Effects of ozone therapy on hematological, biochemical, and oxidative stress parameters of vaquejada athlete horses

Ozone therapy is a technique used in several specialties of equine medicine; however, there are few studies on its use in vaquejada (cowboy competition) athlete horses. This study aims to evaluate the potential effect of ozone gas administered by two different routes on hematological and biochemical values and the oxidative stress marker in vaquejada athlete horses. For this, nine healthy equines that followed a training protocol and underwent two treatments were used with an 8-day wash-out between them. The major ozonated autohemotherapy (MOA) treatment group received a volume of 600ml of the O2-O3 mixture at a concentration of 60 µg/mL, and the rectal insufflation (RI) treatment group received 5mL of gas per kg of body weight at a concentration of 15µg/kg performed every 24h on three consecutive days. Results were significant for RBC, hematocrit, and hemoglobin in the hematological variables, and AST and lactate for biochemical and malondialdehyde variables. No statistically significant differences were found in comparisons between treatment groups. Thus, we can conclude that there is no difference between the two therapies, indicating that the two techniques are effective for the application of ozone therapy in horses competing for vaquejada.


INTRODUCTION
Vaquejada is an equestrian sport traditionally practiced in the northeastern region of Brazil (Santiago et al., 2013;Bastos et al., 2017).The athletic activity developed by vaquejada horses is considered high intensity and short duration, making the anaerobic pathway prevail for energy production (Lopes et al., 2009;Hunka et al., 2017;Sousa et al., 2018).Physical exercises with these characteristics induce changes in hematological and biochemical parameters and the oxidative metabolism of horse athletes (Balogh et al., 2001;Sagai and Bocci, 2011;Sousa et al., 2018).
There is an increase in oxygen consumption by muscle mitochondria during sports activity, which will convert the consumed oxygen into carbon dioxide and water.However, 2% to 5% of this oxygen is not converted and will result in the formation of reactive oxygen species (ROS) (Piccione et al., 2007;Soares et al., 2011;Antunes, 2013;Bottegaro et al., 2018).The body balances ROS production and degradation (Wulf, 2001).However, when ROS production increases to the limit where the organism cannot eliminate or neutralize it, a condition called oxidative stress (OS) occurs (Fernandes et al., 2012).
In vaquejada competitions, horses are exposed to continuous days of exercise with reduced moments of rest, deviating from their regular exercise routine and increasing physical activity during competition days (Lopes et al., 2009).This increased exercise load, and the resulting OS can decrease performance during competition due to muscle tissue injury and decreased skeletal muscle force production (Ott, 2021).
Malondialdehyde (MDA) is the best known and most reliable marker of lipid peroxidation, widely used as evidence of oxidative stress installed in the animal's body (Kerksick and Willoughby, 2005;Sara et al., 2012).
The effects produced by O3 are a result of its action mechanism, which, through a moderate, adequate, and transient oxidative stress, induces the activation of second messengers, such as nuclear factor related to erythroid 2 (Nrf2) and lipid peroxidation products, such as malondialdehyde and 4-hydroxynonenal, which will increase the synthesis of different antioxidant enzymes and, consequently, promote the growth of the total antioxidant capacity of the organism (Smith et al., 2017;Rosseto, 2020;Sciorsci et al., 2020;Farias et al., 2020).
In this context, this study aims to evaluate the effects of ozone therapy administered by different routes on the hematological and biochemical parameters and on the lipid peroxidation index of vaulting athlete horses.

MATERIAL AND METHODS
The research was approved by the Ethics Committee on Animal Use of the Federal University of Alagoas (Ceua/Ufal), under number 02/2021.
The experiment was conducted at Grupo Aliança de Vaquejada, located in the municipality of Viçosa, Alagoas (geographical coordinates: latitude -9.325494 south, longitude -36.303363 west), in October and November 2021.
Nine animals of the Quarter Horse breed were selected for the experiment, six males and three females, with a mean age of 4.2±0.94years and a mean weight of 474.89±30.82kg.The feeding program consisted of 6 kg of commercial concentrate per day (14.0%crude protein, 3.5% ethereal extract, and 2.9 Mcal of digestible energy) supplied in three meals (7 am, 12 noon, and 5 pm), 12kg of Tifton (Cynodon dactylon) based volume also divided into three times, plus mineral salt (EQX Pro -Integral Mix ® ) and water ad libitum.

≤450 >450 7
Presence of injuries/bleeds on the animals caused by the harness, bridle by "hackamore," or recent accidents (identify in the review).
Absent Present 8 Presence of pain or claudication (including dorsal tenderness) through classification according to OBEL (1-5), identifying the region.

Absent Present 9
Presence of stereotypies or aggressiveness, assessing the animal in the stall for two hours to identify which option.

