DNA-PKcs mutations in dogs and horses: allele frequency and association with neoplasia

Previously, spontaneous genetic immunodeficiencies in mice, Arabian foals, and recently in Jack Russell terriers have been ascribed to defects in DNA-PKcs (catalytic subunit of the DNA dependent protein kinase) expression. In severe combined immunodeficiency (SCID) foals, a 5 bp deletion at codon 9480 results in a frameshift and a 967 amino acid deletion from the C terminus (including the entire PI3 kinase domain) and an unstable mutant protein. In SCID mice, a single base pair mutation results in a premature stop codon and deletion of 83 amino acids; as in SCID foals, the mutant protein is unstable. Here, we define the mutation within the canine DNA-PKcs gene that results in SCID. In this case, a point mutation results in a stop codon at nucleotide 10,828 and premature termination at a position 517 amino acids before the normal C terminus resulting in a functionally null allele. Thus, this is the third documentation of a spontaneous germline mutation in the C terminus of DNA-PKcs. Emerging data implicate DNA repair factors as potential tumor suppressors. Here, we have ascertained the carrier frequency of the defective DNA-PKcs genes in Arabian horses and in Jack Russell terriers. Our data indicate (in good agreement with a previous report) that the carrier frequency of the equine SCID allele is approximately 8%; in contrast, the carrier frequency of the canine SCID allele is less than 1.1%. We also assessed the frequency of the equine SCID allele in a series of 295 tumors from Arabian horses. We find a statistically significant correlation between the development of a virally induced tumor (sarcoid) and heterozygosity for the equine SCID allele. These data provide further support for an emerging consensus: that DNA-PK may normally act as a tumor suppressor through its caretaker role in maintaining chromosomal stability.     

SCID in Jack Russell terriers: a new animal model of DNA-PKcs deficiency

We recently described the incidence of a SCID disease in a litter of Jack Russell terriers. In this study, we show that the molecular defect in these animals is faulty V(D)J recombination. Furthermore, we document a complete deficit in DNA-dependent protein kinase activity that can be explained by a marked diminution in the expression of the catalytic subunit DNA-dependent protein kinase catalytic subunit (DNA-PKcs). We conclude that as is the case in C.B-17 SCID mice and in Arabian SCID foals, the defective factor in these SCID puppies is DNA-PKcs. In mice, it has been clearly established that DNA-PKcs deficiency produces an incomplete block in V(D)J recombination, resulting in "leaky" coding joint formation and only a modest defect in signal end ligation. In contrast, DNA-PKcs deficiency in horses profoundly blocks both coding and signal end joining. Here, we show that although DNA-PKcs deficiency in canine lymphocytes results in a block in both coding and signal end joining, the deficit in both is intermediate between that seen in SCID mice and SCID foals. These data demonstrate significant species variation in the absolute necessity for DNA-PKcs during V(D)J recombination. Furthermore, the severity of the V(D)J recombination deficits in these three examples of genetic DNA-PKcs deficiency inversely correlates with the relative DNA-PK enzymatic activity expressed in normal fibroblasts derived from these three species.     

Whole-Genome SNP Association in the Horse: Identification of a Deletion in Myosin Va Responsible for Lavender Foal Syndrome

Lavender Foal Syndrome (LFS) is a lethal inherited disease of horses with a suspected autosomal recessive mode of inheritance. LFS has been primarily diagnosed in a subgroup of the Arabian breed, the Egyptian Arabian horse. The condition is characterized by multiple neurological abnormalities and a dilute coat color. Candidate genes based on comparative phenotypes in mice and humans include the ras-associated protein RAB27a (RAB27A) and myosin Va (MYO5A). Here we report mapping of the locus responsible for LFS using a small set of 36 horses segregating for LFS. These horses were genotyped using a newly available single nucleotide polymorphism (SNP) chip containing 56,402 discriminatory elements. The whole genome scan identified an associated region containing these two functional candidate genes. Exon sequencing of the MYO5A gene from an affected foal revealed a single base deletion in exon 30 that changes the reading frame and introduces a premature stop codon. A PCR–based Restriction Fragment Length Polymorphism (PCR–RFLP) assay was designed and used to investigate the frequency of the mutant gene. All affected horses tested were homozygous for this mutation. Heterozygous carriers were detected in high frequency in families segregating for this trait, and the frequency of carriers in unrelated Egyptian Arabians was 10.3%. The mapping and discovery of the LFS mutation represents the first successful use of whole-genome SNP scanning in the horse for any trait. The RFLP assay can be used to assist breeders in avoiding carrier-to-carrier matings and thus in preventing the birth of affected foals.     

