Speed and Blood Parameters Differ between Arabian and emaitukai Horses during Endurance Racing

ation Equestre Internationale (FEI) has described equine endurance racing as the second largest discipline in the world, above which is only show jumping. The emaitukai is an ancient indigenous Lithuanian horse breed known since the 6th or 7th century. The Arabian horse breed is one of the oldest human-developed horse breeds

changes in horse blood gasometrical and biochemical indicators. Abstract: F²d²ration Equestre Internationale (FEI) has described equine endurance racing as the second largest discipline in the world, above which is only show jumping. The emaitukai is an ancient indigenous Lithuanian horse breed known since the 6th or 7th century. The Arabian horse breed is one of the oldest human-developed horse breeds in the world. Compared with other race horse breeds, the muscle tissue of Arabian horses is characterized by signi cant differences in structure—a predominance of oxidative ber type I is observed in Arabians, making them the prevailing breed in endurance racing. The Arabian horses are recognized as the leading breed in endurance competitions. Speed, pace, and total time in the race strategy have been extensively studied in human sports, and in contrast, this strategy appears to have been virtually ignored in equestrian sport, despite the potential for contributing to performance optimization. In relation to speed and total time in the race, there are limited data on postrace physical, biochemical, and blood gas parameters of endurance horses. Thus, this study was carried out to investigate the effects of speed on the blood parameters of the Arabian and emaitukai horses during an endurance race. Blood samples were taken before and immediately after the exercise. Biochemical and blood gas indicators were analyzed. The study showed signi cant increases in mean blood gasometrical indicators, such as partial carbon dioxide pressure (8.09–15.18%,p< 0.001); base excess in the extracellular uid (14.01%,p< 0.001 in the Arabian horses and 172.01% in the emaitukai breed,p= 0.006); decreases of the blood electrolyte ionized calcium (4.38–8.72%,p< 0.001) and the hematocrit and hemoglobin values (20.05–20.12%,p< 0.001in the Arabian horses and 6.22–6.23% in the emaitukai breed,p= 0.003–0.004); and decreases in the base excess in the blood values (29.24–39.38%,p< 0.001) and lactate (13.45–31.97%,p< 0.001) in the blood of both breeds in the post-competition horses. Signi cant increases after competition were determined for the values of creatinine (21.34–30.82%, p= 0.001–0.004), total bilirubin (50.84–56.24%, PH < 0.001), and albumin (2.63–4.48%,p= 0.048–0.001) for both breeds. For the faster Arabian horse breed, recovering after racing took half the

in the blood values (29.24–39.38%,p< 0.001) and lactate (13.45–31.97%,p< 0.001) in the blood of both breeds in the post-competition horses. Signi cant increases after competition were determined for the values of creatinine (21.34–30.82%, p= 0.001–0.004), total bilirubin (50.84–56.24%, PH < 0.001), and albumin (2.63–4.48%,p= 0.048–0.001) for both breeds. For the faster Arabian horse breed, recovering after racing took half the time that the local emaitukai breed did. Keywords:exercise; horse; endurance; pace; horse breed Animals2021,11, 995.

