Clock gene per 2 daily rhythm: Correlation with the serum level of uncoupling protein 1 (UCP1) in goat and horse.

Recent studies evidenced that the circadian rhythm of Per2 is involved in adaptive thermogenesis by the modulating transcription of uncoupling protein 1 (UCP1). For this purpose, we investigated the linkage between the daily rhythm of Per2 and UCP1 in ruminant and non-ruminant mammalian species. Five clinically healthy, not pregnant, and not lactating Maltese female goats and five clinically healthy, not pregnant, and not lactating Italian Saddle horses were enrolled in the study. All animals were housed under natural photoperiod (sunrise 05:05, sunset 20:55) and environmental temperature and humidity. Goats were kept individually in 3.0 × 2.0 m box, horses were housed individually in 3.5 × 3.5 m box; all boxes were equipped with an opening window. On each subject, blood samples were collected every 4 h for a 48-h period. The Per2 gene expression was determined on blood samples collected in PAX gene Blood RNA Tube, whereas UCP1 concentration was assessed on serum. Per2 and UCP1 levels were statistically influenced by the species (p < 0.0001) and the time of data collection (p < 0.0001), but not by the day of monitoring. Per2 showed daily rhythmicity, statistically different in mesor and amplitude between the two species, diurnal in goats, nocturnal in horses; with the same robustness. UCP1 did not show daily rhythmicity. During the experimental period the two parameters showed a negative correlation in horses. According to the findings herein obtained, we can claim that the role of Per2 in the thermogenesis induced by the beige adipocytes throughout UCP1 activation did not reflect what found in other mammal species, but further studies are required to establish their correlation in equids.

Thermographic ocular temperature correlated with rectal temperature in cats.

Body temperature reflects the animal health and/or disease conditions. During clinical examination, temperature measurement is a basic step in veterinary medicine. The conventional method used is the rectal thermometry, particularly stressful in some subjects, especially for cats. A less stressful alternative method, such as infrared thermal imaging camera, is used in various fields of medicine for diagnosis, prognosis and correct therapeutic approaches. To evaluate the usefulness of infrared thermal imaging for the assessment of ocular temperature, twenty cats of different breeds (European, Siamese and Persian, 4-6 years old, mean body weight 4.3 ± 0.30 Kg) were enrolled in the study. In order to evaluate the applicability of the ocular temperature assessment through thermal imaging as a tool for measuring the animal's body temperature, the obtained values were compared with the rectal temperature values recorded in each cat by means of a digital thermo-camera. There were no differences between left and right eye; and a difference of about 1.19 °C between the ocular and rectal temperature value was recorded (p < 0.0001). Rectal and ocular temperatures were positively correlated (p < 0.0001; r = 0.93). In conclusion, we show that ocular temperature is an alternative method for body temperature measurement that can be used in clinical evaluation of cats, especially in cases where rectal temperature recording is not possible.

Different training schedules influence platelet aggregation in show jumping horses.

Depending on the intensity, duration and type of physical exercise, equine metabolism has to adapt to nervous, cardiovascular, endocrine and respiratory system requirements. In horses, exercise and training are known to have considerable effects on the mechanisms of hemostatic system involving platelet activity. The aim of the present study was to evaluate the effect of different training schedules on platelet aggregation in 15 Italian Saddle jumping horses. Animals were divided into three equal groups: Group A was subjected to a high intensity-training program; group B to a light training program, group C included sedentary horses. From each animal, blood samples were collected by jugular venipuncture at rest on the 1st, 3rd and 5th days, and afterwards, once a week, for a total of 5 weeks data recording, in order to assess the maximum degree of platelet aggregation and the initial velocity of aggregation (slope) platelet aggregation. Two-way analysis of variance (ANOVA) showed a significant effect of the different training schedules on studied parameters. The results revealed a different degree of platelet aggregation and a different initial velocity of platelet aggregation that changes during the different training schedules in horses that could represent a different protective endothelial mechanism. These findings could have an important role for a clearer knowledge of the physiological reference values of platelet aggregation and for a better interpretation of these variations during the training.

Leukocyte modifications during the first month after foaling in mares and their newborn foals.