T lymphocyte development and maturation in horses

Monoclonal antibodies specific for equine T lymphocyte subpopulations were produced and procedures for the continuous culture of equine lymphocytes were developed. These reagents and procedures were used to analyse the appearance, maturation and functions of T lymphocytes in normal horses and in T lymphocyte deficient horses with severe combined immunodeficiency (SCID). T lymphocytes appeared as early as the 75th day of fetal development and were normally distributed prior to birth of normal foals. Analysis of thymic T lymphocyte differentiation in SCID foals revealed the presence of both prothymocytes and mature thymocytes, but a virtual absence of cortical thymocytes. The data obtained support the hypothesis that two distinct pathways of T lymphocyte differentiation exist within the thymus. Although the gene defect in foals with SCID blocks the production of mature B and T lymphocytes, such foals do possess large granular lymphocytes which are cytotoxic following induction with interleukin 2. This suggests that lymphoid cells with natural killer cell activity are spared by the gene defect resulting in SCID in horses.     

A primary production deficit in the thrombocytopenia of equine infectious anemia.

The purpose of this study was to identify the mechanisms responsible for the thrombocytopenia that develops following infection of horses by the lentivirus equine infectious anemia virus (EIAV). Immunocompetent Arabian foals and Arabian foals with severe combined immunodeficiency (SCID), which lack functional B and T lymphocytes, were experimentally infected with EIAV. Levels of viremia and a number of clinical and hematologic parameters were examined prior to and following infection. Thrombocytopenia was not dependent on the immune response: SCID foals were affected as severely as immunocompetent foals. Production of platelets, measured by metabolic incorporation of radioactive label, was significantly reduced. The decrease ranged from 35 to 89% in three SCID and two immunocompetent foals examined. Platelet survival, measured by 51Cr labeling, also declined following infection in both SCID and immunocompetent foals: 51 and 68%, respectively, relative to the preinfection life spans. The difference between immunocompetent and immunodeficient foals was not statistically significant. The number of megakaryocytes (MK) per square millimeter of bone marrow, determined by digitizing morphometry, was not significantly altered in either SCID or immunocompetent thrombocytopenic foals. Numbers of denuded MK nuclei per unit area increased, but the elevation was not statistically significant. No evidence for viral replication in MK was found. Three different parameters of intravascular coagulation (activated prothombin time, fibrin degradation products, and one-step prothombin time) remained normal until after platelet numbers had declined significantly, arguing against an important role for disseminated intravascular coagulation. The findings indicate that EIAV induces thrombocytopenia principally through an indirect, noncytocidal suppressive effect on platelet production, the mechanism of which is unknown. A shortening of platelet life span apparently contributes moderately to the platelet deficit as well. The shortening of platelet life span is multifactorial in origin, including both mechanisms that depend on an active immune response and those that do not.     

Novel insights into Sabino1 and splashed white coat color patterns in horses

Within the framework of genome‐wide analyses using the novel Axiom^® genotyping array, we investigated the distribution of two previously described coat color patterns, namely sabino1 (SBI), associated with the KIT gene (KI16+1037A), and splashed white, associated with the PAX3 gene (ECA6:g.11429753C>T; PAX3^C70Y), including a total of 899 horses originating from eight different breeds (Achal Theke, Purebred Arabian, Partbred Arabian, Anglo‐Arabian, Shagya Arabian, Haflinger, Lipizzan and Noriker). Based on the data we collected we were able to demonstrate that, besides Quarter horses, the PAX3^C70Y allele is also present in Noriker (seven out of 189) and Lipizzan (three out of 329) horses. The SB1 allele was present in three breeds (Haflinger, 14 out of 98; Noriker, four out of 189; Lipizzan one out of 329). Furthermore, we examined the phenotypes of SB1‐ and PAX3^C70Y‐carrier horses for their characteristic white spotting patterns. None of the SB1/sb1‐carrier horses met the criteria defining the Sabino1 pattern according to current applied protocols. From 10 heterozygous PAX3^C70Y‐carrier horses, two had nearly a splashed white phenotype. The results of this large‐scale experiment on the genetic association of white spotting patterns in horses underline the influence of gene interactions and population differences on complex traits such as Sabino1 and splashed white.     

