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Aerobic Training, But Not Creatine, Modifies Longissimus Dorsi Muscle Composition
REFEREED
ORIGINAL RESEARCH Aerobic Training, But Not Creatine, Modifies Longissimus Dorsi Muscle Composition
Flora H.F. D’Angelis, PhD,a Marcı´lio D.S. Mota, PhD,b Eduardo V.V. Freitas, MS,a Guilherme C. Ferraz, MS,a Andre´ R. Abraha ˜o, PhD,b Jose´ C. Lacerda-Neto, PhD,c and Antonio Queiroz-Neto, PhDa
ABSTRACT The goal of this study was to investigate by means of an ultrasound examination the composition of the longissimus dorsi muscle in 12 purebred Arabian horses submitted to aerobic training for 90 days, with and without creatine supplementation. Creatine supplementation was carried out by daily administration of 75 g creatine monohydrate mixed into the ration during 90 days of training. Physical conditioning was conducted on a high-performance treadmill, and training intensity was stipulated by calculating the velocity at which blood lactate reaches 4 mmol/l, determined monthly for each animal. The individual intensity of physical force was established at 80% of velocity that resulted in a blood lactate level of 4 mmol/l. The cross-sectional area and the thickness of the layer of fat of the longissimus dorsi muscle were measured for each group at the end of 8 months of inactivity and after 30, 60, and 90 days of training without (control groups) or with creatine supplementation (experimental groups). The results showed that aerobic training combined with or without creatine supplementation caused significant longissimus dorsi hypertrophy and reduction in the thickness of the fat layer. No beneficial effect from prolonged creatine supplementation on the body composition of horses was detected. Keywords: Aerobic training; Creatine; Horse; Longissimus dorsi; Ultrasound
INTRODUCTION Various ergogenic substances have been employed to improve the athletic performance of horses, including creatine, a substance widely used as a nutraceutical in various From the Departamento de Morfologia e Fisiologia Animal, Faculdade de Cieˆncias Agra´rias e Veterina´rias (FCAV), Sa˜o Paulo State University (UNESP), campus de Jaboticabal, Jaboticabal, SP, Brasil a; Departamento de Melhoramento e Nutric¸a˜o Animal, Faculdade de Medicina Veterina´ria e Zootecnia, UNESP, campus de Botucatu, SP, Brasilb; and Departamento de Clı´nica e Cirurgia Veterina´ria, FCAV, UNESP.c Reprint requests: Antonio Queiroz-Neto, PhD, Via de Acesso Prof. Paulo Donato Castellane, 14884-900, Jaboticabal, SP, Brasil. 0737-0806/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.jevs.2007.01.008
118
equestrian sports. Creatine is an organic compound that is abundant in skeletal muscle and plays a central role in the availability of energy in muscles.1 Creatine is phosphorylated by creatine kinase, forming creatine phosphate, which provides the phosphate group necessary for rephosphorylation of adenosine diphosphate (ADP) to adenosine triphosphate (ATP) and thereby contributes to energy metabolism and muscle contraction.2 Creatine supplementation in humans at high doses for short periods (1 week) results in a greater capacity to achieve intermittent maximum exercise.3,4 Creatine supplementation for a prolonged time has been demonstrated to stimulate muscle hypertrophy during resistance training in sedentary females.4 Creatine also increases body weight, as shown in a study in human athletes.5 Conversely, no influence was observed in the concentration of creatine in plasma or in the transport of creatine in muscles of horses after a daily intake of 50 g creatine monohydrate for 7 days,6 which agreed with previous studies on creatine supplementation in horses.7,8 These findings justify the need for investigations into the evaluation of body composition and creatine supplementation in the diet of athletic horses. Various regions of the body in an animal can be measured in the evaluation of body composition. The longissimus dorsi muscle was chosen for ultrasound measurements in this study because it is easy to access and provides good ultrasonographic images. In addition, this muscle is highly worked during exercise, where it is responsible for the extension and flexion of the vertebral column and, at maximum extension, for maintenance of posture, elevation and support of the head, and supporting the weight of the rider. At least one study9 demonstrated a significant relationship between the size of the cross-sectional area of the longissimus dorsi muscle and the number of victories scored by the animals in races. Finally, the apparent existing contradictions and doubts regarding utilization of creatine as a dietetic supplement clearly indicate the need for additional studies of this substance, before being able to prescribe it as a complementary food for athletic horses. Few studies have been conducted aimed at examining the relationship between ultrasound measurements of the longissimus dorsi muscle and the physical conditioning of Journal of Equine Veterinary Science
March 2007
athletic horses in different sport modalities. Therefore, the objective of the current study was to conduct an ultrasound examination of the longissimus dorsi muscle to determine the cross-sectional area and thickness of the layer of fat in purebred Arabian horses submitted to endurance training with and without creatine supplementation.