Absent Present
The observation of all animals was performed by two researchers interspersed between days to verify the occurrence of stereotypes.The animals were assessed in their environment, keeping a distance between the observer and the animal to prevent interest in human presence.The types of behaviors classified as normal (distracted, alert in the station, lying down, muzzle close to the ground, and neighing) or abnormal (aggression, digging, stereotypical stall walking, repeated head movements, wolf dance or bear syndrome or "weaving", aerophagia, wood biting, coprophagia, and trough licking) were considered.
The animal's inclusion in the research was conditioned to obtaining a score between 0-3 in the sum of the scores of the variables described in Table 1 as proof of good-welfare practices applied in their sporting activities, according to Coelho et al. (2018) (Table 2).Animals submitted to good-welfare practices in their sporting activities.

7-9
Animals with compromised welfare in need of activity interruption and urgent professional intervention.
The athletes were subjected to the same training system, ensuring standardization in their physical conditioning, constituted as follows: day 1-simulation of vaquejada according to the proposal by Santiago et al. (2013), where each horse runs three times with a 2-min interval between each race; day 2-a 60-minute walk in the morning (aerobic work); day 3-same as day 1; day 4-same as day 2; day 5: total rest in paddocks.As described, a training cycle was considered for each set of sequential five-day activities, which was repeated three times during the experimental period.
Blood samples for hematological, biochemical, and malondialdehyde (MDA) evaluations were aseptically collected from peripheral blood by jugular venipuncture.They were then packed into 4mL tubes, two containing ethylenediaminetetraacetic acid tripotassium (EDTA k3) and another containing a clot activator.The tubes were packed in a thermal box with reusable ice packs for storage and transport to the laboratory.
The first collection was performed at baseline (T0), and the animals were at rest for seven days without any physical activity.The next day, after sample collection, the first five-day training cycle was initiated, as described above, with the subsequent inclusion of the animals in the two experimental groups, the first group of greater ozonated autohemotherapy (MOA) and the second group of rectal insufflations (RI).The ozone therapy employed in these groups is fully described in item 2.4.New blood collections were performed after 24h (T1), 48h (T2), 72h (T3), 7 d (T4), and 15d (T5).The wash-out between treatments was eight days, considering seven days of rest for the animals.
The samples collected in one of the tubes with EDTA were used for hematological analyses.The parameters were evaluated in the erythrogram: Red blood cells count (RBC), Hemoglobin level (HG), Hematoctit (HCT), Mean Corpuscular Volume (MCV), Mean Corpuscular Hemoglobin Concentration (MCHC), Platelets count (PLT), plasma total plasma protein concentration (TPP), Fibrinogen (FIB), and in the leukogram, the parameters were evaluated: Leukocytes total count (LEUC), Rods (ROD), Neutrophils (NEU), Lymphocytes (LIN), Monocytes (MON), Eosinophils (EOS), Basophils (BASO).Hematological measurement was performed with the aid of an automated device model BC-30Vet ® , Mindray (Shenzen, China) and differential counting on blood smear stained by the Romanowsky method (Panoptic) using a Nikon microscope (E100 ® ) and an Inbras ® manual cell counter (ALB300CC).The measurement of fibrinogen was performed by the heat precipitation method in a water bath (Kacil bm-03 ® ) at a temperature of 56°C for three minutes, and the result corresponds to the difference between the protein concentrations pre-and post-precipitation and centrifugation of the sample, measured in mg/dL using a portable Instrutherm ® refractometer (RTP-20ATC).
Samples collected in a tube with a clot activator were intended to determine the serum activity of the enzymes: Aspartate Aminotransferase (AST), Alanine Aminotransferase (ALT), Creatine Kinase (CK), Alkaline Phosphatase (ALP), Gamma Glutamyl Transferase (GGT), plasma lactate concentration and the serum concentration of Urea and Creatinine (CREAT).The serum was centrifuged at a speed of (4000 rpm/5 minutes) in a KASVI tube centrifuge (MODEL K14-0815), and the serum was stored in Eppendorf tubes containing 1000 µl each.The concentration of blood lactate and biochemical analyses were measured in the BIOCLIN 2200 ® device using the enzymatic and kinetic UV method, respectively, using Bioclin Quibasa Química Básica Ltda commercial kits.
MDA levels were measured by highperformance liquid chromatography (HPLC) coupled with UV detection at 270 nm.For analyses, samples were stored on ice and transported to the laboratory, where they were centrifuged at 4,000 rpm for 10 minutes at 4°C to separate plasma from RBCs.The plasma was divided into 1.5mL Eppendorf tubes stored in a freezer at -80°C until analysis.
For analysis in HPLC, the solution was filtered through a sterile 0.22μm pore size Durapore membrane filter.A volume of 50 μL of the filtrate was injected into the HPLC, and the reading was performed at 270 nm.The reading lasts a total of six min, where the MDA retention time is around 2'81''.MDA values were assessed from the standard curve using 1,1,3,3tetramethoxypropane (TMP), a precursor compound of MDA, and expressed as nanomol of MDA.mL-1 of blood.
The MOA group's treatment was adapted from the treatment proposed by Tsuzuki et al. (2015).Through left jugular venipuncture, and after trichotomy and antisepsis with alcohol and Arq.Bras.Med.Vet.Zootec., v.74, n.6, p.1024-1038, 2022 iodine at the site, the collection of two CPDA-1 transfusion bags (JP Indústria Farmacêutica SA) was performed.The anticoagulant volume in the bag was adjusted to a 300 ml blood volume per bag.The blood bags were weighed on scales (Toledo Prix 3/14) to standardize the amount of blood collected, and the collection was stopped when the total weight reached 375 g (315 g of blood + 60 g of the package and diluent).The convention that every 1 ml of blood equals 1.05g was used to calculate the total blood weight (YAGI, 2016).
Then, a volume of 300 mL of the oxygen/ozone mixture was infused per bag at a 60 µg/mL concentration, making slight movements to homogenize the mixture.A transfusion line was attached to the bag, and the ozonated blood was slowly reinfused into the animal at a transfusion rate of 3.2mL/kg/hr.
The RI group's treatment methodology was adapted from that proposed by Jaramillo et al. (2020).The animals' rectal ampulla was emptied by palpation to increase the contact surface of the ozone with the mucosa.For O2/O3 gas administration, a urethral probe no.8 was inserted coupled to the ozone generator extender (Ozone & Life ® -São José dos Campos, SP, Brazil).
The O2 flow and the electrical discharge meter of the ozone generating machine were regulated so that 5mL of gas per kg of body weight was administered at a concentration of 15µg/kg.
For administering the treatments, the animals were randomly divided into groups.At first, five animals received the MOA treatment, and four received RI, reversing this division after the wash-out described in item 2.3.Only one administration was performed for the MOA treatment, and one application was performed every 24 hours for three days for the RI treatment.
The data were tested for normality using the Shapiro-Wilk test and presented by the media and its quartiles (first and third quartile).

