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Journal of Equine Veterinary Science 32 (2012) 542-551 Journal of Equine Veterinary Science Original Research Effects of b-Hydroxy-b-Methylbutyrate and g-Oryzanol on BloodBiochemical Markers in Exercising Thoroughbred Race Horses Piotr Ostaszewski PhD, DVM a, Agnieszka Kowalska MSc a, Ewa Szarska PhD b,Piotr Szpota
nski DVM c, Anna Cywinska PhD, DVM d, Bo _zena Ba1asi
Tomasz Sadkowski PhD, DVM a a Faculty of Veterinary Medicine, Department of Physiological Science, Warsaw University of Life ScienceseSGGW, Poland b The General Karol Kaczkowski Military Institute of Hygiene and Epidemiology, Warsaw, Polandc Equine Veterinary Clinic, Łasieczniki, Poland d Faculty of Veterinary Medicine, Department of Preclinical Sciences, Warsaw University of Life Sciences, Poland In both the horse and the man, nutritional ergogenic aids have been used to improve Received 22 June 2011 physical ability in conjunction with an appropriate training regimen. Although training Received in revised form increases physical condition, the ease of taking a nutritional additive to improve training results explains the demand for supplementation, which may increase mechanical energy Accepted 20 January 2012 of work, delay onset of fatigue, or improve neuromuscular coordination. The purpose Available online 6 March 2012 of this study was to determine the effects of oral supplementation of b-hydroxy-b-methylbutyrate (HMB) and g-oryzanol (GO) on indices of exercise-induced muscle damage in Thoroughbred race horses. In this 32-week study, the horses were assigned to either a placebo, GO (3.0 g/d), HMB (15 g/d), or GO and HMB treatment groups. The supplements were administered for the first 16 weeks of the study during the training period before the racing season began. Blood samples were taken at baseline, and then during training, before exercise, immediately after exercise, and 30 minutes after exercise. Heart rate and Biochemical markers speed were monitored in each exercise session. Hematocrit, glucose, lactate (LA), creatinephosphokinase, and aspartate aminotransferase were measured before and after eachexercise session. Analysis of variance showed a significantly greater increase in post-exercise creatine kinase activity in placebo-supplemented group than in the other treat-ment groups, both in the training period and during the racing seasons (P < .05). Blood LAwas higher immediately after exercise in the placebo group compared with the supple-mented groups. In conclusion, supplementation with HMB and GO resulted in decreasedcreatine kinase and LA after exercise. These findings support the hypothesis that HMB andGO supplementation helps to prevent exercise-induced muscle damage.
Ó 2012 Elsevier Inc. All rights reserved.
endogenously in small amounts and has been shown toimprove gains in strength and lean body mass in humans b-Hydroxy-b-methylbutyrate (HMB) and g-oryzanol when associated with resistance training [1,2]. The efficacy (GO) are supplements used to enhance the effects of of HMB has been demonstrated in pathological conditions, training in exercising humans, dogs, and horses. A metab- where it has been reported to reduce muscle wasting olite of the amino acid leucine, HMB is produced associated with AIDS, trauma, and cancer cachexia [3-5].
More recently, HMB has been shown to decrease protein Corresponding author at: Piotr Ostaszewski, PhD, DVM, Faculty of degradation and increase protein synthesis [6]. In decreasing Veterinary Medicine, Department of Physiological Science, Warsaw muscle damage, HMB may also provide a source of cytosolic University of Life Sciences-SGGW, ul. Nowoursynowska 159, 02-776 HMG-coenzyme A for cholesterol synthesis and increase Warsaw, Poland.
E-mail address: [email protected] (P. Ostaszewski).
the availability of cholesterol for cell membrane synthesis.
0737-0806/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved.
doi:10.1016/j.jevs.2012.01.002 Author's personal copy
P. Ostaszewski et al. / Journal of Equine Veterinary Science 32 (2012) 542-551 This may result in an overall reinforcement of the sarco- lemma as well as the provision of valuable substrate for its Analyzed composition and calculated energy density of hay andconcentrates repair following muscle damage or injurious exercise [7].