MATERIALS AND METHODS Animals and Handling Twelve purebred Arabian horses were used, both geldings (n ¼ 5) and mares (n ¼ 7), with an initial body weight (BW) of 391.0 25.4 kg (mean SD) and a mean age of 8.6 3.3 years. The animals selected for study were evaluated by clinical examination and laboratory tests such as complete blood count and blood biochemistry. They had no signs or symptoms compatible with skeletal muscle lesions, and were deloused and dewormed with ivermectin (Ivotan LA, Intervet, Sa˜o Paulo, Brazil) at intervals of 40 and 60 days, respectively. The 12 horses were kept in one paddock for 8 months of inactivity and then were divided randomly into two pre-trained groups (C0 and CR0). Both groups were subjected to aerobic training for 30, 60, and 90 days, but only one group (CR0) received 75 g creatine monohydrate (Vetnil, Louveira, SP, Brazil) per animal per day for the same period (CR30, CR60, and CR90). The other group served as the control group (C30, C60 and C90) and received no supplementation. The animals were kept in a paddock of Brachiaria sp. grass, with ad libitum access to water and supplementation with Cynodon sp. hay and mineralized salt (Agromix, Jaboticabal, SP, Brazil). Creatine was given individually in the stall, and mixed with half of the daily commercial caloric ration (Nutriage MIX Guabi, Campinas, SP, Brazil) followed by the rest of the ration to make sure that all the creatine was ingested by each horse. The fed formulation was: 12% crude protein, 2% ether extract, 12% crude fibers, 12% mineral matter, 1.8% calcium, 0.5% phosphorus, and 2,990 kcal/kg of digestible energy. To determine the daily quantities (2.5–3.8 kg/animal) of commercial diet furnished, the animals were evaluated monthly for BW and body condition score (BCS). BCS was determined by visual appraisal and palpable fat cover at six areas of the body of the horse.10 The areas selected as being indicative of changes in stored body fat were the lumbar spinous processes, ribs, tailhead, area behind the shoulder, neck, and withers. The BCS was evaluated on a scale of 1 to 9, with 1 being extremely emaciated and 9 being extremely fat. The experimental protocol was approved by the university’s Institutional Animal Care and Use Committee. Physical Conditioning Physical conditioning of each animal was performed in a high-performance treadmill (Galloper 5500, Sahinco Ltda, Palmital, Sa˜o Paulo, Brazil). Before initiation of the training program, the horses were submitted to a 30-day adaptation to handling period. The training program was Volume 27, Number 3
conducted exclusively on the treadmill. The intensity (velocity) of the training was set at 80% of Vlac4 (velocity at which the blood lactate concentration reached 4 mmol/l) calculated in an incremental exercise test. At the end of each training period (30 days), a new exercise test was conducted to establish a new Vlac4. After a warm-up period of 4 minutes at 4 m/second, the treadmill was inclined at 10% and the speed was gradually increased at 2-minute intervals to 6, 8, and 10 m/second. The blood samples were collected 15 seconds before the end of each step by means of an intravenous catheter fixed in the left jugular vein. After each blood sampling, the whole system was washed out with 2.5% heparin solution. For this reason, 2 ml blood were discharged at the beginning of each collection. The samples were immediately analyzed for lactate concentration by a portable digital lactometer (YSI 1500 Sport L-Lactate Analyzer, YSI Incorporated, Yellow Springs, OH). The individual Vlac4 was determined graphically using an exponential regression. The training velocity was thereby determined monthly for each animal. The frequency of training consisted of the performance of exercises three times per week (alternate days). In the first month of training, each animal exercised for 10 km in a mean time of 50 minutes. In the second month, the distance run in each exercise was increased to 15 km, with a mean duration of 60 minutes. In the last month, all horses exercised 20 km per session, with a mean duration