Program BR22885681 - Improvement of existing methods for managing the genetic resources of Adai and Kostanay horses while preserving the gene pool of the Kostanay breed

Program title: BR22885681 "Improvement of existing methods for managing the genetic resources of Adai and Kostanay horses while preserving the gene pool of the Kostanay horse breed"

Type of funding: Program-targeted funding of the Ministry of Agriculture of the Republic of Kazakhstan

Applicant: Kazakh Research Institute of Livestock and Fodder Production LLP

Scientific supervisor: A.A. Torekhanov, Doctor of Agricultural Sciences, Professor

Implementation period: 2024-2026

Relevance

One of the most important issues is assessing the significance of the breed composition of horses in the Republic and expanding the range of the domestic Kostanay horse breed.

The Kostanay horse breed was distinguished by its universal direction of use. However, in the process of breed development, under the influence of paratypic factors, its genealogical structure has changed noticeably, and significant changes in conformation, speed, and other breeding qualities are also observed. All this determines the need for its constant monitoring.

Successful breeding work, both with the breed and with individual subpopulations, is impossible without the development of main directions for further development.

In this regard, the development of new, effective technological measures for the Kostanay horse breed has been initiated. In the horse stud farm of "Kazakh Tulpary" LLP and other agricultural formations, additional stages will be developed and introduced into the existing feeding and management technology.

In 2023, within the framework of innovative projects in the agro-industrial complex of Mangistau region, under the 019 budget program "Services for the dissemination and implementation of innovative experience", for the implementation of measures to approve the "Adai" breed under the project "Determination of the genetic structure of the Adai horse population based on modern genotyping technologies, formation and approval of new lines, types and breeds", the results of long-term breeding work were summarized and applications for breeding achievements were submitted to the Intellectual Property Institute: "Rattory-60" factory line, "Bugabai kiik konyir-98" factory line, "Manap sur-93" factory line, "Mangistau intra-breed productive type", "Adai riding intra-breed type" and the "Adai" horse breed.

During the development of this program, employees of "KazRILFP" LLP developed, tested, and implemented a program for analyzing and calculating the breeding value of horses using the BLUP method. The domestic program contains no borrowed code, has patent purity, and is open for modifications.

Objective

Preservation of the gene pool of the Kostanay breed and improvement of existing methods for managing the genetic resources of Adai and Kostanay horses.

Expected and achieved results

Task: "Development of a software product integrating with existing information systems for collecting, storing, and processing data on breeding qualities of Adai and Kostanay horse populations".

In 2024, horses of the Adai and Kostanay populations meeting the desired type will be selected in base farms, with the study of phenotypic and selection-genetic indicators.

Selection and justification of criteria for statistical groups of horse populations based on pedigree records for subsequent analysis in BLUP (analytical model).

Development of a formalized description system of phenotypes of the Kostanay and Adai breeds for machine analysis (database).

Development of a program for selecting animals based on phenotypic traits (program).

Task: "Formation of the Adai horse population of riding and productive directions for the purpose of creating new breeding achievements".

In 2024, outstanding horses of the Adai population of riding and productive directions in Mangistau region will be selected for the formation of new lines. Evaluation, selection, and mating of horses in each genealogical line of stallions.

Preliminary evaluation of stallions by progeny quality.

Task: "Validation of specific genetic markers of the Adai and Kostanay horse populations with screening for the presence of genetically determined diseases".

In 2024, individuals of Adai and Kostanay horses will be selected for study by whole-genome and SNP genotyping methods. Hair follicles will be taken from selected animals from different farms. Genomic DNA will be extracted from these animals and prepared for SNP genotyping using the K80 array. Determination of the frequency of occurrence of specific alleles associated with genetically determined diseases in the Adai and Kostanay horse populations.

Task: "Development of a panel of genetic markers enabling early prediction of endurance, speed, and agility with further validation of the developed methodology on horses of the Adai and Kostanay populations".

In 2024, whole-genome SNP genotyping will be performed using the SNP K80 array on iScan at the molecular genetic analysis laboratory of the Kazakh Research Institute of Livestock and Fodder Production LLP.