This is evidenced by studies demonstrating that HMB leads to decreased markers of muscle damage following mechan- ically strenuous exercise, including lower activity of creatine DM, % of fresh matter kinase (CK), lactate dehydrogenase, and a decrease in muscle Crude protein, % of DM protein breakdown as indicated by serum 3-methylhistidine, Crude fat, % of DM Crude fiber, % of DM a direct marker of muscle protein degradation [8-10]. Other Crude ash, % of DM studies have shown that HMB reduces cancer-induced Net energy, MJ/kg DM muscle weight loss through attenuation of the ubiquitin- DM, dry matter.
proteasome proteolytic pathway [11], suggesting that HMBfunctions predominantly as an anticatabolic, rather thananabolic compound. However, a recent study has shown that 520
50 kg were studied at the Sluzewiec Racetrack HMB supplementation induces muscle hypertrophy in the training center (Warsaw, Poland). The horses were extensor digitorum longus and soleus muscles in rats via privately owned, and the experimental design and all mammalian target of rapamycin pathway [12], in addition to procedures were approved by the Ethical Committee in attenuating the depression in protein synthesis induced by Warsaw and by the owners of horses. The horses were the proteolysis-inducing factor [6]. In geldings fed an alfalfa- selected on the basis of a clinical examination and hema- based supplement containing 10 g of HMB per day during tological analysis, and horses with pathological conditions 6 weeks of low to moderate-intensity training followed by were excluded. The horses were also chosen on the basis of 6 weeks of high-intensity training, HMB supplementation similar racing performance as recorded from the previous resulted in a 10% improvement in treadmill endurance [13].
year's racing records. During a preliminary period of 2 This was followed by a study in racing Thoroughbreds weeks, all the horses were acclimated to a basal diet and where HMB-supplemented horses had reduced serum CK, were individually housed on straw in box stalls under maintained body weight better, needed less recovery time identical conditions. Each horse consumed 5 kg of oats, 1 kg between races, and had a better win rate. These effects were of a complete high-quality commercial feed (daily ration) most likely through a decrease in training and race-related distributed in three feedings, and 6 kg of hay that was muscle damage and increased aerobic ability, which administered daily ad libitum. The concentrate/forage ratio allowed for a quicker recovery after racing.
was 50/50. Table 1 shows the composition of the feedstuffs.
GO is a mixture of ferulic acid esters of sterol and tri- Total net energy intake in the daily ration was 135 MJ/ terpene alcohols extracted from rice bran, and is known to be a powerful inhibitor of iron-driven hydroxyl radicalformation; it has also been reported to possess antioxidantactivity in stabilizing lipids [14]. Because GO is insoluble in 2.2. Supplement and Placebo: Composition and water, a GO emulsion is used in supplementing humans, dogs, and horses. There are few studies in the peer-reviewed literature on GO, despite its apparent use as an The HMB alfalfa-based supplement (150 g/horse/d) was ergogenic aid. One study looked at resistance-weight- distributed in a pelleted form twice daily and provided for trained male athletes supplemented with 500 mg/d GO or a total of 15 g of CaHMB (Metabolic Technologies Inc, Ames, a placebo [15]. However, this study failed to show an effect IA). The placebo for this treatment consisted of 150 g of the of GO on training performance.
pelleted supplement matrix (dehydrated alfalfa meal, cane Thoroughbred horses undergo intensive training start- molasses, soy oil, soy lecithin, caramel flavor) but did not ing at a young age. The results of this training and their race contain HMB. GO was purchased from Oryza oil & Fat performance may be improved by the use of dietary chemical Co., Ltd (Aichi, Japan) as a crystalline powder.
supplementation. The use of supplementation may not only Three grams of GO was suspended extempore in 20 mL of improve performance but also improve muscle recovery rice bran oil and was top-dressed on the morning after a race or heavy training period. Therefore, the main concentrate (once a day). The placebo for this treatment objective of this study was to evaluate the effect of dietary consisted of 20 mL of regular cooking oil and was also given supplementation of HMB and GO on exercise parameters in as a top-dress with the morning concentrate.
horses trained from winter break to their maximal physical A groom verified the consumption of both the supple- performance at the start of the racing season. Our hypoth- ments and the placebo. All treatments were palatable and esis is that one or both supplements, either alone or in consumed by the horses, and no adverse reactions were combination, will decrease muscle damage and improve reported. The riders and trainers, as well as the technicians recovery, and thus improve overall performance.
performing blood sample analyses, were blinded to thetreatment allocation.