of 80 minutes. In addition, a speed play type of training, which involves sudden, rapid, relatively short bursts of speed interspersed throughout the exercise bout, was instituted once per week with the aim of stimulating tissues not addressed by slower exercise and increasing the horse’s metabolic capabilities. The protocol consisted of a warming period of 10 minutes at 4 m/second followed by 10 more minutes with the treadmill inclined (10%) at the speed of 8 m/second. After that, the speed was reduced to 2.5 m/ second for 5 minutes without any inclination of the treadmill, and then it was inclined to 5% and the speed increased to 10 m/second for 5 minutes. After another decrease in the speed and inclination, the speed was increased to 5 m/second at a 10% inclination for 20 minutes. The last increase in the speed to 12 m/second for 3 minutes was performed. A warm-down period of 10 minutes at 2 m/ second without inclination concluded the task. This protocol was adopted because some authors7,11 have recommended strenuous training sessions as part of the endurance training to develop fitness for fast exercise. Ultrasound Measurements The effects of training and creatine supplementation on skeletal muscles were determined by ultrasound measurements of the cross-sectional area (CSA, cm2) and fat layer (FL, mm) of the longissimus dorsi muscle in groups C0, C30, C60, C90, CR0, CR30, CR60, and CR90. These were performed using a Pie Medical-scanner 200 ultrasonograph (model 51B04UM02 3.5/5 Mhz DF TRD Linear, USA) equipped with a specific computer program (Software Eview, Echo Image Viewer, Maastricht, The 119
a C0, animals before training; CR0, animals before training and supplementation with creatine; C90, animals trained for 90 days and not supplemented with creatine; CR90, animals trained and supplemented with creatine for 90 days. Means in the same row followed by different letters (A, B) differ significantly (P < .05) according to Tukey’s test.
2.47B 2.32 2.79B 2.31 3.00B 0.83 4.60A 0.91 4.62A 0.91
3.03B 0.74
2.61B 0.90
2.69B 1.87
6 89.79B 2.32 6 88.81B 2.32 6 88.70B 5.00 6 76.90A 7.20 6 69.60A 1.83
Number of horses Cross-sectional area (cm2) Layer of fat (mm)
6 83.58B 4.35
6 83.23B 2.94
6 85.35B 1.87
CR90 C90 CR60 C60 CR30 C30 CR0 C0 Variable
90 d 60 d 30 d 0d Experimental Groupa
Table 1. Means ( SD) of morphometric variables studied in the longissimus dorsi muscle obtained by ultrasound examination of 12 purebred Arabian horses, submitted to aerobic training for 30, 60, and 90 days, with and without creatine supplementation 120
Netherlands, Copyright Pie Medical, 1996) for muscle studies. This type of apparatus is specialized for B-mode and has the ability to produce images of moving objects almost instantaneously. The measurements were carried out at the height of the last ribs on the left side with the animal in the upright position and the longissimus dorsi muscle relaxed. Initially three images were obtained for each animal at each of the four occasions (0, 30, 60, and 90 days of training). Later, the first images of each of the 12 animals were measured, followed by measures of its second images, and finally the third images were evaluated. This procedure was followed to avoid evaluating the three measures from an animal in sequence, so that the initial measure would not influence evaluation of the subsequent measures. Although the correlations between the measures taken in one animal were relatively high (0.80 for CSA and 0.84 for FL), an average of the three measures in the analysis was used. The same person obtained and measured all images. Statistical Analyses Ultrasound measurements of the longissimus dorsi muscle were studied using a factorial split plot in time design (3 2 þ 2) considering three periods of training (30, 60, and 90 days), two treatments (animals trained, with and without creatine supplementation), and two additional (control) treatments related to the untrained horses. Analysis of variance (ANOVA) was performed considering training split in periods of training. The means were compared by Tukey’s test (P < .05).
