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�v�v�@A4��@A4A4V�;@�<v�����g2٠�����*�<�@IA0yA<xG�2�G�<�<�G�P>|Qa�A4#��&QQQ�@�@�3�QQQyA������������������������������������������������������������������������GQQQQQQQQQf �:	Physiological and hemato-chemical  evaluation of thoroughbred race horse after exercise. 

Allaam M. a, ELseady Y d,Nayel M. a, Elsify A.a, Salama A. b., Hassan H.a,  Hassan M.c and Kamar A. a
aDepartment of animal medicine and infectious diseases - Faculty of veterinary medicine - Sadat City branch - Minufyia University � Egypt.
b Department of animal medicine and infectious diseases- Faculty of veterinary medicine - Obihiro University � Japan.
c Department of animal medicine and infectious diseases - Faculty of veterinary medicine - Benisuef University � Egypt.
d  Department of physiology � Faculty of Vet. Medicine- Mansoura  University 

Abstract

Exercise physiology is the most useful information that make the race horse  such a super athlete and good managed. This study was carried out on twenty one thoroughbred race horses in order to evaluate physical fitness and performance through physical ( Heart rate, Respiratory rate, Body temperature and capillary refilling time ) , hematological ( RBCs, PCV, Hb, total  and Differential leucocytic count) and hematochemical  evaluation  ( TP, Albumin, AST, ALT,CK,LDH, Lactic acid, Glucose, Cholestrol,Na, K, Cl and Urea , Creatinine, Ca, P and Mg ).  Physical  examination and Blood samples were collected before and at 5, 15 and60 min. after 1600 meter  exercised. The results showed significant increase in all physiological , hematological and  hematochemical parameters 5 min after exercise that returned to basal levels after 60 min. rest.  The results can be useful index about horse performance, the effect of exercise on horse metabolism and helpful  in management protocols  of athletic  horses.
Key Words : physiology, hematology, biochemical ,  exercise,  Thoroughbred  horses.  








Introduction
The Thoroughbred racehorse is one of nature�s most gifted athletes, capable of utilizing nearly every muscle in its body when at a full gallop. One of the most important roles of research in equine physiology is to obtain new useful information on characteristics  that make the horse such a super athlete 1. Perhaps the most important change for an athletic horse is in the cardiovascular system, although during acute and intense training other important modifications arise 2. Exercise, in fact, can induce  variations in plasma biochemical constituents 3, 4.   The principal method to assess the efficacy of training is to verify the modifications of blood parameters relatively to the effort . Repetitive exercise induces a multitude of physiologic and  anatomic adaptations in horse,   these adaptive responses act to reduce the effect of the strain induced by the physiologic stressors associated with  exercise 5. In addition to physical modifications such as muscle remodelling, there are changes in blood constituents 6, and  these reflect the metabolic pathways and the functional processes involved in the particular athletic discipline 7.  Over the years, evaluation of   haemogram and plasma or serum biochemistry was used to assess the health status or function of a range of body  systems in the athletic horse 8.  Biochemical alterations are produced by various type of   exercise, and they reflect alterations in the functions of  different systems and in the type of energy utilized  5,9. .The massive metabolic demands of going from rest to a gallop in a matter of seconds require exceedingly high rates of ATP provision. Owing to the sluggishness of the aerobic system to provide the required ATP and the limited ATP that can be supplied via the phosphocreatine  system, the requirement is therefore for very high rates of glycolytic ATP production 10.
       Physical exercise can modify the animal's physiologic metabolism. This is because the different types of training, racing, transport, breed and temperament can produce variations in blood constituents levels 11. However, only few researches studied the responses during a specific  training period 9.  Although the changes occur after exercise were poorly studied so the aim of this research  was planned  to evaluate the modifications of some physiological and  heamatochemical parameters occurring after exercised thoroughbred horse at different interval of rest. 
Material and methods
1-Animals
Twenty one thoroughbred race horses (ten mares and eleven   stallion, the age  ranged from 3 to 5 years weighting  350 to 400 kg. The body condition score is 3 and height 146- 148 cm.). They   were proved to be clinically  healthy by clinical checkup. This study were carried out at shams and Aljazeera equestrian clubs and exercised  by  official trot with average speed 200 m / min. for 1600 m. distance.  Training and general animal care were performed by professional staff not associated with the research team. The horses were fed standard rations, calculated to fulfill all the nutritional requirements according to NRC.  
2- samples:
Blood samples were obtained  in duplicate from jugular vein by sterile needle before and 5, 15 and 60 minutes  after 1600 m. exercise from each animal. The first blood sample was anticoagulated with EDTA  for hematological examination. 
      The second blood sample is collected without anticoagulant in centrifuged tubes for serum collection that  stored at �20�C  for biochemical assay .
METHODS
A- Physiological and clinical parameters:
      Clinical examination and physiological parameters had been done to all tested horses before and 5, 15and 60 minutes after exercise according to the method described by Imren (1997) 12.                                                                                   
       
