First transcriptomic insight into the working muscles of racing pigeons during a competition flight.

BACKGROUND: The currently known homing pigeon is a result of a sharp one-sided selection for flight characteristics focused on speed, endurance, and spatial orientation. This has led to extremely well-adapted athletic phenotypes in racing birds. METHODS: Here, we identify genes and pathways contributing to exercise adaptation in sport pigeons by applying next-generation transcriptome sequencing of m.pectoralis muscle samples, collected before and after a 300 km competition flight. RESULTS: The analysis of differentially expressed genes pictured the central role of pathways involved in fuel selection and muscle maintenance during flight, with a set of genes, in which variations may therefore be exploited for genetic improvement of the racing pigeon population towards specific categories of competition flights. CONCLUSIONS: The presented results are a background to understanding the genetic processes in the muscles of birds during flight and also are the starting point of further selection of genetic markers associated with racing performance in carrier pigeons.

Characteristic of Przewalski horses population from Askania-Nova reserve based on genetic markers.

Przewalski horses are considered the last living population of wild horses, however, they are secondarily feral offspring of herds domesticated ~ 5000 years ago by the Botai culture. After Przewalski horses were almost extinct at the beginning of the twentieth century, their population is about 2500 individuals worldwide, with one of the largest breeding centers in Askania-Nova Biosphere Reserve (Ukraine). The research aimed to establish the maternal variation of Przewalski horses population maintained in Askania-Nova Reserve based on mitochondrial DNA hypervariable 1 and hypervariable 2 regions profiling, as well as, analysis of Y chromosome single nucleotide polymorphism unique for Przewalski horses, and coat color markers: MC1R and TBX3. The mtDNA hypervariable regions analysis in 23 Przewalski horses allowed assigning them to three distinctly different haplotypes, showing the greatest similarity to the Equus caballus reference, the Equus przewalskii reference, and to extinct species-Haringtonhippus. The Y chromosome analysis using fluorescently labelled assays differentiated horses in terms of polymorphism (g731821T>C) characteristic of Equus przewalskii. All male individuals presented genotype C characteristics for Przewalski horses. The polymorphisms within the coat color genes indicated only native, wild genotypes. The Y chromosome and coat color analysis denied admixtures of the tested horses with other Equidae.

The use of the SLC16A1 gene as a potential marker to predict race performance in Arabian horses.

BACKGROUND: Arabian horses are commonly believed to be one of the oldest and the most popular horse breeds in the world, characterized by favourable stamina traits and exercise phenotypes. During intensive training, the rates of lactate production and utilization are critical to avoid muscle fatigue and a decrease in exercise performance. The key factor determining transmembrane lactate transport is the monocarboxylate transporter 1 protein coded for by the SLC16A1 gene. The aim of the present research was to identify polymorphisms in the coding sequence and UTRs in the equine SLC16A1 gene and to evaluate their potential association with race performance traits in Arabian horses. Based on RNA-seq data, SNPs were identified and genotyped using PCR-RFLP or PCR-HRM methods in 254 Arabian horses that competed in flat races. An association analysis between polymorphisms and racing results was performed. RESULTS: Novel polymorphisms in the equine SLC16A1 locus have been identified (missense and 5'UTR variants: g.55601543C > T and g.55589063 T > G). Analysis showed a significant association between the 5'UTR polymorphism and several racing results as follows: the possibility of winning first or second place, the number of races in which horses started and total financial benefits. The analysis also showed differences in genotype distribution depending on race distance. In the studied population, the shorter distance races were only won by TT horses. The GG and TG horses took first and second places in middle- and long-distance races, and the percentage of winning heterozygotes increased from 19.5 to 27% at the middle and long distances, respectively. The p.Val432Ile (g.55601543C > T) polymorphism was not significantly related to the analysed racing results. CONCLUSION: Our results showed that g.55589063 T > G polymorphism affected the possibility of winning first or second place and of competing in more races. The different distribution of genotypes depending on race distance indicated the possibility of using a SNP in the SLC16A1 gene as a marker to predict the best race distance for a horse. The presented results provide a basis for further research to validate the use of the SLC16A1 gene as a potential marker associated with racing performance.