DISCUSSION
The two main administration routes of ozone therapy in athletes are major autohemotherapy and rectal insufflation.In the experimental conditions proposed in this study, no statistical difference was found between the results of the two therapies (p > 0.05), indicating that the two techniques are equally effective for applying ozone therapy to horses competing in cow jumping (vaquejada).
However, the RI route has practical advantages compared to MOA regarding its easy management, low cost, and less invasiveness, corroborating what was described by Moreira (2015).In addition, rectal application of O3 has systemic therapeutic effects, as mentioned by Viebahn-Hänsler et al. (2012).
It is common to observe changes in hematocrit values, total red blood cell count, and hemoglobin concentration in athletic horses Arq.Bras.Med.Vet.Zootec., v.74, n.6, p.1024-1038, 2022 because of catecholamine release and, consequently, splenic contraction due to exercise (Orozco, 2007;Miranda et al., 2011;Pereira, 2015;Mattosinho, 2018).et al. (2016), the elevation of these enzymes is associated with increased membrane permeability and slow clearance from the circulation, not associating these elevations with a muscle injury.
In equine sports medicine, blood lactate concentrations are important for providing information on the physical conditioning of athlete horses (Ferraz, 2006;Botteon, 2012;Masko et al., 2021) Several authors mention supplementation use with antioxidant substances such as Coenzyme Q10, ascorbic acid (vitamin C), beta-carotene (vitamin A), and alpha-tocopherol (vitamin E) to control oxidative stress in horses and humans (Inal et al., 2011;Barbosa, 2012;Picchi, 2015;Svete et al., 2021).However, few studies analyze the effect of ozone therapy on the stimulation of antioxidant capacity and oxidative stress control in horses performing high-intensity, short-duration exercise (Inal et al., 2011;Tsuzuki et al., 2015).
According to Smith et al. (2017), O3 can induce further differentiation of erythroblasts, leading to a progressive increase in the number of erythrocytes.This increase raises oxidative stress resistance due to efficient antioxidant mechanisms, as described by Fernandes et al. (2012).Thus, the ozone therapy used in the animals of this study provided a strengthening of hematological antioxidant defenses, making them more resistant to OS.
At the beginning of therapies, O3 induces a cascade of events producing transient and moderate oxidative stress (Smith et al., 2017).
Ozone has a higher affinity to react with unsaturated fatty acids and antioxidant products (Travagli et al., 2010).Immediately, the body's antioxidant system is consumed to protect the macromolecules that make up the cell membrane, inactivating the ROS formed by the reaction (Bhatt et al., 2016;Di Mauro et al., 2019).In this context, these factors elucidate why MDA values were not elevated in the first moment (24h) after ozone therapies.

Figure 1 .
Figure 1.Quantitative data are given as box plots showing medians, means, and first and third quartiles for comparison between the RI and MOA groups for MDA values.

Table 1 .
Coelho et al. (2018)ological, and biochemical parameters considered for the inclusion criteria of animals in the research, adapted fromCoelho et al. (2018) creatine kinase -CK, considering the scoring range in values higher than 450 IU/L, following observations by Valberg et al.

Table 2 .
Scoring system of the welfare scale for athletic horses, adapted fromCoelho et al.

Table 3 .
Median (minimum, maximum)values of the serum hematological profile, before the treatments