B- Hematological examination:  
      Complete hematological analysis was done according to the  method described by Schalm (1965)13 , while Haemoglobin was measured colorimetrically by using Hb kit that was produced by  Egyptian company for biotechnology according to method described by Tietz (1990) 14.
C- Biochemical analysis :
Total protein and albumin : 
      Total protein and albumin was measured in serum by UV -calorimetric  spectrophotometric method by using total protein kit that was  supplied  by vitro scient company according to method described by Grant et al ( 1987) 15. 
 Lactate dehydrogenase, Alanine aminotransferase and aspartate amino transferase  
alanine aminotransferase and aspartate amino transferase    Were measured in serum Calorimetrically by using kits that were  produced by vitro scient company while lactate dehydrogenase was measured by kinetic  method  by using  LDH kit that supplied  by Egyptian company for biotechnology  according to the method described by  Young ( 1990) 16.
Glucose and cholesterol  :
Serum  glucose was measured by Colorimetric method by using glucose kit that was produced by vitro scient company according to the method described by  Caraway  (1987) 17.
Serum Cholesterol       was measured by CHOD-PAP-enzymatic colorimetric method by using cholesterol kit that was  produced by Egyptian company for biotechnology according to the method described by   Elefson and Caraway (1976) 18.
Urea and creatinine
         Urea and creatinine were  measured in serum by colorimetric method by using urea and creatinine kits that were produced by Egyptian company for biotechnology according to the method described by   Tietz (1990) 14.
Bilirubin:
        Bilirubin was measured in serum by colorimetric method by using bilirubin kit that was produced by BioMed company according to the method described by  Walters et al (1970) 19.
Creatine kinase (CK) , Lactate, Sodium, potassium, Chloride
      Creatine kinase was measured by kinetic method while lactate, sodium, potassium and chloride  Were  measured in serum by colorimetric method by using their kits that were produced by Egyptian company for biotechnology      according to the method described by  Tietz (1999)14.
 7- Magnesium , Calcium and phosphorus .   
      Magnesium , Calcium and phosphorous   were measured in serum by colorimetric method by using their kits that were  supplied vitro scient company  according to the method described by    Thomas (1998) 20.                                                                                                    
D-Statistical Analysis
A p <0.05 was considered statistically significant . All results were expressed as mean � SE . the data were analysed using  ANOVA by using SPSS version 17 computer software package .  
Results
1-Clinical examination of thoroughbred race horse before and after 5, 15,60 minutes of 1600 m. exercise:
       Heart rate was  significantly increase from 42.85 � 0.31 to 180.70 � 0.89 beats/min  after 5 min before returning again to basal levels at 60 min. rest. Similarly, respiratory rate  started to significantly increase from 14.85 � 0.19  to  91.00 � 0.82 cycles/m  after  5 min  rest before returned  to basal value at 60 min rest. By the same way, body temperature significantly increased started from 5 min after exercises to reach to basal value at 60 min rest while  capillary refilling time increased significantly after 5 min. rest to reach   3.75 � 0.00 /second that  returned to normal levels after 60 minutes exercise as shown in (Table. 1).
2-Hematological changes  before and after 1600 m exercise.   

      Hematological data of  thoroughbred race horse after 1600 meters exercise were presented in table (2, 3). Heamogram  inclouding Red blood cells, PCV ,  Hb and total leukocytic count  showed significant increased after 5 min from exercise   before returned back to basal  levels within 60 minutes. In the same respect, differential leucocytic count appeared significant neutrophilia accompanied with lymphocytopenia at 5 min. rest before achieving pre-exercise data at 60 min