A program to reduce the incidence of combined immuno-deficiency

Combined immuno-deficiency (CID) is a common genetic defect of Arabian horses, the incidence of which is being increased both by a tendency to select for the condition and by measures currently taken to identify non-carrier individuals. This paper derives probability statements for the incidence of the condition and suggests a program of record keeping and simple calculations, based on tables, by which stud owners can reduce CID pending development of a test to identify carriers of the gene.     

Linkage of the gene for equine combined immunodeficiency disease to microsatellite markers HTG8 and HTG4; synteny and FISH mapping to ECA9

Equine combined immunodeficiency disease (CID) is caused by homozygosity for an autosomal recessive gene. To identify linked markers for the disease, we studied a family segregating for the equine CID gene. A stallion and 19 of his CID-affected offspring were tested for marker segregation at 23 microsatellite DNA loci. His CID-affected offspring inherited only one of his two alleles at the HTG8 and HTG4 loci, namely HTG8–186 and HTG4–124 , respectively. Lod scores for linkage to the CID gene using a Θ of 0·01were 5·34 for HTG8 and 2·37 for HTG4. The apparent genotypes also suggested linkage disequilibrium between the HTG8–186 allele and the gene for CID. The gene for the DNA protein kinase catalytic subunit ( DNA-PK ) was recently suggested as a candidate gene for equine CID. A defect of this gene causes a disease in mice that is similar to equine CID. Therefore, we investigated whether this gene might be associated with the microsatellite markers. Analysis of a somatic cell hybrid panel demonstrated synteny of DNA-PK with HTG4 and HTG8 (Kentucky Synteny Group 3). Fluorescence in situ hybridization (FISH) studies demonstrated that DNA-PK is located on horse chromosome ECA9p12. This work supports the hypothesis of DNA-PK as the probable cause of equine CID.     

Analyses of TCRB rearrangements substantiate a profound deficit in recombination signal sequence joining in SCID foals: implications for the role of DNA-dependent protein kinase in V(D)J recombination

We reported previously that the genetic SCID disease observed in Arabian foals is explained by a defect in V(D)J recombination that profoundly affects both coding and signal end joining. As in C.B-17 SCID mice, the molecular defect in SCID foals is in the catalytic subunit of the DNA-dependent protein kinase (DNA-PKCS); however, in SCID mice, signal end resolution remains relatively intact. Moreover, recent reports indicate that mice that completely lack DNA-PKCS also generate signal joints at levels that are indistinguishable from those observed in C.B-17 SCID mice, eliminating the possibility that a partially active version of DNA-PKCS facilitates signal end resolution in SCID mice. We have analyzed TCRB rearrangements and find that signal joints are reduced by approximately 4 logs in equine SCID thymocytes as compared with normal horse thymocytes. A potential explanation for the differences between SCID mice and foals is that the mutant DNA-PKCS allele in SCID foals inhibits signal end resolution. We tested this hypothesis using DNA-PKCS expression vectors; in sum, we find no evidence of a dominant-negative effect by the mutant protein. These and other recent data are consistent with an emerging consensus: that in normal cells, DNA-PKCS participates in both coding and signal end resolution, but in the absence of DNA-PKCS an undefined end joining pathway (which is variably expressed in different species and cell types) can facilitate imperfect signal and coding end joining.     

A new allele in the prealbumin system of horse serum markers

A family study of an index case in the Arabian breed of horses demonstrated the presence of a new allele in the prealbumin (Pr) system of electrophoretically determined markers in horse serum which, when homozygous, results in the absence of any recognizable zones in the Pr region. The symbol Pr is proposed for this allele which has an estimated frequency in Arabian horses of 0.09.     

Metabolic investigations of fibroblasts from horses, Equus caballus, with hereditary severe combined immunodeficiency

In an attempt to determine the metabolic defect causing severe combined immunodeficiency (SCID) in horses in which altered purine metabolism has been observed, various parameters of purine and pyrimidine metabolism were evaluated. The activities of nine purine enzymes (adenosine kinase, purine nucleoside phosphorylase, deoxyadenosine kinase, deoxycytidine kinase, 5'-nucleotidase, AMP deaminase, hypoxanthine-guanine phosphoribosyl transferase, and adenine phosphoribosyl transferase were measured in fibroblasts. All activities determined for SCID horses were normal. Uptake of 10 microM adenosine or 2'-deoxyadenosine (a growth inhibitory concentration for SCID fibroblasts) by SCID fibroblasts was identical to that found for normal fibroblasts in the presence of both 1 and 50 microM phosphate. The Km determined for the transport of both adenosine and 2'-deoxyadenosine was 35 microM. In the presence of p-nitrobenzylthioguanosine (a nucleoside transport inhibitor), 2'-deoxyadenosine uptake was inhibited to the same extent in all fibroblast lines tested. To determine if the last step in pyrimidine biosynthesis might be altered in SCID fibroblasts, UMP synthase activities were evaluated but found to be normal (0.5 nmol UMP formed/min/mg protein).     