2. Material and Methods 2.3. Experimental Design After the 2-week acclimation period, four homogenous Twenty-four Thoroughbred racehorses ranging in age groups of six horses were chosen, with three mares and from 3-6 years (12 mares and 12 stallions) and weighing three stallions of similar age assigned within each group.

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P. Ostaszewski et al. / Journal of Equine Veterinary Science 32 (2012) 542-551 Fig. 1. Experimental design and sampling schedule.
The horses were fed the same diet as during acclimation, was excluded after exercise session III and one horse from and the supplement regimens were begun. The placebo HMB group after exercise session IV due to lameness.
group consumed 150 g of pelleted matrix without HMB,and 20 mL of cooking oil; the GO group consumed 150 g of 2.4. Blood Sample Collection and Processing pelleted matrix without HMB, and 3 g of GO suspended in20 mL of rice oil; the HMB group consumed 150 g of pel- Peripheral venous blood samples were taken by leted HMB supplement and 20 mL of cooking oil; and external jugular venipuncture every 4-week intervals, with the GO/HMB group consumed 150 g of pelleted HMB the final sampling at 16 weeks while the horses were in the supplement and 3 g of GO suspended in 20 mL of rice oil.
racing season. The first session had only one blood sample Oral administration of selected supplements was initiated taken in the morning, whereas the remaining sessions 4 weeks before the beginning of training season and las- consisted of three samples taken at the following intervals: ted for 16 weeks. The blood samples were taken between Rerest (before exercise), Eeexercise (after exercise), and February and September, and the exercise data were '30ehalf an hour later after being in an exercise walker collected between March and September.
(recovery). The blood samples were aspirated into 20-mL Four weeks after horses were assigned to dietary syringes and immediately transferred into sterile ethyl- treatments, they began a conditioning program as shown in enediaminetetraacetic acid tubes for hematological tests Fig. 1. During the entire training season, the horses were and into plain tubes for serum analyses. Glucose and lactate exercised 6 d/wk on a training course with a sand footing.
(LA) concentrations were determined immediately by The same exercise intensity was repeated every day. For the ejecting a drop of full blood onto single-use Accu-Chek first 3 weeks, the conditioning program consisted of 10 Active and BM-LA test strips (Roche Diagnostics Corp.
minutes of walking, 15 minutes of trotting at 250 m/min, Indianapolis). The ethylenediaminetetraacetic acid-treated and 4 minutes of cantering at 600 m/min, followed by 30 blood samples were kept in refrigerator (4C) and analyzed minutes of exercise on a horse walker. During the following within 6 hours after collection. Hematocrit (Hct) was weeks, only time and intensity of the exercise were counted with an automated hematology analyzer (Abacus changed so that at week 5, galloping time consisted of 5 Diatron, Hungary). The samples taken for serum were minutes of cantering at 500 m/min followed by 1 minute of promptly centrifuged, and the serum samples immediately galloping at about 750 m/min. Starting week 8, the inten- frozen and kept at 20C until analyzed. The serum sity was again increased to 5 minutes of cantering at 550 m/ samples were analyzed for creatine phosphokinase (CK) min, followed by 1 minute of galloping at 850 m/min. This and aspartate aminotransferase (AST) activity by a kinetic higher intensity conditioning program was continued until method, using a reagent kit (Pointe Scientific, Inc., Canton).
the beginning of the racing season (end of June). After theconditioning program, all horses participated in the races 2.5. Statistical Analysis for the next 12 weeks. During this time (racing season), thehorses were also exercised, but with the intensity modified Results are expressed as means
standard errors of to the racing schedule. Exercise during the racing season mean. Exercise session II (n ¼ 6 for each group) was was designed to maintain the horses' current condition.
considered as beginning of training, and the data from Additional training was withheld on the day of the races.
exercise sessions III, IV, and V were pooled for each group The training was monitored using heart rate (HR) and referred to as the training season. The last exercise monitors RS800 on Polar Equine Wearlink W.I.N.D. (Polar session (VI) was referred to as the racing season. The data Electro Oy, Finland) and G3 GPS. HR monitor consists of were analyzed for differences between blood indices and a receiver and two electrodes placed under the girth. Model HRs at various stages using a repeated-measures analysis of RS800 allows simultaneous control of the HR values and variance, and pairwise comparisons were made using speed of the horse. Twenty-two of the horses successfully Tukey test. Probabilities of P < .05 were considered statis- completed the entire study. One horse from the GO group

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P. Ostaszewski et al. / Journal of Equine Veterinary Science 32 (2012) 542-551 groups (Table 2). Hct was, on average, approximately 41% inall groups. Resting blood glucose and LA levels alsoremained unchanged during the study (Table 3, Fig. 3,respectively).