During early post-partum period both neonatal foals and peripartum mares are most susceptible to diseases. The aim of this study was to establish physiologic modifications of leukogram during the first month after foaling in mares and their newborn foals. To this end blood samples were collected from nine mares and nine foals (T0-T10), every three days from the 1st day until the 30th day after foaling. Samples were analysed for white blood cell (WBC) count and differential leucocyte counts. Two-way repeated measure analysis of variance (ANOVA) showed, in postpartum mares WBC showed significant higher values at T0 (9.02±0.76) in respect to other time points, and at T2 (8.08±0.53) and T3 (7.92±0.59) compared to T1 (6.98±0.43), whereas in foals lower WBC values at T0 (6.11±0.49) compared to other experimental periods except T1 (6.90±0.94), and at T1 compared to T8 (7.95±0.61) and T10 (7.90±0.36) were observed. The differential leucocyte counts showed significant modifications in the percentage of neutrophils (π<0.001) and lymphocytes (p<0.001) both in postpartum mares and in foals during the experimental period. Furthermore ANOVA showed significant differences between postpartum mares and foals (P<0.01) in all studied parameters, and between postpartum mares and control mares in WBC and neutrophils values. The obtained results provide suitable information about the influence of foaling on leukogram of periparturient mares and reveal WBC dynamics in newborn foals during the first month post-partum.

Metabolic profile of broodmares during late pregnancy and early post-partum.

Pregnancy represents a specific physiological status characterized by continuous adjustments that affect maternal metabolism of all nutrients. In the last trimester of pregnancy, mare's nutrient requirements greatly increase and most pregnancy-associated diseases are likely to occur. Therefore, we aimed to assess the metabolic profile of broodmares focusing on the last 3 months of pregnancy and the early post-partum. Fifteen pregnant mares (Group A) were monitored from 263 ± 3 days of pregnancy until 21 days after foaling. Seven non-pregnant mares (Group B) were used as the control group. Blood samples were collected weekly by jugular venipuncture throughout the experimental period, and additional blood samples were collected within 24 ± 12 h of foaling. Obtained sera were analyzed for urea (Ur), creatinine (Cre), total protein (TP), total, direct and indirect bilirubin (tB, dB, iB), triglycerides (TG), total cholesterol (tChol) and ß-hydroxybutyrate (BHB). During pregnancy, Ur (p = 0.015) and dB (p = 0.028) were higher in Group A than Group B. Serum Cre, tB, iB and TG (p < 0.001) decreased after foaling; furthermore, Group A exhibited lower tChol (p < 0.001) and higher BHB (p < 0.001) than Group B during the study. There were significant interactions of time × peripartum on Ur (p = 0.007), Cre (p < 0.001), tB (p = 0.018), TG (p < 0.001) and tChol (p < 0.001). These results can be used in the interpretation of biochemical studies in late gestation mares which develop systemic disease. The improved understanding of the metabolic profile during the peripartum might assist in monitoring the health status of the broodmares to promote foetal growth and well-being.

Electrophoretic serum protein fraction profile during the different physiological phases in Comisana ewes.

The aim of this study was to evaluate the influence of different physiological phases on serum total proteins and their fractions of ten Comisana ewes housed in Mediterranean area. From each animal, blood samples were collected at different physiological phases: late pregnancy, post-partum, early, mid-, end lactation and dry period. On all samples serum total proteins were determined by the biuret method, and albumin, α-globulins, ß(1) -globulins, ß(2) -globulins and γ-globulins concentrations were assessed using an automated system. One-way repeated measures analysis of variance was applied to determine the significant effect of different physiological phases on the parameters studied. During the late pregnancy and post-partum, total proteins, ß1- and ß2-globulins and γ-globulins showed the highest values. Starting from post-partum, α-globulins increased to reach their peaks in mid-lactation. Early lactation was characterized by low γ-globulins values. The increase in serum albumin concentration and the drop in some globulin fractions determined the significant increase in albumin/globulin ratio. The obtained results contributed to improve the knowledge on electrophoretic profile during the different physiological phases in ewes, confirming that pregnancy and lactation periods affect the protein metabolism. Particularly, serum protein fractions pattern could give information about dehydration, plasma volume expansion and hepatic function, which occur during the different physiological phases. Dynamics of the protein profile - from pregnancy to dry period - which are provided by our results, could be considered as guidelines for the management strategies to guarantee the nutritional needs of these animals during the different physiological phases and to avoid a decline of productive performance and consequently an economic loss.