Genetic diversity of Syrian Arabian horses

Although Arabian horses have been bred in strains for centuries and pedigrees have been recorded in studbooks, to date, little is known about the genetic diversity within and between these strains. In this study, we tested if the three main strains of Syrian Arabian horses descend from three founders as suggested by the studbook. We examined 48 horses representing Saglawi (n = 18), Kahlawi (n = 16) and Hamdani (n = 14) strains using the Equine SNP70K BeadChip. For comparison, an additional 24 Arabian horses from the USA and three Przewalski's horses as an out group were added. Observed heterozygosis (H_o) ranged between 0.30 and 0.32, expected heterozygosity (H_e) between 0.30 and 0.31 and inbreeding coefficients (F_is) between ?0.02 and ?0.05, indicating high genetic diversity within Syrian strains. Likewise, the genetic differentiation between the three Syrian strains was very low (F_st < 0.05). Hierarchical clustering showed a clear distinction between Arabian and Przewalski's horses. Among Arabian horses, we found three clusters containing either horses from the USA or horses from Syria or horses from Syria and the USA together. Individuals from the same Syrian Arabian horse strain were spread across different sub‐clusters. When analyzing Syrian Arabian horses alone, the best population differentiation was found with three distinct clusters. In contrast to expectations from the studbook, these clusters did not coincide with strain affiliation. Although this finding supports the hypothesis of three founders, the genetic information is not consistent with the currently used strain designation system. The information can be used to reconsider the current breeding practice. Beyond that, Syrian Arabian horses are an important reservoir for genetic diversity.     

Presentation and binding affinity of equine infectious anemia virus CTL envelope and matrix protein epitopes by an expressed equine classical MHC class I molecule

Control of a naturally occurring lentivirus, equine infectious anemia virus (EIAV), occurs in most infected horses and involves MHC class I-restricted, virus-specific CTL. Two minimal 12-aa epitopes, Env-RW12 and Gag-GW12, were evaluated for presentation by target cells from horses with an equine lymphocyte Ag-A1 (ELA-A1) haplotype. Fifteen of 15 presented Env-RW12 to CTL, whereas 11 of 15 presented Gag-GW12. To determine whether these epitopes were presented by different molecules, MHC class I genes were identified in cDNA clones from Arabian horse A2152, which presented both epitopes. This horse was selected because it is heterozygous for the SCID trait and is used to breed heterozygous females. Offspring with SCID are used as recipients for CTL adoptive transfer, and normal offspring are used for CTL induction. Four classical and three putative nonclassical full-length MHC class I genes were found. Human 721.221 cells transduced with retroviral vectors expressing each gene had equine MHC class I on their surface. Following peptide pulsing, only cells expressing classical MHC class I molecule 7-6 presented Env-RW12 and Gag-GW12 to CTL. Unlabeled peptide inhibition of (125)I-labeled Env-RW12 binding to 7-6-transduced cells demonstrated that Env-RW12 affinity was 15-fold higher than Gag-GW12 affinity. Inhibition with truncated Env-RW12 demonstrated that amino acid positions 1 and 12 were necessary for binding, and single substitutions identified positions 2 and 3 as possible primary anchor residues. Since MHC class I 7-6 presented both epitopes, outbred horses with this allele can be immunized with these epitopes to optimize CTL responses and evaluate their effectiveness against lentiviral challenge.     

