Determination of Diplotypes Associated with Meat Productivity in Cattle Breeds Common in the Territory of the Republic of Kazakhstan

: Technologies for genetic labeling of economically useful traits using polymorphic variants of genes that control the rate of growth and development of animals make it possible to assess the genetic potential of an animal immediately after birth, which significantly reduces time and financial costs. This study aimed to identify paired combinations of the genes growth hormone, growth hormone receptor, and insulin-like growth factor-1 associated with meat productivity in related breeds of cattle meat direction: Hereford, Kazakh white-headed, Aberdeen Angus, Auliekol. The materials for the study were blood and hair follicles samples of animals born in 2015, aged 18 and 24 months. The animals were selected from farms in northern Kazakhstan with equal feeding and housing conditions. The PCR-RFLP method was used to determine the genotypes of animals. According to the results of this study, the associations of phenotypes with the genotypes of the bGH , bGHR , and bIGF-1 genes in the studied cattle breeds were revealed.


Introduction
Animal husbandry in the Republic of Kazakhstan is the main branch of agriculture, which faces the important task of providing the population with food, processing, and light industry with raw materials. To do this, it is necessary not only to improve the technology of raising animals and preparing feed but also to develop and introduce new promising methods for determining and increasing the productivity of animals, which allow them to meet the new requirements for evaluating animals and reduce economic costs by reducing the investment period.
Per the challenges of our time for the intensive development of the meat industry in Kazakhstan, along with the purchase of elite livestock of highly productive breeds, there is an increasing need to develop and master modern breeding methods that allow for forming a highly productive herd of its selection, quickly and efficiently, based on the purchased livestock. This highly productive herd is adapted to the local infectious background, climate, and conditions of keeping, feeding, and breeding.
Such fast and effective tools are provided to breeders of the Republic of Kazakhstan by a marker-accompanying selection. This technology makes it possible to accelerate the rate of selection and reduce the financial cost of classical breeding activities. The MAS -breeding allows for estimating the genetic potential of animal productivity at the early stages of postnatal development (Khare and Khare, 2017;Salisu et al., 2018;Beishova et al., 2019;Dushayeva et al., 2021).
Is a chain of sequential interactions between The Growth Hormone (GH), and somatotropin, which is known to be the most important growth regulator in mammals. The synthesis of somatotropin and the realization of its physiological effects on protein and receptor (somatotropin cascade). Important components of this chain are the growth hormone receptor (bGHR), which transmits the humoral somatotropin 288 signal to target cells, and insulin-Like Growth Factor-1 (bIGF-1), which triggers intracellular responses to somatotropin exposure (Parmentier et al., 1999).
In several cases, the data on the somatotropin cascade genes alleles with productivity traits obtained in different breeds are difficult to compare and contradict each other (Boichard et al., 2003) and such studies have not been performed for a significant part of the identified alleles.
Therefore, research to understand the mechanisms of phenotypic effects of candidate genes in different breeds is of great importance. Of particular interest, in this case, are breeds that are related to each other.
This article presents the results of the analysis of gene associations of bGH-AluI, bGHR-SspI, and bIGF-1-SnaBI with phenotypic data on meat productivity in the most widespread breeds in Kazakhstan-foreign Hereford and Aberdeen Angus breeds and local Kazakh Whitehead and Auliekol breeds.
According to the Ministry of Agriculture of the Republic of Kazakhstan as of 01.01.2021, the number of breeding livestock for meat production in the country is more than 682 thousand. Of these, more than 69 thousand are the Hereford breed, more than 393 thousand the Kazakh whiteheaded, about 113 thousand are Aberdeen Angus breed and more than 60 thousand are the Auliekol breed. Thus, these breeds make up the bulk of beef cattle bred on the territory of the Republic of Kazakhstan and the search for effective ways to increase the productivity of livestock is of strategic importance for the country (MARK, 2022).
This study aimed to identify paired combinations of the genes growth hormone, growth hormone receptor, and insulin-like growth factor-1 associated with meat productivity in related breeds of cattle meat direction: Hereford, Kazakh white-headed, Aberdeen Angus, Auliekol.