RESULTS The main finding of this study was the lack of a significant effect of creatine supplementation on longissimus dorsi muscle response to endurance training. Mean BW did not differ significantly (P ¼ .749, .075, and .873 for 30, 60, and 90 days, respectively) between the groups over the course of training (data shown elsewhere12). Similarly, BCS did not show a significant difference between groups and among times of training. Table 1 shows the means and standard deviation of measurements of the variables CSA (cm2) and thickness of FL (mm) obtained in the ultrasound examination of the longissimus dorsi muscle (Fig. 1). Analysis of the results of the CSA showed a significant increase (P < .05) in this variable in groups C30, C60, and C90 compared with group C0, and in groups CR30, CR60, and CR90 when compared with animals in group CR0. However, a cross-sectional area of groups C30, C60, C90 did not differ significantly (P > .05) from that of groups CR30, CR60, and CR90, respectively. For thickness of the FL, mean values for the C30, C60 and C90 groups were significantly lower (P < .05) compared with C0, and the same was true for the CR30, CR60, and CR90 groups compared with the CR0 group. A comparison of the nonsupplemented and supplemented groups at each Journal of Equine Veterinary Science
March 2007
Figure 1. Ultrasound image of the longissimus dorsi muscle in a purebred Arabian horse. Black arrows delimit the area of the muscle measured, and white arrow indicates the layer of fat.
of the three time periods indicated no significant differences (P > .05) in this parameter.
DISCUSSION AND CONCLUSIONS The low quantity of body fat and the increase in area occupied by skeletal muscle are of great value in estimating the athletic potential of elite competitive horses, in both longdistance events and short-distance races.13 Our study showed an increase in the cross-sectional area of the longissimus dorsi muscle in all experimental groups during the course of training. Training induces this enhancement, characterizing an improved body composition of animals by increasing fat-free mass. Training led to increased muscle cross-sectional area and a decreased layer of fat, which was not influenced by creatine. The lack of effect of creatine was also observed by other authors.12,14 A well-accepted theory is that creatine-mediated water retention increases cell size by modifying the concentration gradient.15,16 This could explain the weight increase seen in humans after the ingestion of dietary creatine.5 However, no evidence was seen of significant weight gain in animals in this study during the course of creatine supplementation, although a horse would have to show considerable weight gain to demonstrate a statistically significant increase.8 The ultrasound measurement of the layer of fat of the longissimus dorsi muscle is generally used to determine the meat tenderness of animals destined for consumption. However, this study proposed the use of this technique to evaluate training, to examine the correlation between cross-sectional area and thickness of the layer of fat in an established anatomic part, using a standard aerobic training program. Cross-sectional area increased and fat layer thickness decreased after the training period, whereas body weight and body conditioning score of the animals did not change (data shown elsewhere12). Thus, during the monthly evaluations, a diminution in adipose tissue and Volume 27, Number 3
an increase in muscle mass were noted as a result of the intensity of aerobic exercise used. This effect is supported by other works13,17 that found a negative correlation between the amount of body fat and the athletic performance of horses in races and in long distance events. Another research group18 studied the influence of fat mass in endurance horses using Arabian and part-Arabian horses. The percentage of body fat in horses competing in a 150-mile race was approximately 7.8%. When performance during the competition was compared with the percentage obtained for this variable, horses that were more successful in the course had lower body fat (6.5%) in relation to animals that did not finish the test (11%). Although body composition is considered an important factor for the healthy animal, few findings have been published referring to this evaluation in horses, particularly athletic horses during sport competitions.13 Some of these procedures are expensive in terms of personnel and equipment and are not used in the field because of their difficult operation, making the technique impractical for veterinary examinations in routine training and during sport competitions. The ultrasound technique was carried out easily and safely in the athletic horses used in these experiments, meanwhile not affecting their daily schedule, mainly training, a very important aspect for the horse in any type of sport. Because of the advantages of ultrasound experienced in this study, this technique can be recommended for routine use during training. Analysis of the data showed that, in the first month of training, the cross-sectional area of the longissimus dorsi muscle increased considerably and the thickness of the layer of fat diminished. ACKNOWLEDGMENTS Supported by Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP), Proc. 01/02207-4, Sa˜o Paulo, SP. The authors thank Dr. A. Leyva for his assistance in the preparation of the manuscript. REFERENCES 1. Evans DL. Training and fitness in athletic horses: rural industries research and development corporation. 2000; Available at: http:// www.rirdc.gov.au/reports/Index.htm. Accessed June 21, 2001. 2. Brannon TA, Adams GR, Conniff CL, Baldwin KM. Effects of creatine loading and training on running performance and biochemical properties of rat skeletal muscle. Med Sci Sport Exer 1996;29: 489–495. 3. Casey A, Constantin-Teodosiu D, Howell S, Hultman E, Greenhaff PL. Creatine ingestion favorably affects performance and muscle metabolism during maximal exercise in humans. Am J Physiol 1996;271:E31–E37. 4. Vandenberghe K, Goris M, Van Hecke P, Van Leemputte M, Vangerven L, Hespel P. Long term creatine intake is beneficial to muscle performance during resistance training. J Appl Physiol 1997; 83:2055–2063.