ACTN3 genotype distribution across horses representing different utility types and breeds.

In horses, the identification of the genetic background of phenotypic variation, especially with regard to performance characteristics and predisposition to effort, has been extensively studied. As α-actinin-3 function is related to the regulation of muscle contraction and cell metabolism, the ACTN3 gene is considered one of the main genetic factors determining muscle strength. The aim of the present study was to assess the genotype distribution of two SNP variants within the equine ACTN3 gene (g.1104G > A and c.2334C > T) across different utility types and horse breeds. The analyses were performed on five breeds representing horses of different types, origins and utilities according to performance (in total 877 horses): primitive (Polish koniks; Hucul horses), draught (Polish heavy draught) and light (Thoroughbred and Arabian horses). Two polymorphisms within the ACTN3 gene locus were genotyped and genotype and allele frequency were compared across populations in order to verify if the identified differences contribute to the phenotypic variation observed in horse breeds. The present study allowed confirmation of the significant differences in genotype distribution of g.1104G > A localized in the promoter region between native breeds and racehorse breeds such as Thoroughbreds and Arabians. The allele/genotype variations between primitive and light breeds confirmed that the analysed variant was under selection pressure and can be correlated with racing ability. Moreover, the significant differences for the c.2334C > T genotype frequency between Arabian horses and other breeds indicate its relationship with endurance and athletic performance. The predominance of the T allele (85%) in Arabians suggests that the T variant was favoured during selection focused on improving stamina and could be one of the genetic factors determining endurance ability. Further research is needed to confirm the association of both polymorphisms with exact racing and/or riding results.

Exercise-induced modification of the skeletal muscle transcriptome in Arabian horses.

It has been found that Arabian and Thoroughbred horses differ in muscle fiber structure and thus in physiological changes occurring in muscles during exercise. The aim of the present study was to identify the global gene expression modifications that occur in skeletal muscle following a training regime to prepare for flat racing. Whole transcriptomes of muscle (gluteus medius) were compared between three time points of tissue collection: T0 (untrained horses), T1 (horses after intense gallop phase), and T2 (horses at the end of racing season), 23 samples in total. The numerous groups of exercise-regulated differentially expressed genes (DEGs) were related to muscle cell structure and signaling and included insulin-like growth factor 1 receptor (IGF1R), insulin receptor (INSR), transforming growth factor beta receptors 1 and 2 (TGFBR1, TGFBR2), vascular endothelial growth factor B (VEGFB); epidermal growth factor (EGF), hepatocyte growth factor (HGF), and vascular endothelial growth factor D (FIGF). In Arabian horses, exercise modified the expression of genes belonging to the PPAR signaling pathway (e.g., PPARA, PPARD, and PLIN2), calcium signaling pathway, and pathways associated with metabolic processes (e.g., oxidative phosphorylation, fatty acid metabolism, glycolysis/gluconeogenesis, and citrate cycle). According to detected gene expression modifications, our results suggested that in Arabian horses, exercise switches energy generation toward fatty acid utilization and enhances glycogen transport and calcium signaling. The use of the RNA-Seq approach in analyzing the skeletal muscle transcriptome allowed for the proposal of a panel of new candidate genes potentially related to body homeostasis maintenance and racing performance in Arabian horses.

Transcriptome profiling of Arabian horse blood during training regimens.