There were no differences in serum CK activity among treatment groups at the beginning of the dietary supple-mentation (Fig. 4). However, after 4 weeks of training,resting serum CK was significantly (P < .05) decreased inthe GO þ HMB-supplemented horses compared with theplacebo-supplemented horses. During the remainder of thetraining and racing seasons, resting serum CK was notsignificantly different from pretraining values for any of thegroups.
Resting AST activity was the same among the groups at the beginning of an experiment (sampling session I, Fig. 5).
After 4 weeks of training, resting AST activity had decreasedin all groups, and a significant 30% decrease was observedin the HMB-supplemented horses (P < .05). During theremainder of the training season, pre-exercise AST activitywas still significantly lower in horses receiving HMB whencompared with the beginning of supplementation (P < .05).
During the racing season, pre-exercise AST activity wasincreased in the GO-supplemented horses when comparedwith the training season (P < .05).
3.3. Supplements Versus Placebo After Exercise Exercise produced a significant increase in Hct levels in all the groups (P < .05); however, no differences wereobserved between the placebo and the supplementedgroups (Table 2). On average, a 29% increase was observedcompared with the resting values at the beginning of die-tary supplementation, a 36% increase was observed duringtraining season, and a 29% increase was observed duringracing season. Exercise did not affect blood glucoseconcentration in any group except at the beginning oftraining (sampling session II), where horses from HMB- Fig. 2. Minimum and peak heart rate (beats/min) as well as maximumspeed (km/hr) determined in Thoroughbred racing horses at the beginning of training (sampling session II; n ¼ 22), during the training season glucose level (Table 3, P < .05). Thirty minutes after exer- (sampling sessions III, IV, and V; n ¼ 66), and during the racing season cise, the glucose level in the HMB-supplemented group (sampling session VI; n ¼ 22). For 16 weeks, the horses were supplemented was still significantly higher than in GO þ HMB group with an oral placebo (n ¼ 6), g-oryzanol (GO, n ¼ 5), 3-hydroxy-3- methylbutyrate (HMB, n ¼ 5), or GO þ HMB (n ¼ 6).
< .05). During the training season, exercise resulted in a significant increase in glucose level only in HMB- supplemented group (P < .05). Exercise also resulted ina significant increase in blood LA concentration in all 3.1. Exercise Monitoring groups (Fig. 3) during the beginning of training (P < .05),except in the HMB-supplemented group. As the training Resting and peak HR as well as maximum speed did not season progressed, the exercise-related increase in LA differ between placebo and experimental groups (Fig. 2).
became significant in the HMB-supplemented group Resting HR during all exercise sessions was in the range of (P < .05). In all horses, 30 minutes after exercise, blood LA 32-37 beats/min. Average peak HR was approximately 200 was only slightly elevated when compared with the cor- beats/min for all exercised horses and was not affected by responding pre-exercise values. During the training season the length of training. Maximal speed increased from (sampling sessions III, IV, and V), the postexercise (E) average 38 km/hr at the beginning of training to about increase in blood LA was significantly lower in GO, HMB, 50 km/hr at the end of the racing season. These data and GO þ HMB groups when compared with the placebo confirm that training exertion was similar and intense in all group (P < .05). Exercise sessions performed at the beginning of training, after 4 weeks of dietary supple-mentation, induced an increase in serum CK activity in all 3.2. Supplements Versus Placebo at Rest groups. However, only in the GOþHMB supplementedgroup was the increase significantly different 30 minutes After 4 weeks of dietary supplementation, no change in following the end of exercise (P < .05). During the training resting hematocrit (Hct) values was observed among the season, a significant exercise-related increase in CK was Author's personal copy
P. Ostaszewski et al. / Journal of Equine Veterinary Science 32 (2012) 542-551 Table 2Hematocrit (%) determined in jugular venous blood of 22 horses at the beginning of training (sampling session II; n ¼ 22), during the training season(sampling sessions III, IV, and V; n ¼ 66), and during the racing season (sampling session VI; n ¼ 22) at rest before (R), immediately after exercise test (E), and30 minutes later ('30); for 16 weeks, horses were treated with an oral placebo (n ¼ 6), GO (n ¼ 5), HMB (n ¼ 5), and GO þ HMB (n ¼ 6) Group Effect Exercise-Time Time Period Nontraining Beginning of Beginning of training R 42.36
4.38o 42.05
3.89o 44.45
5.12o n.s.