4-Biochemical analysis before and 5,15,60 minutes after 1600 meters exercise:
The hemato-chemical parameters of thoroughbred race horse before and after 1600 meter exercise was presented in table (4). The results appeared significant increase in all data reported at 5 min. after exercise than that detected before. All data were returning to basal levels after 60 min. rest. 
Discussion
The characteristics of racing horses have been essentially required for a rapid speed-up and short time to complete the competition. Thoroughbred horses have been selected for this sport for its own inheritance in greatest speed running among all animals. Clinical examination was used to  determine physical fitness and performance of horses before exercise and was done according to method described by Imren (1997)12.
The heart rate and respiratory rate of thoroughbred race horses showed significant increases  (P<0.05)  5 minutes after exercise then decreased gradually after 15 minutes till reach to normal baseline after 60 minutes ,similar result was observed by  Snow and Mackenzie1(997) 22 and Katz et al (2000)23. The increase of heart and respiratory rate  after exercise may be attributed to stimulation of sympathetic nervous system before starting competition included the process of warming-up, resulting in an increase in catecholamine (adrenaline) levels. It  increase metabolic rate, aerobic and anaerobic glycolysis and that the increase in metabolic rate leads to major changes in cardiovascular and respiratory functions in addition to using respiratory system to lose the heat that generated during exercise 24.
The rectal temperature of thoroughbred race horses showed significant increase (P<0.05)  5 minutes  after exercise then decreased gradually after 15 minutes till reach to pre-exercise baseline after 60 minutes . Many studies were recorded similar data  28,26,27. This increases may be attributed to  continues muscular contractions that requires a constant supply of energy that generated from fat metabolism and the Energy derived from the breaking down of ATP molecule is generally used to maintain body temperature, nerve and muscle functions of many vital organs 28,29.

       The capillary refilling of thoroughbred race horses showed significant increase (P<0.05)  5 minutes after  exercise then decreased gradually after 15 minutes till reach to pre-exercise state  after 60 minutes .This result was similar to data observed by Fritzsche and  Coyle (2000) 30, Morgan  and Funquist.(2002) 31 and McKeever (2002)32.  This significant increase in capillary refilling time may be attributed to hyperthermia that occurred when exercise is undertaken in hot and humid ambient conditions where  body temperature rises excessively, the demands of muscle metabolism and skin blood flow for heat dissipation arise concurrently resulting in dehydration as a proved by Lisa( 2011) 33.
        Hematological examination include RBCs count , ,Hb, PCV  total and differential leucocytic count  was used to determine the effects of exercise on the hemogram of thoroughbred race horses. The Red blood cells, hemoglobin and packed cell volume in this study showed significant increase (P<0.05) 5 minutes after  exercise then decreased gradually after 15 minutes till reach to baseline after 60 minutes. This result was compatible with that   recorded by Evans, (1994) 34 ,Andrews et al (1995) 35 and Thompson et al  (2001) 36. These  changes  may be attributed to releasing  of spleenic erythrocytes under the influence of catecholamine during exercise and haemoconcentration resulted from dehydration 36. 
 The  total count of white blood cells in this   showed significant increase at (P<0.05)   5 minutes after  exercise then decreased gradually at 15 minutes till reach to baseline at 60 minutes . This data  agreed with Rose and Hodgson (1982)37 and Snow et al. (1983) 38 , they  reported significant leukocytosis accompanied with exercises. Differential leucocytic count of thoroughbred race horses showed significant  neutrophilia , lymphocytopenia  and increase  in N/L ratio   (P<0.05) 5 minutes after exercise then returned to normal baseline after 60 minutes, this result agreed with Zobba et al (2011)64 and they return this  changes to  corticosteroid release.  The total protein, albumin of thoroughbred race horses showed significant increase (P<0.05) 5 minutes  after  exercise then decreased gradually after 15 minutes till reach to normal baseline after 60 minutes . These results were  the same that reported by  Sommardahl et al  (1994)39 and Stockham, and Scott (2002)40 . They  attributed that to redistribution of fluid and electrolytes from the vascular compartment to the tissue extra-cellular fluid spaces and decrease of plasma volume due to withdrawal of fluid from blood leading to heamoconcentration and dehydration.
       The Creatine kinase (CK) and aspartate aminotransferase (AST)of thoroughbred race horses  showed significant increase (P<0.05)  5 minutes after exercise then decreased gradually after 15 minutes till reach to normal baseline after 60 minutes,  this result was parallel to that observed by Hodgson and Rose, (1994)41 and Kobluk et al (1995 ) 42 . This increase of both serum enzymes (CK and AST ) is due to increase permeability of both enzymes from muscle cells due to muscular stress 43.
        The lactate dehydrogenase (LDH) of thoroughbred race horses showed significant increase (P<0.05) at 5 minutes rest  then decreased gradually at  15 minutes till reach to normal baseline after 60 minutes. Kratz et al (2002a) 44  and Tateo et al (2008) 45. were observed the same results and attributed that to releasing of  LDH from horse tissues after exercise, it is documented that the source have been mostly from muscles.
       The glucose level of thoroughbred race horses showed significant increase (P<0.05)  after 5 minutes rest   then decreased gradually after 15 minutes till reach to normal baseline after 60 minutes,  this result was similar to that reported by  Sim�es et al (1999) 46, and Nakata et al  (1999 ) 47 . These increases may be attributed to hyper activity of  sympathetic system and adrenaline release which activate hepatic glycogenolysis  48.
        The lactate level  of thoroughbred race horses showed significant increase (P<0.05)  5 minutes after  exercise then decreased gradually after 15 minutes till reach to normal baseline after 60 minutes, this result was similar to observed by Gollnik et al (1996) 49, Marlin and Nankervis, (2002 ) 50  and this  may be attributed to   anaerobic glycolysis  that accompanied intense exercise with  decreased ATP/ADP ratio and decreased  oxygen tension 48,51.   
       The sodium level of thoroughbred race horses showed significant increase (P<0.05)  5 minutes after  exercise then decreased gradually after 15 minutes till reach to normal baseline after 60 minutes, those  data opposed to  that reported by Carlson, (1992)52 and Nemec Svete et al  (2008) 53  while McCutcheon and Geor, (1998 ) 54and Goundasheva and Katsarova,( 2008 ) 55  was observed the same result and  they attributed that  to  HYPERLINK "http://en.wikipedia.org/wiki/Aldosterone" aldosterone release as the result of water deficit during exercise.  