BACKGROUND: Arabian horses are believed to be one of the oldest and most influential horse breeds in the world. Blood is the main tissue involved in maintaining body homeostasis, and it is considered a marker of the processes taking place in the other tissues. Thus, the aim of our study was to identify the genetic basis of changes occurring in the blood of Arabian horses subjected to a training regimen and to compare the global gene expression profiles between different training periods (T1: after a slow canter phase that is considered a conditioning phase, T2: after an intense gallop phase, and T3: at the end of the racing season) and between trained and untrained horses (T0). RNA sequencing was performed on 37 samples with a 75-bp single-end run on a HiScanSQ platform (Illumina), and differentially expressed genes (DEGs) were identified based on DESeq2 (v1.11.25) software. RESULTS: An increase in the number of DEGs between subsequent training periods was observed, and the highest amount of DEGs (440) was detected between untrained horses (T0) and horses at the end of the racing season (T3). The comparisons of the T2 vs. T3 transcriptomes and the T0 vs. T3 transcriptomes showed a significant gain of up-regulated genes during long-term exercise (up-regulation of 266 and 389 DEGs in the T3 period compared to T2 and T0, respectively). Forty differentially expressed genes were detected between the T1 and T2 periods, and 296 between T2 and T3. Functional annotation showed that the most abundant genes up-regulated in exercise were involved in pathways regulating cell cycle (PI3K-Akt signalling pathway), cell communication (cAMP-dependent pathway), proliferation, differentiation and apoptosis, as well as immunity processes (Jak-STAT signalling pathway). CONCLUSIONS: We investigated whether training causes permanent transcriptome changes in horse blood as a reflection of adaptation to conditioning and the maintenance of fitness to compete in flat races. The present study identified the overrepresented molecular pathways and genes that are essential for maintaining body homeostasis during long-term exercise in Arabian horses. Selected DEGs should be further investigated as markers that are potentially associated with racing performance in Arabian horses.

Molecular insights into the lipid-carbohydrates metabolism switch under the endurance effort in Arabian horses.

BACKGROUND: Recent studies have shown that in Arabian horse muscle, long-term exercise-induced expression of genes related to fatty acid degradation and the downregulation of genes belonging to the glycolysis/gluconeogenesis and insulin signalling pathways. Long-lasting physical exertion may trigger the metabolism to switch the main energy source from carbohydrates to lipids due to higher caloric content. OBJECTIVES: To describe the metabolism adaptation at the whole transcriptome of blood to endurance effort in Arabian horses. STUDY DESIGN: In vivo experiment. METHODS: Venous blood samples from 10 Arabian horses were taken before and after a 120 km long endurance ride to isolate the RNA and perform the high-throughput NGS transcriptome sequencing. RESULTS: The results, including KEGG (Kyoto Encyclopaedia of Genes and Genomes) and GO (Gene Ontology) analyses, allowed us to describe the most significantly upregulated-ARV1, DGAT2, LIPE, APOA2, MOGAT1, MOGAT2, GYS1, GYS2 and downregulated-ACACA, ACACB, FADS1, FADS2 genes involved in carbohydrate and lipid metabolism. Also, the increased expression of RAF1, KRAS and NRAS genes involved in the Insulin pathway and PI3K-Akt was shown. MAIN LIMITATIONS: Limited sample size, Arabians used for endurance racing were not compared to Arabians from other equestrian disciplines. CONCLUSIONS: This general insight into the processes described supports the thesis of the lipid-carbohydrates metabolism switch in endurance Arabian horses and provides the basis for further research.

Congenital heart defects in Arabian horses and the prospects of genetic testing: A review.

Congenital heart defects (CHDs) can have profound and potentially life-threatening consequences on horses' health and performance capability. While CHDs are rare in the general horse population, the Arabian breed is disproportionately overrepresented and thus is widely suspected to be genetically predisposed. This review discusses the most common CHDs in Arabian horses, including ventricular septal defect (VSD), tetralogy of Fallot (TOF), patent duct arteriosus (PDA), tricuspid valve atresia (TVA) and atrial septal defect (ASD). This review also explores how future research into the genetic factors that likely underpin many CHDs can revolutionise the way these disorders are managed in Arabian horses.

Mitochondrial DNA and Y chromosome reveal the genetic structure of the native Polish Konik horse population.