Training-induced modifications of circadian rhythmicity of peroxidative parameters in horses.

The aim of this study was to evaluate the daily rhythms of peroxidative parameters in untrained and trained horses. Blood samples were collected every 4 h for a 48-h period for the determination of reactive oxygen metabolites test (d-ROMs), antioxidant barrier (Oxy-ads), thiol antioxidant barrier (SHp) and lipid peroxidation (LPO). Two-way anova showed a significant effect of time of day on all parameters studied, except on LPO. Higher values of Oxy-ads and SHp were observed in trained horses during both days of monitoring (p < 0.01). All studied parameters, except for LPO, showed robust daily rhythms. Their acrophases occurred during the day in untrained horses and in the evening in trained horses. Our results showed that training increases antioxidative capacity and demonstrated that the daily rhythms of antioxidative parameters can be modified by training.

Daily variations of serum lipids in Ovis aries under different lighting and feeding conditions.

The aim of this study was to discriminate the impact of lighting and feeding conditions on the regulation of lipid metabolism in Ovis aries. Six clinically healthy female Comisana ewes, not pregnant and not lactating were kept under different environmental conditions: 12:12 light-dark (LD) cycle, constant darkness and fasting. Blood samples were collected at the end of each period of treatment every 4 h for a 24-h period. Blood concentration of triglycerides, total cholesterol and non-esterified fatty acid showed robust daily rhythmicity in ewes maintained under 12:12 LD cycle and fed ad libitum. Conversely, either constant darkness or fasting had a deep impact on all lipid parameters investigated. Our results suggest that lighting and feeding conditions have an impact on daily variations in lipid metabolism in ewes.

Clock genes determination in whole blood in goats housed under a long light cycle.

An innate 24 h circadian clock drives various behavioral processes via expression of clock genes that regulate circadian rhythmicity and temporal signals. Elucidating the gene expression in goats may contribute to improving the knowledge of the regulation of circadian rhythms in this species. Five nonpregnant and nonlactating Maltese goats with no evidence of disease were kept in an indoor pen under the natural long photoperiod (05:05-20:56 h) and natural environmental temperature (23°C and 60% RH). They were fed an Alfalfa hay and concentrate mixture provided twice a day; water was available ad libitum. Blood samples were collected every 4 h over a 48 h period into PAX gene Blood RNA Tubes and stored at -80°C until processing. Clock genes (Clock; Cry1; Cry2; Per2; Per3) were determined using real-time quantitative polymerase chain reaction. During the experimental period, locomotor activity was monitored by an actigraphy-based data logger that records a digitally integrated measure of motor activity as a means to assess indices of discomfort during study and stability of the circadian rhythm. All of the tested genes showed daily rhythmicity in their expression in whole blood. Differences in their circadian parameters were observed. Mesor and amplitude were statistically different among the tested gene (Mesor: F(4.30) = 205.30; p < .0001; amplitude: F(4.30) = 104.80; p < .0001), with each gene showing its acrophase at a different time of day (F(4.30) = 81.17; p < .0001), and differences were observed between the two days of monitoring (F(1.30) = 10.25; p = .003). The application of two-way analysis of variance (ANOVA) on robustness of rhythm values did not show statistical differences among the tested genes (F(4.30) = 1.83; p = .14) and between the two days of monitoring (F(1.30) = 1.16; p = .28). Locomotor activity data recording were in accordance with the data reported in literature, indicating the absence of discomfort or alteration of circadian rhythms during the experimental period. Our results support the presence of a cyclic transcription of clock genes in whole blood of healthy goats housed under a long light natural photoperiod and natural environmental conditions.

Effect of desmopressin on immune-mediated haemorrhagic disorders due to canine monocytic ehrlichiosis: a preliminary study.

To evaluate the possible use of desmopressin acetate (DDAVP) in haemorrhagic disorders consequent to canine monocytic ehrlichiosis (CME), three dogs infected by Ehrlichia canis, with a history of thrombocytopenia and recent bleeding, were studied. The dogs were administered desmopressin (1 µg/kg b.w. s.c.) every 24 h on three occasions. Blood samples were collected immediately before, and after 2 and 48 h the first DDAVP administration, to assess haematological, clinical chemistry and clotting time parameters. Spontaneous bleeding stopped within 1 h after the first DDAVP injection. Buccal mucosa bleeding time (BMBT) was shortened from 9.6 to 2.3 min within 2 h after the treatment. A statistically significant increase in platelet PLT count and fibrinogen, and a statistically significant decrease of PT and aPTT were observed after DDAVP administration. The haemorrhagic disorders caused by CME appear to be quickly corrected by DDAVP administration, giving the clinician the time necessary to administer appropriate chemotherapy.