Quality control of genotyped data and preparation of data for genetic disease analysis and GWAS.

Task 1: "Development of a software product integrating with existing information systems for collecting, storing, and processing data on breeding qualities of Adai and Kostanay horse populations"

Horses of the Adai (317 heads) and Kostanay (179 heads) populations meeting the desired type were selected in base farms, with the study of phenotypic and selection-genetic indicators.

An expeditionary survey of the Kostanay horse population with breeding status was conducted in Kostanay, Akmola, and Karaganda regions. Horses were identified by electronic chip, brand, color description, and markings in breeding certificates issued by breed chambers.

The criteria for statistical groups of horse populations were selected and justified based on pedigree records for subsequent analysis in BLUP.

Statistical groups are manifested in the macro-assessment (population-level assessment) of the hereditary factor in the BLUP linear unbiased prediction method. Application of this method to horse populations requires a clear understanding of the causes of genetic contribution variance to animal productivity in the presence of several statistical groups. This leads to the need to estimate variances of individual groups. At another level, point molecular-genetic characteristics of individual animals are also studied.

A database for formalized description of phenotypes of the Kostanay and Adai breeds for machine analysis was developed.

For this task, a database was developed on the KazRILFP website with the ability to enter information from primary material (region, district, farm name, breed, type, sex, identification number, nickname, brand number, color, date of birth, evaluation date, height at withers, oblique body length, chest circumference, cannon bone circumference, live weight, assessment by type and origin, measurements and weight, exterior, milk production, adaptive qualities, and progeny quality), photometric material, data segmentation by various indicators, which can be obtained in the form of a tabular report.

A program for selecting animals by phenotypic traits was developed.

Data analyses were performed in the NEL software environment developed at KazRILFP. This software platform has several releases designed for different types of livestock. Several versions were developed within each release, which was driven by the need to increase the number of functions performed due to the expansion of the range of tasks. For the execution of work on this task, the NEL.meat.hr release, developed for working with meat-direction horse populations, was used.

Task 2: "Formation of the Adai horse population of riding and productive directions for the purpose of creating new breeding achievements"

Outstanding horses of the Adai population of riding and productive directions in Mangistau region were selected for the formation of new lines.

As a result of the expeditionary survey of 9 farms in Mangistau region engaged in breeding Adai horses of riding and productive directions, outstanding horses were selected for the formation of new lines. Total population of 317 heads, including: stallions - 20, mares - 255, colts - 5, fillies - 37.

Evaluation, selection, and mating of horses in each genealogical line of stallions was performed.

As a result of evaluation, selection, and mating, work began on forming a new genealogical line of the stallion Karakus-2005 of the Adai riding intra-breed type, with his sons Almastory-2015, Naizagai-2017, and Kulager-2020.

A preliminary evaluation of stallions by progeny quality was performed.

As a result of the preliminary evaluation of stallions by progeny quality, the highest average score was observed in the progeny of stallion Naizagai-2017. This is most likely due to the higher variability in live weight (8.56) among the mares of his herd.

Thus, the results of the preliminary evaluation of stallions by progeny quality made it possible to determine further breeding measures, where the progeny of stallions Almastory-2015 and Naizagai-2017 will be monitored for growth and development to continue line breeding. The progeny quality of stallion Kulager-2020 will be evaluated over the next two years.

In order to determine the speed performance indicators of Adai riding type horses, together with representatives of the Association of Legal Entities "ADAI" breeding horse breed, individual entrepreneurs, and regional agricultural authorities, participation in the organization and conduct of a 100 km distance horse ride according to FEI rules was ensured.

Horses of various breeds participated in the competition, including:

Adai breed - 18 heads, crossbreeds of thoroughbred breeds - 3 heads.

Institute employees were included in the judging panel: from Oman, Yemen, Egypt, Syria, and UAE - 15 people, Kazakhstan - 3 people. Two employees of "KazRILFP" LLP received the qualification of FEI international veterinary judges.