The materials for the study were blood and hair follicles samples of animals born in 2015, aged 18 and 24 months. The animals were selected from farms in northern Kazakhstan with equal feeding and housing conditions. Animal productivity data were obtained from the records of the farms that provided the samples. The PCR -RFLP method was used to identify the genotypes of animals. Table 1 shows the primer sequences and PCR conditions for the analysis of each polymorphism.
Amplified PCR products of the bovine bGHR gene (182 bp) were digested using the restriction enzyme SspI. The digested bGHR-SspI FF PCR product exhibited two fragments of 158 and 24. For the bGHR-SspI FY genotype were exhibited three fragments of 182, 158, and 24 bp. For the bGHR-SspI YY genotype exhibited undigested one fragment of 182 bp.

Results
Appendix 1 shows that the ratio of genotypes of the same polymorphism differs considerably between breeds. On the one hand, the observed phenomenon may testify in favor of their association with economically useful traits and the pressure of artificial selection. On the other hand, it allows one to assess the prospects for the use of genetic markers in breeding programs.
The generally accepted minimum value of the relative frequency of a minor allele in the population for the genetic marker is 5%. From the data in Appendix 2, it can be seen that the alleles of all three polymorphisms can be used as genetic markers in breeding programs if a statistically significant association with the trait of live weight is found. The exception is the allele bGHR-SspI Y in animals of the Auliekol breed.
The data presented in Appendix 3 and Fig. 1 suggest that there is a difference in live weight performance between animals at the age of 18 and 24 months in the Kazakh whiteheaded and Auliekol breeds of polymorphisms bGH-AluI, bGHR-SspI, and bIGF-1-SnaBI. In turn, the same cannot be said for Hereford and Aberdeen Angus animals.
From the data shown in Table 3, a significant difference between the animals' groups of bIGF-1-SnaBI polymorphism in Kazakh white-headed and Auliekol animals at the age of 18 and 24 months should be noted.
Since the character of the distribution of the trait in the studied groups was different from the normal one and in the future, groups with less than 20 animals were included in the analysis of diplotypes, a nonparametric method for calculating 95% confidence intervals for the median was used to assess the difference between the group and the sample. For a group of animals of the Kazakh white-headed breed, interval estimation was also carried out relative to the total sample (Fig. 2).
The results of the assessment of the nature of the association of the genotype association concerning the total sample for the Auliekol animals are shown in the diagrams in Fig. 3.
The paired-combination analysis in the breed study followed the same research pattern as the individual genotype analysis.
From the data given in Table 4, it can be noted that the diplotype bGH-AluI LL -bIGF-1-SnaBI AA has the most pronounced decreasing effect on the live weight of the Kazakh white-headed breed at the age of 24 months, which is characterized by the decrease in the indicator concerning the standard by 37 kg (9.00%). The frequency of diplotype in the studied sample is approximately 3.05%.
Graphical evaluation of the statistical significance of phenotypic effects of paired combinations of the somatotropin cascade genes in the Auliekol animals at the age of 24 months revealed the diplotypes marking both increased and decreased body weight (Fig. 5).

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In the phenotypic effects' analysis of paired combinations of the studied polymorphisms on the trait of live weight at 24 months in Aberdeen-Angus animals, a significant upward phenotypic effect was found for the paired combination of bGH-AluI VV -bIGF-1-SnaBI BB . Figure 6 shows that the boundaries limits of the confidence interval for the median of the diplotype group from 420 to 431 kg and fall outside the confidence interval for the median sample of 415-418 kg for this trait.
A comparison with the Aberdeen Angus breed standard revealed that the live weight of animals with the bGH-AluI VV -bIGF-1-SnaBI BB diplotype at the age of 24 months exceeded the live weight of the Aberdeen Angus breed standard by 45 kg (12%) and 12.52 kg (3%) when compared with the total sample (Table 4). This indicates that the bGH-AluI VV -bIGF-1-SnaBI BB diplotype is the recommended genetic marker to increase the live weight of Aberdeen Angus animals at the age of 24 months.