Animals2021,11, 995 2 of 13 1. Introduction F²d²ration Equestre Internationale (FEI) has described equine endurance racing as the second largest discipline in the world, above which is only show jumping [1]. Endurance races are described as long-distance competitions (40–160 km) that are organized into loops, which are arranged over variable terrain, and as a rule, these competitions are completed in one day. In endurance competitions, horse and rider combinations are required to complete the racecourse in good condition, aiming to win [2]. Endurance can be described as an exercise performed at a moderate speed whilst covering a long distance [3]. A horse is an interesting physiological model in this context, because different breeds can be used in all types of physical exercise. For example, the Arabian breed is recognized as well adapted to endurance racing, because the Arabian horses are able to run at an average speed of 20 km/hor greater for up to 160 km (in bouts of 30–40 km) [4]. This level of performance is based on aerobic metabolism, adaptation of the cardiorespiratory system, effective body heat dissipation, and maintenance of homeostasis. The Arabian horse breed is one of the world's oldest breeds. This breed is distinguished by its natural beauty, graceful movement, and athletic endurance. Since Arabian horses originated from the Middle East, they feature the unique ability to thrive in a hot, dry environment [5]. The standard height of the Arabian horse is from 145 to 155 cm (standing) [6]. The standard weight is between 360 and 450 kg; chest girth—113.2 cm; body length—145 cm; cannon bone girth— 18.5 cm. Compared with other race horse breeds, the muscle tissue of Arabian horses is characterized by signi cant differences in structure—the predominance of oxidative ber type I is observed in Arabians, [7,8]. In an Arabian horse's muscle, the higher proportion of oxidative type I bers (characterized by a low glycogen content and high triglyceride storage capability) results in a greater use of fat for energy [9]. Due to their exceptional qualities, Arabian horses have been extensively used for over 100 years by horse breeders for the improving

is observed in Arabians, [7,8]. In an Arabian horse's muscle, the higher proportion of oxidative type I bers (characterized by a low glycogen content and high triglyceride storage capability) results in a greater use of fat for energy [9]. Due to their exceptional qualities, Arabian horses have been extensively used for over 100 years by horse breeders for the improving of other horse breeds [10]. Arabian horses are often used not only for re nement of other horse breeds, but also for their endurance abilities [11]. The studies of the phenotypic traits of the emaitukai showed the average body measurements were: wither height—133.6 cm; chest girth—173.2 cm [12]; body length—142.2 cm; cannon bone girth—17.7 cm; weight, 360–420 kg [13]. The emaitukai is a pony by height and type [14]. However, the wide chest, high indices of extension, and massiveness indicate that these horses might have substantial draughting power. Due to thin, strong legs, a low bony index, round hips, a wide trot, good jumping technique, energetic temperament, and mobility, the emaitukai horses are considered as very suitable for quick trotting and jumping. Thanks to the aforementioned qualities, the emaitukai breed is highly valued and used as a versatile horse breed [13]. Based on the results obtained in our last study, it was stated that the horses of the emaitukai breed are suitable for endurance competing [15]. To maximize performance during a race, an athlete has to regulate speed over the entire course of a race. The distribution of energy expenditure during the race is de ned as “pacing strategy.” Pacing strategy is considered to be a key factor determining overall performance in endurance racing. It is considered that the best strategy in a race is maintaining an even pace, and sometimes gradual declining in speed [16]. The aim of every athlete is to reach a certain distance in the shortest time possible [17]. In endurance athletes, an increased aerobic capacity allows skeletal muscle to metabolize more fat (use energy from fatty acids), while at the same time using carbohydrates as an energy source [18]. An aerobic conditioning program in endurance-type

sometimes gradual declining in speed [16]. The aim of every athlete is to reach a certain distance in the shortest time possible [17]. In endurance athletes, an increased aerobic capacity allows skeletal muscle to metabolize more fat (use energy from fatty acids), while at the same time using carbohydrates as an energy source [18]. An aerobic conditioning program in endurance-type athletes induces an increase in the activity of oxidative metabolism and a decrease in anaerobic metabolism. The adaptive response to exercise is associated with changes in gene expression, metabolism, muscle cell cycle progression, and protein homeostasis. However, the exact mechanisms that occur in equine muscles during exercise related to skeletal muscle endurance in high-intensity training are not well understood [19]. Extensive studies of speed, pace, and total time in racing strategy have been carried out in human sports, whereas in equestrian sport, pacing strategy has been practically ignored, although it is one of the key factors to be analyzed when seeking to optimize horse performance [10]. As regards speed, data on postrace physical, biochemical,