54.55
7.57p 54.62
6.13p 55.07
5.76p n.s.
'30 42.98
2.74o 44.60
4.55o 45.73
4.56o n.s.
42.06
2.74o 40.45
3.09o 42.58
4.63o n.s.
58.03
6.31p 55.28
5.23p 56.22
5.40p n.s.
'30 46.26
4.63r 44.93
3.26r 46.34
4.62o n.s.
44.00
3.96o n.s.
51.55
11.57 54.93
6.55p 58.32
7.02p n.s.
‘30 46.63
4.13 46.27
3.18op 46.75
4.83op 49.68
5.38o n.s.
GO, g-oryzanol; HMB, b-hydroxy-b-methylbutyrate.
Data are shown as means
SEM.
For general comparison, P values of group supplements or time effect are indicated in separate columns. Differences between the placebo and experimentaltreatment groups receiving supplements are significant (P  .05) if the first superscript is different (a, b, or c for within-line comparisons); differencesbetween sampling time (R, E, ‘30) during each exercise session are significant (P  .05) if the first superscript is different (o, p, or r for within columncomparisons); differences between training periods (beginning of training, training, and racing seasons) are significant (P  .05) if the first superscript isdifferent (x, y, or z for within column comparisons).
* Significant for P  .05; n.s., nonsignificant.
observed in the placebo, GO, and GO þ HMB groups activity 30 minutes after exercise than the placebo- (P < .05), but not in the HMB-treated horses. The placebo- supplemented horses (sampling sessions III, IV, and V, supplemented group had the largest postexercise increase, Fig. 5). During the racing season, serum AST activity 30 45% above pre-exercise values (Fig. 4, P < .05), which minutes after exercise was significantly higher in horses fed was also significantly higher than the HMB- and GO- GO when compared with the same horses during the supplemented groups 30 minutes after exercise (P < .05).
training season (P < .05).
Additionally, during the racing season (sampling sessionVI), CK activity remained significantly greater than that of the placebo-supplemented group 30 minutes after exercise(P < .05).
The present study was the first study to determine Exercise did not significantly affect serum AST activity in whether dietary supplementation with either HMB or GO any treatment group; however, placebo-supplemented alone, or in combination, would affect indirect markers of horses tended to have increased postexercise AST activity muscle damage and fatigue in Thoroughbred horses during during the beginning of training as well as during a training 16 weeks of training in preparation for the racing season.
season (Fig. 5). During the training season, horses supple- Currently, little data are available on the effects of these mented with both GO and HMB had significantly lower AST supplements in horses, with only one study describing the Table 3Glucose (mmol/L) determined in jugular venous blood of 22 horses at the beginning of training (sampling session II; n ¼ 22), during the training season(sampling sessions III, IV, and V; n ¼ 66), and during the racing season (sampling session VI; n ¼ 22) at rest before (R), immediately after exercise test (E), aswell as 30 minutes later ('30); for 16 weeks, horses were treated with an oral placebo (n ¼ 6), GO (n ¼ 5), HMB (n ¼ 5), and GO þ HMB (n ¼ 6) Beginning of training '30 4.75
0.34ab 4.55
0.31ab 5.00
0.46a 4.71
0.51op 4.71
0.39 Data are shown as means
SEM.
For general comparison, P values of group supplements or time effect are indicated in separate columns. Differences between the placebo and experimentaltreatment groups receiving supplements are significant (P  .05) if the first superscript is different (a, b, or c for within-line comparisons); differencesbetween sampling time (R, E, '30) during each exercise session are significant (P  .05) if the first superscript is different (o, p, or r for within columncomparisons); differences between training periods (beginning of training, training, and racing seasons) are significant (P  .05) if the first superscript isdifferent (x, y, or z for within column comparisons).