The frequency of diplotype in the studied sample is approximately 5.08%.
Studies have shown that there were no marking diplotypes found in the Hereford breed.

Discussion
Any population that is in constant flux. Breeding processes occurring in herds and populations through gene migration, their elimination due to various circumstances, as well as emerging mutations and recombination, change their genotypic structure, which makes it possible to analyze and control the breeding process.
The selection of animals for economically useful traits directly or indirectly leads to changes in the gene pool of 293 animals and its structure (Piccoli et al., 2017) and, in turn, the genetic structure of the population determines the possibility of using marker-associated selection in breeding programs, as well as to assume the effectiveness of such measures.
Analysis of bGH-AluI polymorphism showed a similar distribution of allele frequencies in the studied breeds (Appendix 2). The bGH-AluI L allele occurs with a frequency of 68.43 in the Hereford breed, 51.3 in the Aberdeen-Angus breed, and 66.61 in the Auliekol breed. In the Kazakh white-headed breed its frequency is significantly higher -83.05. The data obtained agree with the results of Selionova et al. (2017), which established a high occurrence of bGH-AluI L allele (0.64; 0.78-0.89) in Hereford, Kalmyk and Kazakh white-headed breeds; Sedykh (2017), who established the frequency of bGH-AluI L allele 0.684 and 0.731, respectively, in Herefords and Limousins; Sharipov et al. (2015), who established the frequency of bGH-AluI L allele 0.61 and 0.85 in Herefords and Limousines breed in Tatarstan; Krasnopiorova et al. (2012) who found the frequency of bGH-AluI L allele in Charolais, Herefords and Simmentals to be 0.850; 0.900 and 0.563, respectively; Lee et al. (2013), who found a high incidence of the bGH-AluI L allele of 0.907 in Hanwoo; Hartatik et al. (2013), who found the frequency of the bGH-AluI L allele of 0.830 and 0.910 in Limousins (Limousin); Kostenko and Starodub (2011), who established the frequency of the bGH-AluI L allele in Polis Sian Beef at 0.807; Mokhnachova et al. (2016), who found the frequency of the bGH-AluI L allele in Ukrainian Grey cattle to be 0.980. It should be noted that the low frequency of the bGH-AluI L allele is observed in different breeds bred in Russia. This may be due to the long-term selection of local cattle populations. Thus, Sulimova et al. (2011) the low frequency of the bGH-AluI L allele in Kalmyk, Kazakh White-headed, and Mongolian breeds -0,117; 0,209, and 0,125 were established. According to the bGHR-SspI polymorphism, all the studied breeds showed a clear advantage of the bGHR-SspI F allele (Appendix 2). Thus, its frequency was 95.10 in the Auliekol breed, 91.36 in the Kazakh whiteheaded breed, and 78.39 in the Aberdeen Angus breed. In the Hereford breed, the frequency of the bGHR-SspI F allele was slightly lower and amounted to 62.12. The data obtained are from the data of other researchers. In their research works the frequency of occurrence of the F allele of the bGHR gene in cattle of different breeds was 0,667 (Fedota et al., 2017), Jersey-0,850 (Komisarek et al., 2011), Holstein-0,840 (Rahmatalla et al., 2011), Romanian Black-and-White-0.770 and Romanian Grey Steppe-1.0, respectively (Carsai et al., 2013). The bIGF-1-SnaBI polymorphism showed a similar distribution of allele frequencies among the studied breeds (Appendix 2). Thus, the frequency of the bIGF-1-SnaBI B allele in the Kazakh white-headed breed was 66.10, in the Auliekol breed-61.54, and the Hereford breed-60.61. The exception was the Aberdeen-Angus breed, where the bIGF-1-SnaBI B allele was less common (42.19). Several studies have been devoted to determining the bIGF-1-SnaBI polymorphism in cattle. In their works, the frequency of a bIGF-1-SnaBI B allele in Holstein cattle is 0.562 (Yousef et al., 2010), in commercial lines -0,56 (Li et al., 2004), in Aberdeen Angus breed-0,64 (Ge et al., 2001). In M. Szewczuk; E. Siadkowska the frequency of the bIGF-1-SnaBI B allele was lower and was 0.33 for Montbeliarde cattle (Szewczuk, 2016) and 0.48 for the Holstein-Friesian breed (Siadkowska et al., 2006).