Polish Konik remains one of the most important horse breeds in Poland. The primitive, native horses with a stocky body and mouse-like coat color are protected by a conservation program, while their Polish population consists of about 3,480 individuals, representing 16 dam and six sire lines. To define the population's genetic structure, mitochondrial DNA and Y chromosome sequence variables were identified. The mtDNA whole hypervariable region analysis was carried out using the Sanger sequencing method on 233 Polish Koniks belonging to all dam lines, while the Y chromosome analysis was performed with the competitive allele-specific PCR genotyping method on 36 horses belonging to all sire lines. The analysis of the mtDNA hypervariable region detected 47 SNPs, which assigned all tested horses to 43 haplotypes. Most dam lines presented more than one haplotype; however, five dam lines were represented by only one haplotype. The haplotypes were classified into six (A, B, E, J, G, R) recognized mtDNA haplogroups, with most horses belonging to haplogroup A, common among Asian horse populations. Y chromosome analysis allocated Polish Koniks in the Crown group, condensing all modern horse breeds, and divided them into three haplotypes clustering with coldblood breeds (28 horses), warmblood breeds (two horses), and Duelmener Pony (six horses). The clustering of all Wicek sire line stallions with Duelmener horses may suggest a historical relationship between the breeds. Additionally, both mtDNA and Y chromosome sequence variability results indicate crossbreeding before the studbooks closure or irregularities in the pedigrees occurred before the DNA testing introduction.

Equine Metabolic Syndrome: A Complex Disease Influenced by Multifactorial Genetic Factors.

Equine metabolic syndrome (EMS) has become an important issue in modern veterinary medicine and is linked to the common, extremely painful, most-of-the-time performance-terminating hoof laminitis. The growing knowledge in the field of genetic background, inducing environmental factors, diagnosis, treatment and maintenance of affected equines led us to summarise the available information to be used not only for scientific purposes but for fieldwork. In horses, the clinical presentation of EMS includes: obesity or local fat deposition, bilateral lameness or hoof rings attributed to ongoing or previous (pasted) laminitis with the key feature of the occurrence of insulin dysregulation, disturbing the homeostasis within insulin, glucose and lipid metabolism. The management of EMS is based on dietary and fitness discipline; however, intensive research is ongoing in the field of regenerative medicine to develop modern and promising therapies.

Refining the evolutionary tree of the horse Y chromosome.

The Y chromosome carries information about the demography of paternal lineages, and thus, can prove invaluable for retracing both the evolutionary trajectory of wild animals and the breeding history of domesticates. In horses, the Y chromosome shows a limited, but highly informative, sequence diversity, supporting the increasing breeding influence of Oriental lineages during the last 1500 years. Here, we augment the primary horse Y-phylogeny, which is currently mainly based on modern horse breeds of economic interest, with haplotypes (HT) segregating in remote horse populations around the world. We analyze target enriched sequencing data of 5 Mb of the Y chromosome from 76 domestic males, together with 89 whole genome sequenced domestic males and five Przewalski's horses from previous studies. The resulting phylogeny comprises 153 HTs defined by 2966 variants and offers unprecedented resolution into the history of horse paternal lineages. It reveals the presence of a remarkable number of previously unknown haplogroups in Mongolian horses and insular populations. Phylogenetic placement of HTs retrieved from 163 archaeological specimens further indicates that most of the present-day Y-chromosomal variation evolved after the domestication process that started around 4200 years ago in the Western Eurasian steppes. Our comprehensive phylogeny significantly reduces ascertainment bias and constitutes a robust evolutionary framework for analyzing horse population dynamics and diversity.

Mitochondrial Whole D-Loop Variability in Polish Draft Horses of Sztumski Subtype.