Task 3: "Validation of specific genetic markers of the Adai and Kostanay horse populations with screening for the presence of genetically determined diseases"

Individuals of Adai and Kostanay horses were selected for study by whole-genome and SNP genotyping methods. Hair follicles were taken from selected animals from different farms. Genomic DNA was extracted from these animals and prepared for SNP genotyping using the K80 array.

In accordance with the project objective, the Adai and Kostanay horse breeds were genotyped using the Equine 80K single nucleotide polymorphism array and statistical analysis was performed. For whole-genome DNA extraction, 496 hair follicle samples were collected from the tails of Adai and Kostanay horses.

SNP genotyping was performed using the Equine 80K SNP chip on the iScan genetic apparatus. Primary quality control of SNPs was performed using GenomeStudio software, and the Plink 1.9 program was additionally used for final results. SNP errors exceeding 10% were removed from genotype data. Individuals with low genotyping rates were removed. Minor allele frequencies below 5% were excluded from the final genotypic dataset.

Broad genomic SNP genotyping was performed using the Equine 80K SNP array at the molecular genetic laboratory of the Kazakh Research Institute of Livestock and Fodder Production. The Illumina iScan genetic analyzer was used for SNP genotyping of horses. As a result, 61,161 SNP markers were obtained in Adai breed horses, and the overall genotyping rate was 0.99%. After filtering SNPs with a 2% threshold, 15 variants were removed due to missing genotype data. Data quality control for missing sample genotypes was set at 0.02, and no individuals were removed. As a result, 61,146 variants remained in Adai horses.

As a result of the primary quality control, 61,161 variants remained in the genotypic data of Kostanay breed horses. More than 2% of missing SNP genotype data and samples were excluded for further studies of Kostanay breed horses. After checking the genotype data quality, the Kostanay breed had 58,478 SNPs.

The frequency of occurrence of specific alleles associated with genetically determined diseases in the Adai and Kostanay horse populations was determined.

Based on the results of the literature review, a list of the main markers and genetically determined diseases of horses was compiled.

Task 4: "Development of a panel of genetic markers enabling early prediction of endurance, speed, and agility with further validation of the developed methodology on horses of the Adai and Kostanay populations"

Whole-genome SNP genotyping was performed using the SNP K80 array on iScan at the molecular genetic analysis laboratory of the Kazakh Research Institute of Livestock and Fodder Production LLP.

Minor allele frequency values were determined within various categories in horses of the Kostanay and Adai breeds.

It was determined that the myostatin gene consists of 4,973 nucleotide sequences, located on chromosome 18 of Equus Caballus, whose main function is the regulation of muscle fibers, on the correct expression of which muscle hypertrophy and hypotrophy depend. The mutation located at position 1444, which represents a nucleotide substitution of Thymine to Cytosine, is the most studied and has a connection with endurance and speed of horses. The TT genotype is found in more enduring horses, and the CC genotype in faster horses. These alleles require additional research.

Quality control of genotyped data and preparation of data for genetic disease analysis and GWAS was performed.

In the work, 2 separate datasets were used for genetic disease analysis and GWAS. GWAS to determine the association of genes with endurance and speed of horses will be conducted exclusively on new genotyping data of selected Adai and Kostanay breed horses. Genetic disease analysis will be performed on the dataset expanded with data from the previous project.

For the formation of the new dataset, hair follicle samples were collected from 317 representatives of the Adai breed from 9 farms in 6 administrative districts of Mangistau region, and 179 from representatives of the Kostanay horse breed from 12 agricultural formations in 8 districts of Kostanay, Karaganda, and Akmola regions.

Two articles published:

1. Kapas T., Akhmetov U.A., Kulboldin T.S., Zetova A.A., Nurmakhanbetov D.M., Dosybayev K.Zh. Analysis of domestic horse breeds based on whole-genome SNP genotyping. pp. 332-341. Bulletin of Korkyt Ata Kyzylorda University, Agricultural Sciences, No. 4 (71), 2024. https://doi.org/10.52081/bkaku.2024.v71.i4.212
2. Brel-Kiseleva I.M., Amandykova A.B., Safronova O.S. Improvement of factory lines in the Kostanay horse breed. pp. 62-68. Multidisciplinary Scientific Journal of Kostanay Regional University named after Akhmet Baitursynov "3i: intellect, idea, innovation", No. 4, December 2024. https://doi.org/10.52269/22266070_2024_4_62

A final report for 2024-2026 was submitted in accordance with GOST 7.32-2017.