Depletion of natural killer cells does not result in neurologic disease due to Sarcocystis neurona in mice with severe combined immunodeficiency

Sarcocystis neurona is an apicomplexan parasite that is the primary etiologic agent of equine protozoal myeloencephalitis in horses. Protective immune responses in horses have not been determined, but interferon-gamma (IFN-gamma) is considered critical for protection from neurologic disease in mice. The role of adaptive and innate immune responses in control of parasites was explored by infecting BALB/c, IFN-gamma knockout (GKO), and severe combined immune deficient (SCID) mice with S. neurona (10(4) sporocysts/mouse). Immune competent BALB/c mice eliminated parasites within 30 days, with no sign of neurologic disease, whereas GKO mice developed fulminant neurologic disease. In contrast, SCID mice remained healthy throughout the experimental period despite the persistence of parasite at low levels in some mice. Treatment with anti-IFN-gamma antibody resulted in neurologic disease in infected SCID mice. Although SCID mice lack adaptive immune responses, they have natural killer (NK) cells capable of producing significant quantities of IFN-gamma. Therefore, SCID mice were infected with sporocysts of S. neurona and treated with anti-asialo GM1. Depletion of NK cells, confirmed by flow cytometry, did not result in neurologic disease in SCID mice. These results indicate that IFN-gamma mediates protection from neurologic disease in SCID mice. Protective levels of IFN-gamma may originate from a low number of nondepleted NK cells or from a non-T cell, non-NK cell population.     

Genome-wide linkage and association analysis identifies major gene Loci for guttural pouch tympany in arabian and german warmblood horses

Equine guttural pouch tympany (GPT) is a hereditary condition affecting foals in their first months of life. Complex segregation analyses in Arabian and German warmblood horses showed the involvement of a major gene as very likely. Genome-wide linkage and association analyses including a high density marker set of single nucleotide polymorphisms (SNPs) were performed to map the genomic region harbouring the potential major gene for GPT. A total of 85 Arabian and 373 German warmblood horses were genotyped on the Illumina equine SNP50 beadchip. Non-parametric multipoint linkage analyses showed genome-wide significance on horse chromosomes (ECA) 3 for German warmblood at 16-26 Mb and 34-55 Mb and for Arabian on ECA15 at 64-65 Mb. Genome-wide association analyses confirmed the linked regions for both breeds. In Arabian, genome-wide association was detected at 64 Mb within the region with the highest linkage peak on ECA15. For German warmblood, signals for genome-wide association were close to the peak region of linkage at 52 Mb on ECA3. The odds ratio for the SNP with the highest genome-wide association was 0.12 for the Arabian. In conclusion, the refinement of the regions with the Illumina equine SNP50 beadchip is an important step to unravel the responsible mutations for GPT.     

Deletion of 2.7 kb near HOXD3 in an Arabian horse with occipitoatlantoaxial malformation

In the horse, the term occipitoatlantoaxial malformation (OAAM) is used to describe a developmental defect in which the first cervical vertebra (atlas) resembles the base of the skull (occiput) and the second cervical vertebra (axis) resembles the atlas. Affected individuals demonstrate an abnormal posture and varying degrees of ataxia. The homeobox (HOX) gene cluster is involved in the development of both the axial and appendicular skeleton. Hoxd3‐null mice demonstrate a strikingly similar phenotype to Arabian foals with OAAM. Whole‐genome sequencing was performed in an OAAM‐affected horse (OAAM1) and seven unaffected Arabian horses. Visual inspection of the raw reads within the region of HOXD3 identified a 2.7‐kb deletion located 4.4 kb downstream of the end of HOXD4 and 8.2 kb upstream of the start of HOXD3. A genotyping assay revealed that both parents of OAAM1 were heterozygous for the deletion. Additional genotyping identified two of 162 heterozygote Arabians, and the deletion was not present in 371 horses of other breeds. Comparative genomics studies have revealed that this region is highly conserved across species and that the entire genomic region between Hoxd4 and Hoxd3 is transcribed in mice. Two additional Arabian foals diagnosed with OAAM (OAAM 2 and 3) were genotyped and did not have the 2.7‐kb deletion. Closer examination of the phenotype in these cases revealed notable variation. OAAM3 also had facial malformations and a patent ductus arteriosus, and the actual malformation at the craniocervical junction differed. Genetic heterogeneity may exist across the HOXD locus in Arabian foals with OAAM.     