The Polish draft horse (PDH) breed is a result of crossing local mares with imported cold-blooded stallions, such as Belgians, Ardennes, Fjords, and others. A part of the broodmare stock investigated in this study was also imported from various countries, such as Denmark. In this study, we investigate the genetic composition of the PDH by analyzing the whole mitochondrial d-loop variability and comparing it to previously demonstrated whole d-loop sequences of other cold-blooded breeds: Ardennais, Belgian, Breton, Clydesdale, Noriker, Norwegian Fjord, Percheron, and Suffolk. Our results show high nucleotide diversity within the PDH population (π = 0.011), and the existence of two main haplogroups: one of relatively concise origin, with strong kinship to the Belgian breed, and the second showing close relation to the majority of other analyzed cold-blooded breeds. Some of the PDH maternal strains clustered separately, which can be a result of the influence of other unidentified breeds that served as a foundation stock for the present population. This present study explains the genetic relationship of the PDH to other cold-blooded breeds and indicates the high genetic diversity of the breed.

Profiling of genetic markers useful for breeding decision in Selle Francais horse.

Genetic disorders are recognised as hereditary diseases with the most significant economic impact on horse breeding, causing important foal losses, costs of treatments of horses, and maintenance of the mare during the pregnancy. The Selle Francais horses are recognized in many countries and are showing great results in equestrian sports around the world (dressage, show jumping and eventing). The study aimed to detect the presence of three mutant alleles associated with inherited diseases including Fragile Foal Syndrome (FFS), Cerebellar Abiotrophy (CA), Polysaccharide Storage Myopathy (PSSM1) and variant impacting gait type in DMRT3. This trait is important for breeding decision in Selle Francais horses and sheds new light on genetic potential and risks on this breed. The genotyping was performed on 91 Selle Francais horses using PCR-RFLP (for POLD1; GYS1 and DMRT3 genes) and PCR-ACRS (TOE1 gene) methods. The presented report indicated the presence of mutant allele A casual for PSSM1 and allele T associated with FFS syndrome occurrence, in 4% and 6% of analysed horses, respectively. Regarding CA, the present survey did not register any cases of this genetic disorder in Selle Francais horses. Our results show also that about 1% of all the Sell Francais horses studied carry the A allele of DMRT3 gene. The present findings have provided data for these fulness of monitoring genetic diseases and gait type in the investigated breed to avoid losses of offspring.

Assessment and Distribution of Runs of Homozygosity in Horse Breeds Representing Different Utility Types.

The present study reports runs of homozygosity (ROH) distribution in the genomes of six horse breeds (571 horses in total) representing three horse types (primitive, light, and draft horses) based on the 65k Equine BeadChip assay. Of major interest was the length, quantity, and frequency of ROH characteristics, as well as differences between horse breeds and types. Noticeable differences in the number, length and distribution of ROH between breeds were observed, as well as in genomic inbreeding coefficients. We also identified regions of the genome characterized by high ROH coverage, known as ROH islands, which may be signals of recent selection events. Eight to fourteen ROH islands were identified per breed, which spanned multiple genes. Many were involved in important horse breed characteristics, including WFIKNN2, CACNA1G, STXBP4, NOG, FAM184B, QDPR, LCORL, and the zinc finger protein family. Regions of the genome with zero ROH occurrences were also of major interest in specific populations. Depending on the breed, we detected between 2 to 57 no-ROH regions and identified 27 genes in these regions that were common for five breeds. These genes were involved in, e.g., muscle contractility (CACNA1A) and muscle development (miR-23, miR-24, miR-27). To sum up, the obtained results can be furthered analyzed in the topic of identification of markers unique for specific horse breed characteristics.

Y-Chromosomal Insights into Breeding History and Sire Line Genealogies of Arabian Horses.

The Y chromosome is a valuable genetic marker for studying the origin and influence of paternal lineages in populations. In this study, we conducted Y-chromosomal lineage-tracing in Arabian horses. First, we resolved a Y haplotype phylogeny based on the next generation sequencing data of 157 males from several breeds. Y-chromosomal haplotypes specific for Arabian horses were inferred by genotyping a collection of 145 males representing most Arabian sire lines that are active around the globe. These lines formed three discrete haplogroups, and the same haplogroups were detected in Arabian populations native to the Middle East. The Arabian haplotypes were clearly distinct from the ones detected in Akhal Tekes, Turkoman horses, and the progeny of two Thoroughbred foundation sires. However, a haplotype introduced into the English Thoroughbred by the stallion Byerley Turk (1680), was shared among Arabians, Turkomans, and Akhal Tekes, which opens a discussion about the historic connections between Oriental horse types. Furthermore, we genetically traced Arabian sire line breeding in the Western World over the past 200 years. This confirmed a strong selection for relatively few male lineages and uncovered incongruences to written pedigree records. Overall, we demonstrate how fine-scaled Y-analysis contributes to a better understanding of the historical development of horse breeds.