The highest live weight according to the bGH-AluI polymorphism in the Kazakh white-headed breed at the age of 18 months was observed in the group of animals with the bGH-AluI LV genotype (395±27), animals with a lower live weight are observed in the group with the bGH-AluI LL genotype (342±29 kg) (Appendix 3). At the same time, according to Fedota et al. (2016), animals of Aberdeen-Angus breed with bGH-AluI LL genotype were superior to animals with bGH-AluI LV and BGH-AluI VV genotypes in growth performance. According to Lee et al. (2013), Thomas et al. (2007), Hartatik et al. (2020) and Gill et al. (2009) animals with the bGH-AluI LL genotype had the highest live weight at birth. In cattle, the bGH-AluI L allele is associated with higher body weight and marbling score. However, Hartatik et al. (2020) suggested that the bGH-AluI V allele may be a potential genetic marker in cattle.
The bGHR-SspI polymorphism in animals of the Kazakh white-headed breed was the preferred bGHR-SspI YY genotype (376±31 kg) (Appendix 3). This agrees with the previously published data of other authors. For example, according to (Fedota et al., 2017) the bGHR-SspI YY genotype was also preferred for Aberdeen-Angus breed animals. Thus, the group with this genotype was characterized by a higher Average Daily Gain (ADG) and live weight at 8 months of age (Fedota et al., 2017). The known SNP F279Y is associated with dairy performance and is included in the current Marker-Assisted Selection (MAS) for commercial purposes in several countries (Olenski et al., 2010).
In animals of the Auliekol breed, the difference in live weight in groups with different genotypes is characteristic of the bIGF-1-SnaBI polymorphism and the genotype bIGF-1-SnaBI AA is preferred (387±11 kg) (Appendix 3). Other researchers have previously reported that the bIGF-1-SnaBI polymorphism affects the growth rate and meat productivity of Angus, Beefbooster, Holstein-Friesian, and Charolais cattle (Li et al., 2004;Siadkowska et al., 2006;De la Rosa Reyna et al., 2010;Ge et al., 2001).
According to Daniela do Amaral Grossi et al. (2015), the bIGF-1-SnaBI A allele was found to be associated with higher live weight at birth, weaning, 12 and 18 months of age in Canchim beef cattle. A. Rogberg-Muñoz et al. (2011) identified an association of the bIGF-1 gene with live weight at weaning in commercial and experimental groups of Hereford breeds.
The trend of increased or decreased live weight in animals of the Kazakh white-headed and Auliekol breeds by preferred or undesired genotypes persists at the age of 24 months. Data for the other genotypes were not considered, since they were approximately equal to the weight of the breed standard.
The diagrams in Fig. 2 show that the groups with the bIGF-1-SnaBI AA , bIGF-1-SnaBI AB , and bIGF-1-SnaBI BB genotypes are indeed significantly different from each other. However, the group with the genotype is deconsolidated and its range of traits is outside both the upper and lower confidence intervals of the median of the total sample. Moreover, this trend can be traced in all age categories.
Thus, it should be stated that it is not appropriate to use the bIGF-1-SnaBI BB genotype as a genetic marker for cows of the Kazakh white-headed breed.
The average live weight of animals at 18 months is 365±10 kg and at 24 months 413±7 kg, which is 4 and 3 kg below the breed standard (Appendix 3). Taking into account that this genotype in the studied sample is 43.36%, a decrease in its frequency of occurrence in the course of breeding programs could yield a very tangible result. Thus, it is obvious that the bIGF-1-SnaBI BB genotype is a genetic marker of the low live weight of the Auliekol breed animals at the age of 18 and 24 months. Work with this genetic marker should not be based on the selection of the preferred genotype but the elimination of the negative bIGF-1-SnaBI BB genotype.