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5. Balson PD, So¨derlund K, Sjo¨din B, Ekblom B. Skeletal muscle
11. Ridgway KJ. 1994. Training endurance horses. In: Hodgson DR,
metabolism during short duration high-intensity exercise influence
Rose RJ, eds. The athletic horse. Philadelphia: W.B. Saunders; 1994:
of creatine supplementation. Acta Physiol Scand 1995;154: 303–310.
409–417. 12. D’Angelis FHF, Ferraz GC, Boleli IC, Lacerda-Neto JC, Queiroz-
6. Sewell DA, Harris RC. Effects of creatine supplementation in the Thoroughbred horse. Equine Vet J 1995;18(suppl.):239–242. 7. E´ssen-Gustavsson B, Lindholm A, Persson SG. Circulatory and mus-
13. Kearns CF, Mckeever KH, Abe T. Review. Overview of horse body
cle metabolic responses in the horse to a standardized exercise test
composition and muscle architecture: Implications for performance.
with and without creatine supplementation. Proceedings 10th
Vet Rec 2002;164:224–234. 14. Balsom PD, Harridge SDR, So¨derlund K, Sjo¨din B, Ekblom B.
International Conference of Racing Analysts and Veterinarians,
Neto A. Aerobic training, but not creatine supplementation, alters the gluteus medius muscle. J Anim Sci 2005;83:579–585.
Stockholm, Sweden; 1994:85–89. 8. Schuback K, Esse´n-Gustavsson B, Persson SG. Effect of creatine
Creatine supplementation per se does not enhance endurance exer-
supplementation on muscle metabolic response to a maximal treadmill exercise test in standardbred horses. Equine Vet J 2000;32:
15. Greenhaff PL. Review: the nutritional biochemistry of creatine. Nutr Biochem 1997;8:610–618.
533–540.
16. Powers ME, Arnold BL, Weltman AL, Perrin DH, Mistry D, Kahler
9. Dobec RL, Borger ML, Neal SM. Correlation of real-time ultrasonic
cise performance. Acta Physiol Scand 1993;149:521–523.
DM, et al. Creatine supplementation increases total body water
measure of fat and longissimus muscle area in standardbred horses
without altering fluid distribution. J Athl Train 1992;38:44–50.
with lifetime racing records and lifetime winnings. J Anim Sci
17. Sparling PB, Cureton KJ. Biological determinants of the sex difference
1994;72(suppl. 2):45.
in 12-min run performance. Med Sci Sports Exerc 1983;15:218–223.
10. Henneke DR, Potter GD, Krieder JL. Relationship between condi-
18. Lawrence LM, Jackson S, Kline K, Moser L, Biel M. Observations on
tion score, physical measurements and body fat percentage in mares. Equine Vet J 1983;15:371–372.
body weight and condition of horses in a 150-mile endurance ride. Equine Vet Sci 1992;12:320–324.