* Significant for P  .05; n.s., nonsignificant.

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P. Ostaszewski et al. / Journal of Equine Veterinary Science 32 (2012) 542-551 Fig. 3. Blood lactate (mmol/L) in 22 horses at the beginning of training(sampling session II; n ¼ 22), during the training season (sampling sessionsIII, IV, and V; n ¼ 66), and during the racing season (sampling session VI; n ¼22) at rest before (R), immediately after exercise test (E), and 30 minuteslater ('30). For 16 weeks, the horses were treated with an oral placebo (n ¼6), GO (n ¼ 5), HMB (n ¼ 5), or GO þ HMB (n ¼ 6).
Fig. 4. Serum creatine phosphokinase (CK) activity in 22 horses at the effect of HMB on the physiological response to exercise in beginning of training (sampling session II; n ¼ 22), during the training horses. Miller et al. [13] fed horses an alfalfa-based season (sampling sessions III, IV, and V; n ¼ 66), and during the racing supplement twice daily containing a daily dosage of 10 g season (sampling session VI; n ¼ 22) at rest before (R), immediately after of CaHMB. In our study, we administered 15 g of CaHMB exercise test (E), and 30 minutes later ('30). For 16 weeks, the horses weretreated with an oral placebo (n ¼ 6), GO (n ¼ 5), HMB (n ¼ 5), or GO þ HMB daily, split between the morning and evening rations. This dosage provided w30 mg CaHMB/kg bw d1, as the horsesweighed approximately 500 kg, and is closer to the rec-ommended dosage of 38 mg/kg bw d1 for humans [1].
between training stress and recovery. If the next training Because there were no data concerning the daily require- session is applied without sufficient time for recovery, ment for GO in horses, we decided to use 3 g of GO decreases in the performance occur in the form of earlier administered once daily. This was similar to the 500-mg onset of fatigue within each session [16,17]. In our study, GO/d dosage previously administered to humans [15].
the total length of the training program before competitionwas 16 weeks. This was longer than the 4-6 weeks rec- 4.1. Supplements Versus Placebo at Rest and After Exercise ommended for horses aged >3 years [16], but we wanted toachieve a moderate pace in improvement in performance.
The theory of intense training is that a single exercise The horses were exercised 6 d/wk, with the intensity of session will lead to fatigue and cellular damage, which in training increasing as the study progressed. The data turn results in a short-term adaptive response [16]. When indicated that our methods used for training the Thor- exercise is performed regularly, and the training stimulus is oughbreds under field conditions were satisfactory for increased gradually, the adaptation that occurs during the maximization of physiological adaptations within the recovery period of a single exercise session leads to an animal's body. HRs at rest ranged from 32 to 37 beats/min overall improvement in performance. When training is too and were similar to the values obtained by Harris et al. [18].
vigorous and/or rest periods between training sessions are Peak HRs during cantering/galloping were approximately too short, performance is reduced because of an imbalance 200 beats/min and remained the same during both the

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P. Ostaszewski et al. / Journal of Equine Veterinary Science 32 (2012) 542-551 Fig. 5. Serum aspartate aminotransferase (AST) activity in 22 horses at the beginning of training (sampling session II; n ¼ 22), during the training season(sampling sessions III, IV, and V; n ¼ 66), and during the racing season (sampling session VI; n ¼ 22) at rest before (R), immediately after exercise test (E), and 30minutes later ('30). For 16 weeks, the horses were treated with an oral placebo (n ¼ 6), GO (n ¼ 5), HMB (n ¼ 5), or GO þ HMB (n ¼ 6).
training and the racing seasons. At this value, most horses accompanied by an increase in maximal speed, which in are close to the point of onset of blood LA accumulation, turn indicates a physiological adaptation to the exercise.
and it is suggested as a reference point for comparison of Because the horses in the present study were in a familiar cardiovascular capacity [19,20]. This sustained peak HR was environment and were handled and ridden by the regular Author's personal copy
P. Ostaszewski et al. / Journal of Equine Veterinary Science 32 (2012) 542-551 full-time staff, the risk of excitement influencing the HR using an internal conversion factor. The present study was minimized.