Animals2021,11, 995 3 of 13 and blood gas parameters of endurance horses are limited. Therefore, the aim of this study was to investigate the impacts of breed on speed and the blood parameters of the Arabian and emaitukai horses during an endurance race. 2. Materials and Methods 2.1. Location, Animals, and Experimental Design The research was carried out in accordance within the provisions of the Law of the Republic of Lithuania—order number 8–500 on the protection, keeping and use of animals, of 6 November 1997 (the Of cial Gazette “Valstyb es inios” number 108–6595, dated 28 November 1997), order number 4–361 of 31 December 1998 of the State Veterinary Service of the Republic of Lithuania on breeding, care, transportation of laboratory animals, and order number 4 of 18 January 1999 of the State Veterinary Service of the Republic of Lithuania on the use of laboratory animals for scienti c tests. The study approval number was PK012868. The study was conducted on 60 (27 female and 33 male) clinically healthy Arabian horses and 52 (34 female and 18 male) clinically healthy emaitukai horses at six endurance competitions (45 km races) in Lithuania and at the Lithuanian University of Health Sciences Veterinary Academy. All horses were 10 4 years old, with an average body weight of 408 41 kg . The horses were transported from different locations, having been delivered to the competitions at least two hours prior to their rst veterinary examinations. Veterinary inspections were carried out according to FEI regulations. After each loop of the races, all competing horses underwent veterinary inspections, and the physical parameters of each horse were recorded. All horses successfully passed veterinary inspections, which were performed prior to the start of the race, during the race, and after the race. For a horse to be considered t enough to continue the event, its HR must be below 65 bpm within 20 min of arrival. The criterion for inclusion of horses was successful completion of the race, and only the nishers were included in the further research. All horses were trained for endurance racing and had participated

and after the race. For a horse to be considered t enough to continue the event, its HR must be below 65 bpm within 20 min of arrival. The criterion for inclusion of horses was successful completion of the race, and only the nishers were included in the further research. All horses were trained for endurance racing and had participated in similar competitions before; however, such factors as the degree of competition experience, the number of competition kilometers completed, and the number of successful races over the recent period were different for each animal. All researched horses were dewormed and vaccinated at a similar time and were not receiving any medications and/or suffering from any infections in the preceding three weeks. All animals were housed in a similar environment and fed a similar diet with adequate mineral and vitamin supplementation; the amounts of salt and water were not limited in the diet. 2.2. Analytical Procedures Blood samples were taken from each animal by applying the technique of jugular venipuncture with 1.6 mL heparinized vacutainer blood collection tubes for blood gas (Terumo Europe, Belgium) and a 5.0 mL tube without anticoagulant for serum biochemistry (Terumo Europe, Belgium), 30 min before the start and no later than 30 min after the nish. None of the horses showed stress during the blood sampling, which was carried out in less than 30 s for each sample. The samples were taken immediately after the rst and last veterinary inspections; then they were identi ed and stored in an ice bath for a maximum period of 30 min until processing. Using EPOC blood gas analyzers (EPOC, Canada, Ottawa), the following indices were analyzed: hydrogen potential (pH), partial carbon dioxide pressure (pCO2, kPa), partial oxygen pressure (pO2,kPa), base excess in the blood (BE, mmol/L), base excess in the extracellular uid (BE ecf mmol/L), bicarbonate (HCO3, mmol/L), oxygen saturation (sO2, %), total carbon dioxide in the blood (tCO2, mmol/L), hematocrit (HCT, L/L), hemoglobin concentration (Hgb, mmol/L), glucose (Glu, mmol/L), sodium (Na + , mmol/L), potassium (K + , mmol/L), calcium (Ca ++ ,mmol/L), and lactate (Lac, mmol/L). Serum

base excess in the blood (BE, mmol/L), base excess in the extracellular uid (BE ecf mmol/L), bicarbonate (HCO3, mmol/L), oxygen saturation (sO2, %), total carbon dioxide in the blood (tCO2, mmol/L), hematocrit (HCT, L/L), hemoglobin concentration (Hgb, mmol/L), glucose (Glu, mmol/L), sodium (Na + , mmol/L), potassium (K + , mmol/L), calcium (Ca ++ ,mmol/L), and lactate (Lac, mmol/L). Serum urea (Urea, mmol/L), glucose (Glu, mmol/L), aspartate aminotransferase (AST, U/L 1), iron (Fe, umol/L), creatinine (CREA, mol/L), magnesium (Mg, mmol/L), total protein (TP, g/L), total bilirubin (TB, mol/L 1), albumin (ALB, g/L), cuprum (Cu,