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Journal of Equine Veterinary Science
March 2007
ORIGINAL RESEARCH Aerobic Training, But Not Creatine, Modifies Longissimus Dorsi Muscle Composition
Flora H.F. D’Angelis, PhD,a Marcı´lio D.S. Mota, PhD,b Eduardo V.V. Freitas, MS,a Guilherme C. Ferraz, MS,a Andre´ R. Abraha ˜o, PhD,b Jose´ C. Lacerda-Neto, PhD,c and Antonio Queiroz-Neto, PhDa
ABSTRACT The goal of this study was to investigate by means of an ultrasound examination the composition of the longissimus dorsi muscle in 12 purebred Arabian horses submitted to aerobic training for 90 days, with and without creatine supplementation. Creatine supplementation was carried out by daily administration of 75 g creatine monohydrate mixed into the ration during 90 days of training. Physical conditioning was conducted on a high-performance treadmill, and training intensity was stipulated by calculating the velocity at which blood lactate reaches 4 mmol/l, determined monthly for each animal. The individual intensity of physical force was established at 80% of velocity that resulted in a blood lactate level of 4 mmol/l. The cross-sectional area and the thickness of the layer of fat of the longissimus dorsi muscle were measured for each group at the end of 8 months of inactivity and after 30, 60, and 90 days of training without (control groups) or with creatine supplementation (experimental groups). The results showed that aerobic training combined with or without creatine supplementation caused significant longissimus dorsi hypertrophy and reduction in the thickness of the fat layer. No beneficial effect from prolonged creatine supplementation on the body composition of horses was detected. Keywords: Aerobic training; Creatine; Horse; Longissimus dorsi; Ultrasound
INTRODUCTION Various ergogenic substances have been employed to improve the athletic performance of horses, including creatine, a substance widely used as a nutraceutical in various From the Departamento de Morfologia e Fisiologia Animal, Faculdade de Cieˆncias Agra´rias e Veterina´rias (FCAV), Sa˜o Paulo State University (UNESP), campus de Jaboticabal, Jaboticabal, SP, Brasil a; Departamento de Melhoramento e Nutric¸a˜o Animal, Faculdade de Medicina Veterina´ria e Zootecnia, UNESP, campus de Botucatu, SP, Brasilb; and Departamento de Clı´nica e Cirurgia Veterina´ria, FCAV, UNESP.c Reprint requests: Antonio Queiroz-Neto, PhD, Via de Acesso Prof. Paulo Donato Castellane, 14884-900, Jaboticabal, SP, Brasil. 0737-0806/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.jevs.2007.01.008
118
equestrian sports. Creatine is an organic compound that is abundant in skeletal muscle and plays a central role in the availability of energy in muscles.1 Creatine is phosphorylated by creatine kinase, forming creatine phosphate, which provides the phosphate group necessary for rephosphorylation of adenosine diphosphate (ADP) to adenosine triphosphate (ATP) and thereby contributes to energy metabolism and muscle contraction.2 Creatine supplementation in humans at high doses for short periods (1 week) results in a greater capacity to achieve intermittent maximum exercise.3,4 Creatine supplementation for a prolonged time has been demonstrated to stimulate muscle hypertrophy during resistance training in sedentary females.4 Creatine also increases body weight, as shown in a study in human athletes.5 Conversely, no influence was observed in the concentration of creatine in plasma or in the transport of creatine in muscles of horses after a daily intake of 50 g creatine monohydrate for 7 days,6 which agreed with previous studies on creatine supplementation in horses.7,8 These findings justify the need for investigations into the evaluation of body composition and creatine supplementation in the diet of athletic horses. Various regions of the body in an animal can be measured in the evaluation of body composition. The longissimus dorsi muscle was chosen for ultrasound measurements in this study because it is easy to access and provides good ultrasonographic images. In addition, this muscle is highly worked during exercise, where it is responsible for the extension and flexion of the vertebral column and, at maximum extension, for maintenance of posture, elevation and support of the head, and supporting the weight of the rider. At least one study9 demonstrated a significant relationship between the size of the cross-sectional area of the longissimus dorsi muscle and the number of victories scored by the animals in races. Finally, the apparent existing contradictions and doubts regarding utilization of creatine as a dietetic supplement clearly indicate the need for additional studies of this substance, before being able to prescribe it as a complementary food for athletic horses. Few studies have been conducted aimed at examining the relationship between ultrasound measurements of the longissimus dorsi muscle and the physical conditioning of Journal of Equine Veterinary Science
March 2007
athletic horses in different sport modalities. Therefore, the objective of the current study was to conduct an ultrasound examination of the longissimus dorsi muscle to determine the cross-sectional area and thickness of the layer of fat in purebred Arabian horses submitted to endurance training with and without creatine supplementation.
