Studies have shown that the physiological responses to exercise on a treadmill and experimental conditions cannot replicate the responses in the field [7]. The current study presents favourable points that include the use of high-performance athletic horses, considered a risk group for EIPH, evaluated under the real conditions of official races and, consequently, under the maximum level of effort requirements. In addition, endoscopy exams and genetic tests were performed.
Conducting field studies provide the opportunity to evaluate real stress conditions among healthy athletic animals with high-performance capacities. However, due to the high zootechnical value of horses, using more sensitive techniques that can be performed in vivo, such as bronchoalveolar lavage, is a challenge. Because these techniques are more invasive, they demand substantial collaboration between trainers, owners, jockeys and the scientific community; thus, it is not feasible for the individuals involved to make their animals available for analysis [12, 13]. For this reason, trachea-bronchial endoscopy was used in this study, which is a widely used technique for diagnosing EIPH [14–16].
The genotype frequencies of the g.290A > G and g.338G > A mutations were equal across all horses. That is, all heterozygous, homozygous or wild-type horses for the g.290A > G mutation also had the same genotype for the g.338G > A mutation. This finding reinforces the possible existence of a link between these two mutations [8].
Consistent with a previous study [8], the results revealed a high prevalence of g.290A > G and g.338G > A; 96.1% of horses in the EIPH group had at least one allelic alteration. However, 94.7% of the horses in the control group analysed in the present study also presented a heterozygous or homozygous genotype for these same mutations, while the result found in the control group in the previous study was 53%. A possible explanation for this finding is the use of different diagnostic methods; Boudreaux et al. [8] used histopathological samples, while the present study used trachea-bronchial endoscopy.
In the present study, none of the animals in the control group and 3.9% of horses in the EIPH group had the g.291A > T mutation. On the other hand, the horse population assessed in a previous study [8] presented a prevalence of at least one mutated allele in 4.5% of horses with other types of bleeding and 38% of EIPH horses. Thus, the authors of the study cited above suggested that there may be a negative selection for this alteration. Furthermore, the g.464G > A polymorphism present in the ENTPD2 gene was less frequent (84.4% of genotypes were wild-type) in the control group and the EIPH group (75% wild-type). These data corroborate the findings of a previous study [8] in which a high prevalence of wild-type genotypes was observed in affected (62%) and healthy (71.2%) animals; the occurrence of negative selection for this polymorphism was also suggested by these authors.
Notably, the genetic base of the Thoroughbred horse is narrow, with 78% of the alleles in the current population deriving from 30 sires, of which 27 are males, with a single stallion representing 95% of sire lines and 10 mares representing 72% of dam lines [17].
The breeds in which the four mutations studied by Boudreaux et al. [8] were found at a higher frequency were Thoroughbreds and Quarter Horses, which are genetically related. In contrast, despite the small number of evaluated animals of other breeds, g.290A > G and g.338G > A mutations were not found in Rocky Mountain and Belgian horses, the g.291A > T mutation was not present in Oldenburg, Paint, Saddlebred, Tennessee Walking Horse, Warmblood and Belgian horses, and the g.464G > A mutation was not found in Oldenburg, Paint, Rocky Mountain, Saddlebred, Standardbred and Tennessee Walking Horse breeds [8].
Thus, a breed factor may influence the prevalence of these mutations, especially in breeds with greater genetic proximity to Thoroughbreds. This fact could explain the high rate of bleeding in Thoroughbred horses and the high prevalence of mutations found in this study. However, it was not possible to affirm this hypothesis since we only assessed Thoroughbreds, the breed most affected by EIPH.
Moreover, although the low reactivity to ADP shown by horses affected by EIPH has been reported for at least 39 years [2, 10, 11], one of the main challenges in this study was the limitation of data available in the literature about the role of nucleotides in the haemostatic changes that occur in this disease. Therefore, the emphasis of this study was on the possible association between phenotype and genotype that presents mutations in the ENTPD1 and ENTPD2 genes since these genes encode important enzymes in haemostasis regulation in the context of ADP metabolism.