Investigation of Cerebellar Abiotrophy (CA), Lavender Foal Syndrome (LFS), and Severe Combined Immunodeficiency (SCID) Variants in a Cohort of Three MENA Region Horse Breeds.

Genetic disorders in horses are mostly fatal or usually cause significant economic losses for breeders and owners. Here we studied a total of 177 Arabian, Barb and Arab-Barb horses from the Middle East and North Africa (MENA) using Sanger Sequencing and PCR-ACRS (polymerase chain reaction-artificially created restriction site) approaches to examine the genetic disorders in the studied horse breeds. We identified the genetic variations related to Cerebellar Abiotrophy (CA), Severe Combined Immunodeficiency (SCID) occurrence, and the studied population was free of the mutant allele determined Lavender Foal Syndrome (LFS). Overall, presented data showed that 15 of the studied horses are carriers of two genetic disorders; the investigated horse population showed that five Arabian horses were heterozygous for the CA-associated SNP (rs397160943). The SCID-deletion TCTCA within PRKDC was detected in ten horses (nine Arabian horses and one Arab-Barb horse). This investigation shows the importance of testing these breeds for genetic disorders to avoid further spread of deleterious variants.

Variability of ACOX1 Gene Polymorphisms across Different Horse Breeds with Regard to Selection Pressure.

The ACOX1 gene encodes peroxisomal acyl-coenzyme A oxidase 1, the first enzyme in the fatty acid ß-oxidation pathway, which could be significant for organisms exposed to long periods of starvation and harsh living conditions. We hypothesized that variations within ACOX1, revealed by RNA Sequencing (RNA-Seq), might be based on adaptation to living conditions and had resulted from selection pressure. There were five different horse breeds used in this study, representing various utility types: Arabian, Thoroughbred, Polish Konik, draft horses, and Hucul. The single-nucleotide polymorphism (SNP) located in the ACOX1 (rs782885985) was used as a marker and was identified using the PCR restriction fragment length polymorphism method (PCR-RFLP). Results indicated extremely different genotype and allele distributions of the ACOX1 gene across breeds. A predominance of the G allele was exhibited in horses that had adapted to difficult environmental conditions, namely, Polish Konik and Huculs, which are considered to be primitive breeds. The prevalence of the T allele in Thoroughbreds indicated that ACOX1 is significant in energy metabolism during flat racing.

Variation in the SLC16A1 and the ACOX1 Genes Is Associated with Gallop Racing Performance in Arabian Horses.

Arabian horses are not only one of the most ancient breeds in the world, but they are also one of the most appreciated racehorse breeds today. The breed generates attention for their phenomenal endurance ability and their capability for gallop racing. Consequently, genetic testing to select the best individuals is attracting ever increasing interests from the Arabian industry. As such, the aim of this study was to further investigate associations between performance and variation at candidate genes suspected of having a key role in Arabian gallop racing performance. Generalized linear models were fit to test associations between eight candidate gene variants and a variety of gallop racing performance traits in a sample of Arabian racehorses (n = 287). Two genes, solute carrier family 16 member 1 (SLC16A1) and acyl-CoA oxidase 1 (ACOX1), were significantly associated with multiple gallop racing performance traits, whereas another gene, actinin alpha 3 (ACTN3) was associated with best race distance. Previously established associations between these three genes and equine metabolism strongly suggest further investigation of these genes, and their relationship with Arabian horse performance is warranted.