MATERIALS AND METHODS Animals and Handling Twelve purebred Arabian horses were used, both geldings (n ¼ 5) and mares (n ¼ 7), with an initial body weight (BW) of 391.0 25.4 kg (mean SD) and a mean age of 8.6 3.3 years. The animals selected for study were evaluated by clinical examination and laboratory tests such as complete blood count and blood biochemistry. They had no signs or symptoms compatible with skeletal muscle lesions, and were deloused and dewormed with ivermectin (Ivotan LA, Intervet, Sa˜o Paulo, Brazil) at intervals of 40 and 60 days, respectively. The 12 horses were kept in one paddock for 8 months of inactivity and then were divided randomly into two pre-trained groups (C0 and CR0). Both groups were subjected to aerobic training for 30, 60, and 90 days, but only one group (CR0) received 75 g creatine monohydrate (Vetnil, Louveira, SP, Brazil) per animal per day for the same period (CR30, CR60, and CR90). The other group served as the control group (C30, C60 and C90) and received no supplementation. The animals were kept in a paddock of Brachiaria sp. grass, with ad libitum access to water and supplementation with Cynodon sp. hay and mineralized salt (Agromix, Jaboticabal, SP, Brazil). Creatine was given individually in the stall, and mixed with half of the daily commercial caloric ration (Nutriage MIX Guabi, Campinas, SP, Brazil) followed by the rest of the ration to make sure that all the creatine was ingested by each horse. The fed formulation was: 12% crude protein, 2% ether extract, 12% crude fibers, 12% mineral matter, 1.8% calcium, 0.5% phosphorus, and 2,990 kcal/kg of digestible energy. To determine the daily quantities (2.5–3.8 kg/animal) of commercial diet furnished, the animals were evaluated monthly for BW and body condition score (BCS). BCS was determined by visual appraisal and palpable fat cover at six areas of the body of the horse.10 The areas selected as being indicative of changes in stored body fat were the lumbar spinous processes, ribs, tailhead, area behind the shoulder, neck, and withers. The BCS was evaluated on a scale of 1 to 9, with 1 being extremely emaciated and 9 being extremely fat. The experimental protocol was approved by the university’s Institutional Animal Care and Use Committee. Physical Conditioning Physical conditioning of each animal was performed in a high-performance treadmill (Galloper 5500, Sahinco Ltda, Palmital, Sa˜o Paulo, Brazil). Before initiation of the training program, the horses were submitted to a 30-day adaptation to handling period. The training program was Volume 27, Number 3
conducted exclusively on the treadmill. The intensity (velocity) of the training was set at 80% of Vlac4 (velocity at which the blood lactate concentration reached 4 mmol/l) calculated in an incremental exercise test. At the end of each training period (30 days), a new exercise test was conducted to establish a new Vlac4. After a warm-up period of 4 minutes at 4 m/second, the treadmill was inclined at 10% and the speed was gradually increased at 2-minute intervals to 6, 8, and 10 m/second. The blood samples were collected 15 seconds before the end of each step by means of an intravenous catheter fixed in the left jugular vein. After each blood sampling, the whole system was washed out with 2.5% heparin solution. For this reason, 2 ml blood were discharged at the beginning of each collection. The samples were immediately analyzed for lactate concentration by a portable digital lactometer (YSI 1500 Sport L-Lactate Analyzer, YSI Incorporated, Yellow Springs, OH). The individual Vlac4 was determined graphically using an exponential regression. The training velocity was thereby determined monthly for each animal. The frequency of training consisted of the performance of exercises three times per week (alternate days). In the first month of training, each animal exercised for 10 km in a mean time of 50 minutes. In the second month, the distance run in each exercise was increased to 15 km, with a mean duration of 60 minutes. In the last month, all horses exercised 20 km per session, with a mean duration of 80 minutes. In addition, a speed play type of training, which involves sudden, rapid, relatively short bursts of speed interspersed throughout the exercise bout, was instituted once per week with the aim of stimulating tissues not addressed by slower exercise and increasing the horse’s metabolic capabilities. The protocol consisted of a warming period of 10 minutes at 4 m/second followed by 10 more minutes with the treadmill inclined (10%) at the speed of 8 m/second. After that, the speed was reduced to 2.5 m/ second for 5 minutes without any inclination of the treadmill, and then it was inclined to 5% and the speed increased to 10 m/second for 5 minutes. After another decrease in the speed and inclination, the speed was increased to 5 m/second at a 10% inclination for 20 minutes. The last increase in the speed to 12 m/second for 3 minutes was performed. A warm-down period of 10 minutes at 2 m/ second without inclination concluded the task. This protocol was adopted because some authors7,11 have recommended strenuous training sessions as part of the endurance training to develop fitness for fast exercise. Ultrasound Measurements The effects of training and creatine supplementation on skeletal muscles were determined by ultrasound measurements of the cross-sectional area (CSA, cm2) and fat layer (FL, mm) of the longissimus dorsi muscle in groups C0, C30, C60, C90, CR0, CR30, CR60, and CR90. These were performed using a Pie Medical-scanner 200 ultrasonograph (model 51B04UM02 3.5/5 Mhz DF TRD Linear, USA) equipped with a specific computer program (Software Eview, Echo Image Viewer, Maastricht, The 119
a C0, animals before training; CR0, animals before training and supplementation with creatine; C90, animals trained for 90 days and not supplemented with creatine; CR90, animals trained and supplemented with creatine for 90 days. Means in the same row followed by different letters (A, B) differ significantly (P < .05) according to Tukey’s test.