Effect of different workload and hydrocortisone in vitro on platelet aggregation in athletic horse.

The objective of the present study was to evaluate the platelet response to different workload in athletic horse and the influence of hydrocortisone on this response. For this purpose, six Sella Italiana gelding athletic horses, ranging in age from 8 to 10 years and clinically healthy, were subjected to four different exercises: horse-walker, treadmill, obstacle course of 1.00 m and obstacle course of 1.25 m. From all the horses, citrated plasma samples were collected at rest, immediately after exercise and 30 min after the end of the exercise to assess the maximum degree of platelet aggregation and the initial velocity of aggregation (slope) after platelet activation with 10 microM ADP and 20 microM ADP. Successively, the effect of 20 minutes pre-incubation with hydrocortisone in ADP-induced aggregation was determined. One-way repeated measures analysis of variance (ANOVA) was applied to determine significant differences due to experimental conditions (P < 0.05) and, for each sampling time, one-way analysis of variance was applied to determine significant differences due to workload (P < 0.05). Bonferroni's test was applied for post-hoc comparison. Our results revealed a statistically significant effect of experimental conditions only after obstacle course of 1.00 m and 1.25 m. These findings showed an inhibition of platelet aggregation during different workload in athletic horses that probably represents a protective endothelial mechanism through the production of nitrix oxide, and a kinetic measurement of the reaction that changes in relation to percentage of aggregation. The mechanism of hydrocortisone had no effect on platelet receptor expression in vitro in athletic horses, so it might be useful to evaluate the effect of different anti-inflammatory steroids in order to confirm, or note any differences, with drugs that have the same mechanism of action.

Daily rhythm of tear production in normal dog maintained under different Light/Dark cycles.

This study was conduced to assess the daily rhythm of tear production in clinically healthy dog. For our study eight female purebred Beagles were subjected to three different Light/Dark schedules: 12/12L/D, 24/0L/D and 0/24L/D cycles. In all subjects Schirmer tear test I was performed at 4h intervals over a 24h period. A statistical significant effect of photoperiod was observed comparing the three different L/D schedules, and a statistical significant difference was observed comparing left and right eye during the 12/12L/D schedule. We demonstrated daily variation of tear production in dogs maintained under an L/D cycle. We also provided strong evidence that the rhythm of tear production is endogenously generated because it persisted in constant darkness. Although the range of excursion of the daily/circadian oscillation in STT I (about 2mm/min) is likely too narrow to be of clinical significance, it is statistically significant and may have physiological implications not yet appreciated.

Serum homocysteine and oxidative stress evaluation during exercise in horse.

The aim of the present work was to evaluate the influence of exercise on serum homocysteine and oxidative stress in Thoroughbreds during official races. For our study, ten Thoroughbred mares, 5 years old and clinically healthy, were used. The horses were trained to take part in an official 2100 meter race. Reactive oxygen species (dROMs), antioxidant barrier (Oxy-adsorbent), thiol antioxidant barrier (SHp) and homocysteine (sHcy) were investigated. Blood samples, collected from each horse at rest, immediately after the race, and 30 and 180 min after the end of the race, were collected by jugular venipuncture using vacutainer tubes with no additive for the assessment of dROMs, Oxy-adsorbent and SHp by means of a UV spectrophotometer, while serum total Hcy values were determined by high performance liquid chromatography with fluorimetric detection and isocratic elution. Statistical analysis, one way repeated measures analysis of variance (ANOVA), followed by Bonferroni's test, showed statistical differences (P<0.05 was considered statistically significant) for all parameters studied. Moreover, a coefficient of linear correlation (r) was computed for values of dROMs and sHcy for all sampling times but a significant linear regression (r = 0.94) was found only after the race. The variations of sHcy, dROMs, Oxy-adsorbent and SHp during the experimental period considered suggest the important role of oxidative status in the athletic horse. The systematic analysis of oxidative stress and its influence on homocysteine levels contribute to the clinical evaluation and assessment of the athletic performance of the horse.