Animals2021,11, 995 4 of 13 umol/L), zinc (Zn, umol/L), and gamma-glutamyl transferase (GGT, U/L) were measured using an automated analyzer, Hitachi 705 (Hitachi, Japan). Horse speed and recovery time data were taken from the electronic time-keeping system (ECR v.7.01 Systems, Kaunas, Lithuania) designed for endurance events. Speed data were calculated by dividing the length of the course by the time taken for the horse to complete the course (subtracting hold times). Recovery time is the time that horse spends in the recovery area after crossing the end line of a loop until it crosses the line into the vetting area to be presented for its horse inspection. The races were held in Lithuania, in accordance with the FEI rules. The competitions were carried out on variable terrain with some muddy and some rm areas, and with slight elevation changes ( 300 m). The environmental conditions during the competitions varied, with the mean temperature being 22.5 C (within the range of 12.50–26.5 C) and the mean relative humidity being 73.20% (within the range of 52–96%). For a horse to be considered t enough to continue the event, its HR must be below 65 bpm within 20 min of arrival. 2.3. Data Analysis and Statistics The data were analyzed using the IBM SPSS Statistics v20.5 for Windows. The dis- tributions of the evaluated traits were used to carry out the assessment according to the Kolmogorov–Smirnov test. The mean (M) value and standard error of the mean (SE) were calculated. To compare blood parameters before and after competition, a pairedt-test was used; to compare the differences between the breeds before and after competition, the Student'st-test for independent samples was employed. The relationships between speed and recovery time and the blood parameters of a horse were assessed using the Spearman method. The results were considered to be signi cant atp< 0.05. 3. Results 3.1. The Indices of Biochemical Parameters of the Arabian and emaitukai Horses before and after Competition The analysis of biochemical parameters (Table) showed signi cant increases in the values of CREA (21.34–30.82%,p= 0.001–0.004), TB (50.84–56.24%,p< 0.001), and ALB (2.63–4.48%,p= 0.048–0.001) after

horse were assessed using the Spearman method. The results were considered to be signi cant atp< 0.05. 3. Results 3.1. The Indices of Biochemical Parameters of the Arabian and emaitukai Horses before and after Competition The analysis of biochemical parameters (Table) showed signi cant increases in the values of CREA (21.34–30.82%,p= 0.001–0.004), TB (50.84–56.24%,p< 0.001), and ALB (2.63–4.48%,p= 0.048–0.001) after competition in both breeds. Before and after competition, signi cantly higher levels of Fe (p= 0.001 andp< 0.001), CREA (p< 0.001), TP and TB (p= 0.023 andp< 0.001), and ALB (p< 0.001) were observed in the Arabian horses compared to those in the emaitukai breed, for which higher AST (p< 0.001), Cu (p= 0.002 andp< 0.001), and GGT (p< 0.001) were found. 3.2. The Indices of Acid–Base Balance in the Arabian and emaitukai Horses before and after Competition The study showed signi cant decreases in mean blood gasometrical indicators, such as pCO2(8.09–15.18%,p< 0.001) and BE (efc) (14.01%,p< 0.001 in the Arabian horses and 172.01% in the emaitukai breed,p= 0.006) and in the blood electrolyte Ca ++ (4.38–8.72%, p< 0.001); and there were increases in HCT and Hgb (20.05–20.12%,p< 0.001 in the Arabian horses and 6.22–6.23% in the emaitukai breed,p= 0.003–0.004), BE (b) (29.24– 39.38%,p< 0.001), and Lac (13.45–31.97%,p< 0.001) in the blood of both breeds in the post-competition horses compared to those measured before competition (Table).