The most pronounced increasing effect is observed in groups of animals of the Kazakh white-headed breed with the diplotype bGH-AluI LV -bIGF-1-SnaBI AB (Fig. 4). In this group of animals, the live weight at the age of 24 months is in the range of 435-492 kg and the most pronounced decreasing phenotypic effect is observed in animals of the Kazakh white-headed breed with the diplotype bGH-AluI LL -bIGF-1-SnaBI BB (the median value of live weight according to a group from 374 to 397 kg). While the live weight of animals of the total sample of the Kazakh white-headed breed at the age of 24 months is in the range of 405-420 kg.
The diplotype bGH-AluI LV -bIGF-1-SnaBI AB , which is characterized by an increase in the indicator relative to the standard by 57 kg (14.33%), has the most pronounced increasing phenotypic effect on the live weight of the Kazakh white-headed breed animals at the age of 24 months (Table 4). The diplotype frequency in the sample studied is approximately 9.49%. Table 4 shows that the greatest economic effect will be given by animals with the increasing diplotype bGH-AluI LV -bIGF-1-SnaBI AB and bGH-AluI LV -bIGF-1-SnaBI BB . In this case, animals with the above diplotypes should be left in the herd for further selection.
The most pronounced decreasing effect is observed in the group of animals with diplotype bGH-AluI LL -bIGF-1-SnaBI BВ, the live weight of animals aged 24 months is in the range of 365-383 kg (Fig. 5). This is significantly lower compared to the general sample, in which the median live weight is in the range of 405-423 kg. The most pronounced enhancing phenotypic effect was observed in the group of animals with diplotype bGH-AluI LV -bIGF-1-SnaBI BB , where the live weight of animals at 24 months of age is in the range of 428-513 kg, which is considerably higher compared to the total sample, in which the median live weight is in the range of 405-423 kg.
From the data given in Table 4, it follows that the diplotype bGH-AluI LL -bIGF-1-SnaBI BВ is characterized by the most pronounced decreasing effect on the Auliekol breed animals at the age of 24 months concerning the standard of the breed by 40 kg (9.73%). The diplotype frequency in the studied sample is approximately 9.44%.
The diplotype bGH-AluI LV -bIGF-1-SnaBI BB has a markedly pronounced increasing phenotypic effect on the live weight of the Auliekol breed animals at the age of 24 months, which is characterized by a 47 kg (14.40%) increase in the index concerning the standard. The frequency of the diplotype in the studied sample is approximately 3.85%. Animals with this diplotype, as in the case of the Kazakh white-headed breed, should be kept in the herd for further breeding, and animals with the diplotype bGH-AluI LL -bIGF-1-SnaBI BВ should be excluded from reproduction.

Conclusion
According to the results of this study, the associations of phenotypes with the genotypes of the bGH, bGHR, and bIGF-1 genes in the studied cattle breeds were revealed.
We recommend using the identified diplotypes with decreasing or increasing effects on live weight in the studied breeds in the selection of animals within the framework of breeding programs. At the same time, we propose to keep animals with an increasing type of diplotypes in the herd for breeding work and to exclude animals with diplotypes having decreasing effects from reproduction.

Funding Information
The work was carried out within the framework of a project of program-targeted financing of the Ministry of 295 Agriculture of the Republic of Kazakhstan for 2021-2023 BR10764981 "Development of technologies for effective management of the selection process of preserving and improving genetic resources in the beef cattle breeding" and the grant funding for young scientists on scientific and (or) scientific and technical projects of the Ministry of Education and Science of the Republic of Kazakhstan for 2020-2022 AP08052960 "Specific QTL-marking of meat productivity of cattle from Auliekol and Kazakh white-headed breeds based on genome-wide SNP-chipping".