showed a significant increase in blood LA at the beginning In our experiment, increases in LA concentrations were of exercise in all treatment groups except the groups sup- evident after all training sessions. Thus, the intensity of plemented with HMB. Throughout the training, post- exercise during training sessions in our study was high and exercise blood LA remained lower in GO, HMB, and GO þ may be compared with the values reported in the literature HMB groups when compared with the placebo group, and [20,21]. This observation was confirmed by changes in Hct continued to be lower during racing season. These data levels, which reflect mobilization of splenic erythrocytes.
support the concept of a protective role of HMB and/or GO Hct levels increased significantly after all training sessions in counteracting the accumulation of LA; however, from the and did not differ between horses from either the placebo present study, it is impossible to tell whether this was the or experimental groups. The extent of the increase in Hct result of a concomitant reduction of intramuscular acidosis values depends on exercise intensity and has a linear or a shortage of fuel, especially depletion of glycogen stores.
relation to the speed of the exercise, up to a maximum of In fact, 30 minutes after exercise, blood LA concentrations 60%-65% [22]. Our study saw similar significant exercise- returned to near pre-exercise values in all horses indicating related increases in Hct levels.
fast LA removal from blood.
Changes in blood glucose concentration are also CK is a muscle-specific enzyme with a relatively short dependent on the intensity of exercise. Although plasma half-life in serum, that is, w2 hours. Increased serum CK glucose concentration decreases during prolonged exercise activity is used as an indicator of muscle damage or injury.
(>3 hours), studies with short intense exercise have shown Resting plasma level in horses should not exceed 200 U/L.
both decreases and increases, depending on exercise The CK activity increases and decreases through larger intensity and training and feeding status of the horse [23].
ranges earlier at the beginning of training season, and The exercise-associated increase in hepatic glucose output tightens into a narrower range when the horse is attending is mainly mediated through a decrease in the insulin:glu- good fitness levels required for racing. Several studies have cagon ratio, whereas the rate of uptake and utilization in shown a direct relationship between the levels of HMB exercising muscle is restrained by increases in circulating achieved after supplementation and improved nitrogen epinephrine resulting in an elevation in blood glucose retention. In humans, Nissen et al. [8] demonstrated a dose- concentration [24]. In our study, postexercise blood glucose dependent response to oral administration of HMB given at concentration remained within reference ranges in horses; either 1.5 or 3 g/d, with the higher dosage resulting in however, HMB supplementation significantly increased a greater decrease in serum CK. Nissen et al. also observed blood glucose concentration during the training and racing a decrease in serum 3-methylhistidine, an indicator of seasons. This may suggest a glucose-sparing effect of HMB.
muscle protein breakdown. Dietary supplementation of Anaerobic energy production is essential for the production 3.0 g HMB/d in individuals undergoing intense endurance of muscular tension when the demand for energy is greater exercise resulted in decreased CK and LA dehydrogenase than can be provided aerobically, or when oxygen is in after a prolonged 20-km run [9]. In our studies, 15 g of short supply. The largest source of anaerobic energy during CaHMB/d was administered to horses and resulted in the intensive exercise of short duration is from the a significant attenuation of the exercise-related increase of glycolytic pathway. Although the yield of adenosine-5'- plasma CK, an indicator of an alteration in membrane triphosphate from 1 mole of degraded glucose is only 2-3 permeability. Taken together, the aforementioned studies moles, muscle has a high glycolytic capacity, and the two indicate a benefit of having more HMB available to the end products of the glycolytic reactions, pyruvate and muscles during intense training in horses.
hydrogen, combine to form LA. After cessation of exercise, AST has a much longer half-life than CK, approximately the rate of oxygen consumption remains elevated, and 1 week, and therefore reflects muscle changes over several blood LA concentration continues to increase [23]. There is days or even weeks. The activity of AST in horses is much a point where LA efflux mechanisms are probably saturated higher than in other animals; AST is less specific for muscle and rapid accumulation of intracellular LA leads to than for CK, as AST is found in many tissues and organs.
muscular acidosis [25]. Further high-intensity contractions Muscle use affects AST level, and an increase in plasma AST cause loss of intracellular K with the accumulation of activity has been observed in response to exercise [31]. This extracellular Kþ, which is associated with muscle fatigue.
