of foreign selection cattle, the content of triglycerides among animals of all genotypes was increased, compared with indigenous individuals.The concentration of glucose in the blood serum in all groups corresponded to the physiological norm and varied in the range of 2.21-2.94mmol/l.According to N. Wullepit et al. (2009), high glucose levels were associated with high GPx activity [2].But in our study, there is an inverse association between glutathione peroxidase and glucose among highly productive cows: with an increase in GPx activity, the level of glucose in the blood decreases.Such a trend has not been established in the population of cows of the Mukhametshin Z.Z.PFE, but glucose takes high values in groups of animals with homozygous CC and TT genotypes.
The active aspartate aminotransferase (AST) and alanine aminotransferase (ALT) enzymes act as catalysts for the most important processes in the body associated with protein metabolism, and are involved in the opposing transfer reaction of amino acid amino groups to ketoacids, as well as in the synthesis of amino acids.In the population of indigenous cattle, the activity of AST was several times higher than that of imported cattle.In the context of genotypes in both populations, there was a decrease in the level of activity from group to group with CC ˃ TC ˃ TT genotypes.Cows of the Mukhametshin Z.Z.PFE had the difference between genotypes which is 8.74-29.88U/l (25.4-55.8%).Cows with the CC genotype in the Holstein cattle of indigenous selection had higher ALT, 5.4 U/l (15.2%) which is higher than TC, and 4.4 U/l (12.4%) which is higher than TT genotypes.In the groups of foreign selection cattle, the highest ALT level was found in cows with TT genotype -33.99 U/l, the average value in cows with TC genotype -31.42 U/l.These indicators were higher than CC genotype individuals had by 4.09 and 1.52 U/l (12.0 and 4.8%).
Cows of indigenous selection had the content of total protein in blood serum, which characterizes the level of protein nutrition in general, in the range of 88,291.9g/l, in groups of imported cattle this indicator increased slightly and amounted to 76.1-83.3g/l.TC genotypes cows had the highest level of total protein among indigenous selection cows, significantly exceeding this indicator in the CC group by 3.7 g/l (4.0%).TT-individuals were characterized by a high content of total protein in the blood serum in the Holstein imported cattle.Their advantage in this indicator over TC and CC genotypes cows was 6.8 g/l (8.2%) and 7.2 g/l (8.6%), respectively.
The level of albumin fraction, a plastic material that determines the possibilities for the synthesis of milk protein and muscle tissue, at the Lenin Breeding Farm IAPC was 40.5-42.6g/l.In terms of albumin fraction content in blood serum, CC and TT genotypes cows of the Mukhametshin Z.Z.PFE outperformed TC genotype individuals by 0.6 and 0.5 g/l (1.8 and 1.5%; p < 0.05).

Conclusion
As a result of the Tatarstan population of Holstein cattle of indigenous and foreign selection research, all possible variants of alleles and genotypes of the GPX-1 gene were identified, which indicates the genetic biodiversity and polymorphism of the studied populations.The difference in the genetic structure of the populations of indigenous and foreign selection can be caused by the use of bulls' sperm production of different origin.
Cows of indigenous selection, compared with imported cattle had higher: GPx activity, AST, ALT, the level of cholesterol, total protein and albumin fraction.Analysis of the relationship between the polymorphism of the GPX-1 gene and the level of the glutathione peroxidase enzyme indicates that depending on the genotype, the activity of this enzyme changes, and there is also a variation in the biochemical parameters of the Holstein cattle blood serum.However, all indicators were within the physiological norm, which indicates the absence of oxidative stress.

Table 3 .
Blood serum's biochemical parameters of cows with different genotypes of the GPX-1 gene