A pedigree-based study of mitochondrial D-loop DNA sequence variation among Arabian horses

Through DNA sequence comparisons of a mitochondrial D-loop hypervariable region, we investigated matrilineal diversity for Arabian horses in the United States. Sixty-two horses were tested. From published pedigrees they traced in the maternal line to 34 mares acquired primarily in the mid to late 19th century from nomadic Bedouin tribes. Compared with the reference sequence (GenBank X79547), these samples showed 27 haplotypes with altogether 31 base substitution sites within 397 bp of sequence. Based on examination of pedigrees from a random sampling of 200 horses in current studbooks of the Arabian Horse Registry of America, we estimated that this study defined the expected mtDNA haplotypes for at least 89% of Arabian horses registered in the US. The reliability of the studbook recorded maternal lineages of Arabian pedigrees was demonstrated by haplotype concordance among multiple samplings in 14 lines. Single base differences observed within two maternal lines were interpreted as representing alternative fixations of past heteroplasmy. The study also demonstrated the utility of mtDNA sequence studies to resolve historical maternity questions without access to biological material from the horses whose relationship was in question, provided that representatives of the relevant female lines were available for comparison. The data call into question the traditional assumption that Arabian horses of the same strain necessarily share a common maternal ancestry.     

Evidence for a single pedigree source of the hyperkalemic periodic paralysis susceptibility gene in Quarter Horses

The pedigree origin of a base pair substitution in the horse muscle sodium channel gene that confers susceptibility to the muscle disease hyperkalemic periodic paralysis (HYPP) was investigated with a set of 978 Quarter Horses. The horses were chosen at random, based on a collection of blood samples taken between 1989 and 1991 to meet parentage testing requirements, primarily but not exclusively from breeding stallions. The frequency of Quarter Horses positive for the base pair substitution, all heterozygotes, was 4-4%, which corresponds to an allelic frequency of 0.02. All horses positive for the gene traced to a single previously identified stallion as first, second or third generation descendants. A higher frequency of the HYPP susceptibility trait than expected by random occurrence was found among his descendants in this study.     

Multifactorial inheritance of common white markings in the Arabian horse

The results of a previous study were compatible with the hypothesis that common white facial markings in the Arabian horse have a multifactorial mode of inheritance. I expanded that study to (1) include the legs and therefore obtain insight into the heritability of common white markings in all peripheral regions (face and legs) of the Arabian horse and (2) investigate the influence of sex and the genotypes that produce the bay and chestnut phenotypes on the variation in common white markings. Both studies were based on computerized data obtained from the Arabian Horse Registry of America, Inc. Each leg of a horse was scored from 0 to 5 depending on the amount of whiteness present, and the four leg scores were added to obtain the total leg score for each horse. The facial region was divided into five areas, and each horse was given a score from 0 to 5 according to the number of areas with whiteness. Sire families were analyzed in which each sire family consisted of a sire, his foals, and the dams of those foals. There was a correlation between white facial scores and white leg scores, suggesting that both types of white markings are influenced by the same genetic mechanism. Sire-foal and dam-foal regression analyses were compatible with the hypothesis that common white leg markings also show multifactorial inheritance. Although the results support the model that additively acting genes (polygenes) influence the presence and extent of common white markings, the results also show that males are slightly more marked than are females and that chestnut horses are more heavily marked than are bay horses.(ABSTRACT TRUNCATED AT 250 WORDS)     

Implementation of field cardio-respiratory measurements to assess energy expenditure in Arabian endurance horses

Measurements of respiratory exchanges in genuine exercise conditions are undoubtedly of interest to further define the energy needs of endurance horses. However, the equine K4b², the gas exchanges portable device validated for equines, has not been used in Arabian endurance horses yet. Therefore, the objective of this study was to implement field cardio-respiratory measurements in such horses using the equine K4b² in order to assess energy expenditure (EE). Measurements of heart rate (HR), oxygen uptake (VO₂), carbon dioxide production (VCO₂), respiratory frequency (RF), tidal volume (VT) and minute expired volume (VE) were carried out at rest and during a 20-min submaximal incremental field exercise in five trained Arabian endurance horses equipped with the K4b² system. The relationship between HR and VO₂ was determined for each horse. EE of the exercise session was calculated from direct VO₂ measurements and individual HR-VO₂ regression. Out of the five horses, four tolerated the equipment. Respiratory and metabolic variables at rest and during exercise, as well as EE measured at the different gaits, were consistent with reported values in exercising horses: VO₂ ranged from 4.8 to 54.1 ml/min per kg from rest to canter, respectively, and EE from 82 to 1095 J/min per kg BW. The 20-min exercise session EE accounted for 6258 and 6332 J from direct VO₂ measurements and individual HR-VO₂ regression, respectively, which did not differ significantly. Providing an adaptation period and several technical adjustments, the present equine K4b² could be used to assess EE in Arabian endurance horses in a controlled environment. The prediction of EE from the individual VO₂–HR relationship might be an alternative method to evaluate EE when VO₂ measurements are not possible.