Animals2021,11, 995 5 of 13 Table 1. The indices of minerals, trace elements, and biochemical parameters in the blood of the Arabian and emaitukai horses before and after competition. Blood Parameter Breed Pre-Race Post-Race Changes (Pre-Race—Postrace) Normal Ranges [20] M SE p M SE p Difference (%) p Urea mmol/L Arabian 6.06 0.181 <0.001 7.29 0.311 0.675 20.30 <0.001 2.9–9.6 emaitukai7.05 0.181 7.11 0.311 0.85 0.802 AST U/L Arabian 301.02 12.403 <0.001 336.93 11.862 <0.001 11.93 <0.001 205–555 emaitukai434.36 12.403 410.09 11.862 5.59 0.027 Fe umol/L Arabian 31.34 0.849 <0.001 32.99 0.920 <0.001 5.25 0.127 20–45 emaitukai27.52 0.849 25.60 0.920 7.00 0.002 CREA mol/L Arabian 133.002.539 <0.001 161.38 3.012 <0.001 21.34 0.001 53.1–159.2 emaitukai107.772.539 144.15 3.012 30.82 0.004 Mg mmol/L Arabian 0.75 0.031 0.336 1.42 0.248 0.048 89.33 0.058 0.6–1.7 emaitukai0.71 0.031 0.72 0.248 1.41 0.892 anorg P mmol/L Arabian 1.03 0.250 0.994 1.05 0.249 0.958 1.28 0.006 0.8–1.3 emaitukai1.04 0.250 1.03 0.249 0.85 0.492 Total Ca mmol/L Arabian 0.67 0.036 <0.0010.67 0.030 <0.001 0.00 0.953 0.59–0.74 Zemaitukai0.76 0.036 0.76 0.030 0.00 0.874 TP g/L Arabian 78.77 0.870 0.023 83.62 1.098 <0.001 6.16 <0.001 56–76 emaitukai75.93 0.870 75.63 1.098 0.40 0.643 TB (bilirub) mol/L Arabian 31.61 1.467 <0.001 47.68 2.095 <0.001 50.84 <0.001 25–42 emaitukai15.95 1.467 24.92 2.095 56.24 <0.001 ALB g/L Arabian 41.28 0.336 <0.001 43.13 0.417 <0.001 4.48 0.001 26–41 emaitukai36.93 0.336 37.90 0.417 2.63 0.048 Cu umol/L Arabian 10.40 1.141 0.002 10.91 1.428 0.001 4.92 0.177 7.9–39.5 emaitukai12.61 1.141 13.41 1.428 6.39 0.122 Zn umol/L Arabian 26.99 0.817 0.010 28.07 0.683 0.230 3.98 0.112 12–45 emaitukai28.64 0.817 27.62 0.683 3.56 0.001 GGT U/L Arabian 17.18 2.328 <0.001 16.70 2.390 <0.001 2.79 0.353 6–32 emaitukai34.13 2.328 32.70 2.390 4.19 <0.001 M—mean;p—calculated probability; SE—standard error of the mean; Urea—serum urea; AST—aspartate aminotransferase; Fe—iron; CREA—creatinine; Ca—calcium; Mg—magnesium; TP—total protein; TB—total bilirubin; ALB—albumin; Cu- cuprum; Zn—zinc; GGT— gamma-glutamyl transferase; ND—no data. Table 2. The indices of electrolyte and acid–base balance in the Arabian and emaitukai horses before and after competition. Blood Parameter Breed Pre-Race Post-Race Changes (Pre-Race—Postrace) Normal Ranges of Venous Blood [21,22] M SE p M SE p Difference (%)

The study compares blood parameters and speed between Arabian and emaitukai horses in endurance racing.