       The potassium level of thoroughbred race horses showed significant increase (P<0.05) 5 minutes after  exercise then decreased gradually at 15 minutes till reach to baseline at 60 minutes , this data  was compatible to that recorded by Harris And Snow (1986)56 ,  Hodgson and Rose, (1994) 41 . They returned these changes to releasing of potassium from exercising muscles to extra- cellular space 
       The chloride level of thoroughbred race horses showed significant decrease (P<0.05) 5 minutes after  exercise then increased gradually after 15 minutes till reach to normal baseline after 60 minutes , this result was the same observed by  Mckeever et al (1991) 57. This may be attributed to depletion and are often a predisposing factor, along with dehydration, in fatigue, muscle cramps, colic, synchronous diaphragmatic flutter (�thumps�), diarrhea and other symptoms of exhausted horse syndrome  53. 
       The urea and creatinine of thoroughbred race horses  showed significant increase (P<0.05)  5 minutes after exercise then decreased gradually after 15 minutes till reach to normal baseline after 60 minutes and this result similar to that observed by Snow et al., (1983) 38, Hodgson and Rose, (1994)41 and Pringle, (1995) 58. This increase may be attributed to  extensive fluid loss in the sweat, the reduction in renal blood flow and glomerular filtration rate that leads to elevation of urea concentrations after exercise 59. In the same respect Hartlova et al (2010) 60  return this changes to increased production of creatinin from working muscle. So the change in serum creatinin cannot used as indicator for reduced glomerular filteration rate  59 .
The calcium and magnesium  levels of thoroughbred race horses showed significant decrease at (P<0.05)  5 minutes after exercise then increased gradually at 15 minutes exercise till reach to baseline at 60 minutes exercise this result agreed with Schryver et al. (1978)61 and this may be attributed to action of calcitonin possibly persisted, which might decrease the serum Ca concentration in this period and another alternative factor that affects serum Ca concentration in exercising horses indicated that approximately of Ca was lost through sweat during exercise. 
The phosphorous level  in this study   showed significant increase at (P<0.05)  5 minutes after exercise then decreased gradually at 15 minutes till reach to baseline at 60 minutes this result agreed with Yamada et al (1996) 62 and Arslan et al (2002)63 and they attribute this change to escaping  of phosphate from muscles during break down of high energy phosphate (ATP) during exercise.

Conclusion
Equine exercise physiology is important science for sport horses for evaluation of horse fitness. Because physiological and haematochemical parameters change with the performance, the interpretation of the values of athletes cannot be limited to the comparison with a static normal range, but in relation to the dynamic evolution of the values with the progression of training. Therefore, the present data can be useful to assess the status of an athlete and the degree of its training adaptability providing an opportunity to modify the training schedule to achieve the desired performance.
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64. Zobba R. Ardu M. Niccolini S. Cubeddu F. Dimauro C. Bonelli P. Physical, haematological, and biochemical responses to acute intense exercise in polo horses, JEVS, 2011, xxx 1-7.






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