Participants in the program implementation:

• 3 Doctors of Science;
• 7 Candidates of Science;
• 1 PhD;
• 8 Masters of Science;
• 2 doctoral students;
• 4 master's students.

Expected results for 2025:

 

In accordance with the assigned tasks, upon completion of the Program activities, the following results will be obtained:

Task: "Development of a software product integrating with existing information systems for collecting, storing, and processing data on breeding qualities of Adai and Kostanay horse populations".

In 2025, mathematical and computational methods for solving the BLUP equation system with statistical groups of horse populations based on pedigree and genome were developed. Development of the problem of heterogeneous variance in the mixed (pedigree and/or genome) BLUP model was carried out (algorithm and program).

One article was published in a domestic publication recommended by CCSMES.

Task: "Formation of the Adai horse population of riding and productive directions for the purpose of creating new breeding achievements".

In 2025, tests were conducted to determine the endurance and speed of Adai horses in distance horse rides. The results of Adai horses in all official horse rides conducted according to the rules approved by FEI-2002 were analyzed. Phenotypic and selection-genetic characteristics of genotypes of the Adai horse population of riding and productive directions were systematized and studied.

One article was published in a domestic publication recommended by CCSMES.

Task: "Validation of specific genetic markers of the Adai and Kostanay horse populations with screening for the presence of genetically determined diseases".

In 2025, bioinformatic analysis was performed to predict the high risk of the presence or absence of alleles associated with genetic diseases in the studied horses.

One article was published in a domestic publication recommended by CCSMES.

Task: "Development of a panel of genetic markers enabling early prediction of endurance, speed, and agility with further validation of the developed methodology on horses of the Adai and Kostanay populations".

Quality control of genotyped data and preparation of data for genetic disease analysis and GWAS.

 

In 2025, genome-wide association studies were conducted to identify significant SNP markers for endurance, speed, and agility in Adai and Kostanay horses.

One article was published in a domestic publication recommended by CCSMES.

 

1. 3 Doctors of Science, 1 PhD Doctor, 3 Candidates of Science, and 6 Masters participated in the program implementation.

 

List of publications with full bibliographic description for 2025:

1. Torekhanov A.A., Kozhanov Zh.E., Seisenova Zh.M., Dosybayev K.Zh., Akhmetov U., Kapasuly T., Bekmanov B.O. Analysis of the genetic structure of the Adai horse breed using SNP markers // Science and Education. - No. 2-5 (79). Part 5. - pp. 134-143. https://doi.org/10.52578/2305-9397-2025-2-5-133-143
2. Torekhanov A.A., Nurmakhanbetov D.M., Kozhanov Zh.E., Akhmetov U.A. Formation of a new stallion line in the Adai horse breed riding type // Bulletin of Korkyt Ata Kyzylorda University, No. 3 (74), pp. 278-285. https://agro.korkyt.edu.kz/downloads/arch/journal_author_AGsoTXDn7ZD63nc.pdf
3. M.K. Nauryzbayev, E.I. Imangaliyev. Bulletin of the National Engineering Academy of the Republic of Kazakhstan. No. 3 (97), pp. 187-202. https://journal.neark.kz/category/zhurnal/%e2%84%96-3-97-2025/
4. Torekhanov A.A., Kozhanov Zh.E., Kaiyipbai B.B., Talzhanov E.A. Genetic monitoring of hereditary diseases in Kostanay and Adai horses: frequency of carriage of SCN4A, GYS1, GBE1, PPIB, EDNRB and STX17 mutations // Journal "3i: intellect, idea, innovation" of Kostanay Regional University named after Akhmet Baitursynov, No. 3, Part 1, pp. 255-263. https://ojs.ksu.edu.kz/index.php/3i/article/view/1573/739