Features of the daily rhythms of blood melatonin and glucose in goats during different natural photoperiod.

The scope of the present study was to explore the features of the daily rhythms of plasma melatonin and blood glucose in goats during different natural photoperiod imposed by the four seasons. Five Sarda breed pluriparus female goats, 2 years old, with a mean body weight 40.5 ± 2.3 kg, lead to a natural pasture, were cannulated the day before the start of the study for sample collection performed during spring and autumn equinoxes, and winter and summer solstices for a 48-h period. On blood samples, melatonin and glucose concentrations were assessed. Our results indicate a daily rhythm of both parameters studied which characteristics were influenced by the different photoperiod due to season. During the experimental period, the two parameters showed a negative correlation, despite the onset of melatonin concentration was linked to the sunset, whereas the onset and offset of glucose were not linked to the photoperiod. In conclusion, we can claim that the two parameters are driven by different entrainable systems, but further studies are necessary to establish their correlation.

Dynamic modulation of platelet aggregation, albumin and nonesterified fatty acids during physical exercise in Thoroughbred horses.

The effect of exercise on platelet aggregation, albumin and nonesterified fatty acids (NEFAs) values and the correlation among these parameters were evaluated in ten clinically healthy and regularly trained Thoroughbred horses. All horses were subjected to two simulated races. Blood samples were collected by jugular venipuncture before and after the first simulated race (T0PRE and T0POST), every 7 days at rest condition for a month (T1R-T2R-T3R), and before and after the second simulated race (T4PRE and T4POST) in order to assess platelet aggregation, albumin and nonesterified fatty acids (NEFAs) levels. One-way analysis of variance showed a significant effect of exercise (P<0.01) on platelet aggregation, albumin and NEFAs values. A negative correlation between platelet aggregation and albumin or NEFAs values, and a positive correlation between albumin and NEFAs values, were found both at T0POST and T4POST (P<0.05). These findings are likely related to dynamic physiological adaptations to exercise that allow re-establishment of the homeostatic equilibrium of the organism.

Serum levels of mitochondrial uncoupling protein 1, leptin, and lipids during late pregnancy and the early postpartum period in mares.

The aim of this study was to investigate the changes of serum mitochondrial uncoupling protein 1 (UCP1) and leptin levels as well as of lipid and lipoprotein profiles in mares during the peripartum period. Ten pregnant mares (group A) were monitored from 15 ± 3 days of pregnancy until 15 days after foaling, and 10 nonpregnant nonlactating mares constituted the control (group B). In group A, blood sampling was performed on Days 15 ± 3 and 7 ± 3 before foaling, on the day of foaling, and on Days 7 and 15 after foaling. In group B, blood sampling was performed on the same days as in group A. Serum levels were determined for UCP1, leptin, total lipids, phospholipids, triglycerides, total cholesterol (Total-Chol), high-density lipoproteins, low-density lipoproteins (LDLs), and very low-density lipoproteins (VLDLs). Two-way repeated-measures ANOVA was applied to evaluate the effects of peripartum period and group membership. All studied parameters except phospholipid levels (P > 0.05) showed significant changes in group A over the peripartum period (P < 0.0001). A significant effect of pregnancy was found on all studied parameters (P < 0.001), which showed lower levels in group A than in group B for most of the time points considered. Significant negative correlations were found between UCP1 and total lipids, triglycerides, VLDLs, Total-Chol, and LDL values. Positive correlations were found between leptin and total lipids, triglycerides, VLDLs, Total-Chol, and LDLs. These changes observed in mares during the peripartum period could represent a response to hormonal and metabolic adaptations occurring during specific physiological conditions such as late pregnancy and early postpartum. These changes should compensate for the energy loss occurring during these particular life phases and ensure a good body condition to protect mares against negative energy balance.

Iron profile in Thoroughbreds during a standard training program.