increase is related either to overt damage or to a change in Therefore, LA itself cannot be considered as an indicator of the muscle fiber membrane, causing a transient increase in fatigue, as has been stated in some earlier studies [26]. LA, permeability. Increased AST has been shown to occur however, contributes to fatigue by increasing muscular without any tissue destruction. Moreover, working horses acidosis. Usually, the highest LA concentrations are seen have approximately 60% higher AST activity compared with 2-10 minutes after exercise. Blood LA concentration has horses that are at rest for several days [32]. At the begin- been used as an indicator of training intensity and perfor- ning of our study, resting serum AST activity was typical for mance [27]. Blood LA assessment in the athletic horse is race horses (300-360 U/L). Four weeks of dietary supple- necessary to evaluate the onset of LA accumulation as an mentation resulted in a significant decrease of resting AST indicator of the aerobic capacity [28] and is useful for activity in horses receiving HMB. We observed trends for assessing fitness in equine athletes [29]. In equines, LA may a postexercise increase in AST activity only in horses be stored in red blood cells [30], and so measuring LA in receiving the placebo supplement, which may indicate the whole blood is necessary for determining total LA exercise-associated minor muscle damage in that group. In production. We used the Accusport technique to determine contrast, a significant decrease in resting AST activity in plasma LA, which was then converted to whole blood LA horses fed HMB may suggest the potential effectiveness of Author's personal copy
P. Ostaszewski et al. / Journal of Equine Veterinary Science 32 (2012) 542-551 this leucine metabolite to reduce muscle damage or protein Recently, Kreider et al. [36], in the official position paper of the International Society of Sports Nutrition, rated In our study, GO and HMB administered either together HMB in the second highest category of possible effective or separately did not counteract the postexercise increase in plasma CK activity during the training season. This enhancement in human athletes. Although HMB has gained increase was, however, significantly less than in exercised the reputation of an effective dietary supplement for horses from placebo group. Similar differences were training humans, more studies are needed to confirm the observed during the racing season. Therefore, both GO and effectiveness of its supplementation in sport horses. In the HMB may attenuate muscle CK leakage; however, their same paper, based on available literature, GO was classified mechanism of action may be different. Oxidative stress is as an apparently not effective dietary supplement.
a detrimental imbalance in the oxidativeeantioxidativesystem in cells and may damage DNA and cell membranes, Increased oxidation during exercise may be related to This field study, performed on 22 trained Thoroughbred muscle enzyme leakage and microtrauma, hydration status, horses, is the first showing that dietary supplementation and animal welfare. Therefore, the use of GO as a potent with GO and HMB may significantly improve training antioxidant for human athletes as well as for horses is results by decreasing muscle damage caused as a result of widespread. So far, two studies in horses have failed to the intensity of the training. The current study has shown demonstrate any reduction in CK after supplementation that GO does not significantly affect performance-related with the antioxidants vitamin C [33] and a-tocopherol [34].
physiological parameters in training Thoroughbred race Similarly, Piercy et al. [35] found no attenuation in the CK horses; however, when GO is supplemented with HMB, the increase in exercising sled dogs as a result of feeding an training results in increased performance outcomes, such antioxidant supplement containing vitamins C, E, and b- as decreased muscle damage and improved recovery.
carotene. The results of the present study would be the first Further studies investigating the effects of ergogenic to indicate that GO, a powerful inhibitor of reactive oxygen supplements in training and racing horses should be species formation, may reinforce cell membrane and conducted to validate the use of these ergogenic supple- decrease its permeability with less CK released into the ments in horses.
plasma. The results of the present study would seem to bein agreement with our observations that horses receiving GO had significantly lower postexercise total antioxidantstatus and thiobarbituric acid reactive substance level than The study was supported by the State Committee for horses from other groups (unpublished data).
Scientific Research, Poland (grant number N N308 3076 33 In contrast to GO, HMB is not considered as an antiox- for P.O.). The authors thank Mrs Ma1gorzata Podgurniak idant but attenuates the loss of muscle mass and function and Dr John C. Fuller for their assistance with the manu- in various conditions such as resistance exercise training, script preparation.
cancer, and AIDS [3,4,8]. It is also a substrate for cellmembrane cholesterol synthesis, and therefore HMB may help stabilize cell membranes during intense exercise.
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