Bioluminescent sport horse saliva test: prospects for use

Assessment of the physiological state of horses in training is a relevant problem of sport horse breeding worldwide. Existing clinical methods do not provide reliable parameters of the functional state of animals at rest and in physical activity. The reported standards of the physiological state of horses and veterinary guidelines for clinical diagnosis and equestrian sports are contradictory. Therefore, objective tests are necessary to assess the body's response to physical activity. We propose simple, fast, non-invasive method based on an inhibitory effect of saliva on the enzyme activity of luminous bacteria as a screening testing. Changes in the luminescence of the bioluminescent enzymatic system under influence of small amounts of saliva can reveal changes in the body of sport horses as a response to the maximum permissible loads. This study proves for the first time that the bioluminescent enzyme test can track changes in the body condition of sport horses during training. The method uses the integral bioluminescent indicator which depends on the biochemical composition of saliva. The aim of this study is to substantiate suitability of the bioluminescent-based method for testing the functional state of sports horses in training. Trakenen sport horses ( Eguus caballus ) for dressage ( n = 12) kept under standard conditions in the Training Center of Horse Breeding Complex (Krasnoyarsk State Agrarian University) were subjected to low, medium and high intensity training before the competition (February-June 2019-2020). Saliva and blood were sampled before (in the morning) and after training. The respiratory rate (RR) and heart rate (HR) were measured. The electrocardiography (ECG) was carried out according to a common method, including assessmsnt of the heart rhythm parameters and the ventricular systolic functional parameters. Hematological test were performed, and blood concentrations of protein and glucose were measured. The saliva was tested by colorimetric, chemi-, and bioluminescent methods. As the intensity of physical activity increased, there was an increase in heart rate, respiration rate, atrial excitation and a decrease in the time of cardiac diastole while hematological and biochemical blood parameters varied within normal limits. The effect of saliva on the intensity of bioluminescence depended on the physical activity. The residual luminescence signal decreased under low and medium intensity training and increased under high intensity training. During low intensity training, a high percentage of luminescence inhibition correlated with an increase in the total blood protein concentration ( r = 0.6, p = 0.05) and a decrease in the blood glucose content ( r = -0.7, p = 0.05) and the number of erythrocytes ( r = -0.6, p = 0.05). Under moderate physical activity, an increase in bioluminescent fluorescence correlated with an increase in RR ( r = 0.5, p = 0.1) and in the QRS interval ( r = 0.8, p = 0.05). Under high intensity training, a low percentage of luminescence inhibition correlated with the lactate concentration in saliva (r = -0.58, p = 0.1), a reduction in catalase activity in saliva ( r = -0.7, p = 0.05), and a higher amplitude of the P wave on the electrocardiogram ( r = 0.8, p = 0.05). Therefore, the bioluminescent analysis of saliva using a coupled enzyme system, NADH:FMN-oxidoreductase and bacterial luciferase can detect the effect of stressful physical activity during horse training of various intensity. The inhibition of bioluminescence can be an indicator of a horse performance in training. The test can be also applicable in sport horse breeding to prevent overtraining.