OBJECTIVES: The aim of this study was to evaluate the modifications of some haematological parameters and iron profile during an 80-day training program and their relationship with athletic performance of performance horses. METHODS: During the training period, 20 Thoroughbreds were subjected to a standardised exercise test, previously standardised for gallopers to evaluate athletic performance. The test was performed immediately before the beginning of the training program (T0) and after 30 days (T30), 60 days (T60) and 90 days (T90). For each horse, their velocities at blood lactate concentrations of 2 and 4 mmol/L (V2 and V4) and a heart rate of 200 beats/min (V200) were calculated to assess the effect of the training program on athletic performance. Blood samples were collected at 20-day intervals over a period of 80 days from the beginning of the training program. RESULTS: One-way repeated measures analysis of variance (ANOVA) showed a statistically significant effect of days of training (time) on iron, ferritin, transferrin, total iron-binding capacity, transferrin saturation and unsaturated iron-binding capacity (P < 0.001 for all). CONCLUSIONS: The results showed the influence of the intensity and duration of exercise on iron metabolism. These findings may be useful for evaluating the athletic status of horses during training and to assess the degree of training adaptability, providing an opportunity to modify the training schedule to achieve the desired performance.

Hemostatic profile during late pregnancy and early postpartum period in mares.

Hemostasis is a physiological process that prevents excessive blood loss and represents a protective mechanism at the time of delivery. Peripartum hemorrhage is a recurring hazardous condition to mare's health; therefore, we aimed to study mares' hemostatic profile to investigate whether physiological adjustments occur during late pregnancy and early postpartum. Fifteen pregnant mares have been monitored from the 34th week of pregnancy until the third week after foaling. Fifteen nonpregnant mares were used as control group. Jugular blood samples were analyzed for platelet count (Plt), prothrombin time (PT), activated partial thromboplastin time (aPTT), and fibrinogen (Fb). Platelet count showed significant changes at foaling (P < 0.05) and a negative correlation (r = -0.968; P = 0.032) with postpartum. Prothrombin time changed (P < 0.05) showing a significant correlation (r = 0.675; P = 0.016) with late pregnancy. Fibrinogen concentrations changed throughout the experimental period (P < 0.0001). The linear regression model revealed a positive correlation (r = 0.9210; P < 0.0001) between Fb and late pregnancy and a negative correlation (r = -0.9583; P = 0.042) between Fb and early postpartum. The shortening in PT recorded in the imminence of parturition along with the increase in Plt and Fb at foaling might reflect a physiological hypercoagulable state that constrains excessive bleeding, enhancing mares' odds of surviving. Our research improves the knowledge about blood coagulation in periparturient mares providing specific information on routine coagulation tests that may support in monitoring mare's hemostatic profile during late pregnancy and early postpartum.

Changes in blood coagulation induced by exercise training in young athletic horses.

Prothrombin Time (PT), Activated Partial Prothrombin Time (APTT), Fibrinogen concentration (Fbg) and Platelet number (Plt) were evaluated in 20 young athletic horses during a training program. A standardized exercise test (SET) was performed every month for three months. The V4 variations (the speed, in m/min, reached at the blood lactate concentration of 4 mmol/l) obtained for each test were calculated to assess the effect of training program on athletic performance. Blood samples were collected at 20-day intervals over a period of 80 days from the beginning of the training program. The V4 (P < 0.001), PT (P < 0.001), APTT (P < 0.01), Fbg (P < 0.01) and Plt (P < 0.001) varied throughout the training period showing that the modifications of clotting mechanism in response to training period may be considered as a normal physiological response of the hemostatic system to training exercise.

ADP-induced platelet aggregation after addition of tramadol in vitro in fed and fasted horses plasma.

Adenosine diphosphate (ADP)-induced platelet aggregation in fed and fasted horses after addition of tramadol hydrochloride was evaluated in vitro. On 10 horses citrated blood samples were collected 2h after feeding (fed animals) and 21 h after feeding (fasted animals). Final concentrations of ADP 1 and 0.5 µM, and tramadol hydrochloride (1, 15, 30, 45 and 60 min after the addition of tramadol) were used to determine the maximum degree and initial velocity of platelet aggregation. Repeated measures multifactor analysis of variance (MANOVA) was used to evaluate the effect of feeding/fasting condition, ADP concentration and addition of tramadol. Findings showed statistical differences (P≤0.05) on studied parameters after addition of tramadol to different ADP concentrations in fed and fasted horses. The clinical relevance of these results is that tramadol provides many advantages as a therapeutic option; in fact, it is an inexpensive and a relatively new analgesic in equine veterinary medicine. Further investigations would be appropriate to compare the effects of different opioids but also using different concentrations of tramadol associated with other drugs in order to have substances which can regulate the functional activity of the platelets and to extend the knowledges on equine platelet aggregation.