2.47B 2.32 2.79B 2.31 3.00B 0.83 4.60A 0.91 4.62A 0.91
3.03B 0.74
2.61B 0.90
2.69B 1.87
6 89.79B 2.32 6 88.81B 2.32 6 88.70B 5.00 6 76.90A 7.20 6 69.60A 1.83
Number of horses Cross-sectional area (cm2) Layer of fat (mm)
6 83.58B 4.35
6 83.23B 2.94
6 85.35B 1.87
CR90 C90 CR60 C60 CR30 C30 CR0 C0 Variable
90 d 60 d 30 d 0d Experimental Groupa
Table 1. Means ( SD) of morphometric variables studied in the longissimus dorsi muscle obtained by ultrasound examination of 12 purebred Arabian horses, submitted to aerobic training for 30, 60, and 90 days, with and without creatine supplementation 120
Netherlands, Copyright Pie Medical, 1996) for muscle studies. This type of apparatus is specialized for B-mode and has the ability to produce images of moving objects almost instantaneously. The measurements were carried out at the height of the last ribs on the left side with the animal in the upright position and the longissimus dorsi muscle relaxed. Initially three images were obtained for each animal at each of the four occasions (0, 30, 60, and 90 days of training). Later, the first images of each of the 12 animals were measured, followed by measures of its second images, and finally the third images were evaluated. This procedure was followed to avoid evaluating the three measures from an animal in sequence, so that the initial measure would not influence evaluation of the subsequent measures. Although the correlations between the measures taken in one animal were relatively high (0.80 for CSA and 0.84 for FL), an average of the three measures in the analysis was used. The same person obtained and measured all images. Statistical Analyses Ultrasound measurements of the longissimus dorsi muscle were studied using a factorial split plot in time design (3 2 þ 2) considering three periods of training (30, 60, and 90 days), two treatments (animals trained, with and without creatine supplementation), and two additional (control) treatments related to the untrained horses. Analysis of variance (ANOVA) was performed considering training split in periods of training. The means were compared by Tukey’s test (P < .05).
RESULTS The main finding of this study was the lack of a significant effect of creatine supplementation on longissimus dorsi muscle response to endurance training. Mean BW did not differ significantly (P ¼ .749, .075, and .873 for 30, 60, and 90 days, respectively) between the groups over the course of training (data shown elsewhere12). Similarly, BCS did not show a significant difference between groups and among times of training. Table 1 shows the means and standard deviation of measurements of the variables CSA (cm2) and thickness of FL (mm) obtained in the ultrasound examination of the longissimus dorsi muscle (Fig. 1). Analysis of the results of the CSA showed a significant increase (P < .05) in this variable in groups C30, C60, and C90 compared with group C0, and in groups CR30, CR60, and CR90 when compared with animals in group CR0. However, a cross-sectional area of groups C30, C60, C90 did not differ significantly (P > .05) from that of groups CR30, CR60, and CR90, respectively. For thickness of the FL, mean values for the C30, C60 and C90 groups were significantly lower (P < .05) compared with C0, and the same was true for the CR30, CR60, and CR90 groups compared with the CR0 group. A comparison of the nonsupplemented and supplemented groups at each Journal of Equine Veterinary Science
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Figure 1. Ultrasound image of the longissimus dorsi muscle in a purebred Arabian horse. Black arrows delimit the area of the muscle measured, and white arrow indicates the layer of fat.
of the three time periods indicated no significant differences (P > .05) in this parameter.
DISCUSSION AND CONCLUSIONS The low quantity of body fat and the increase in area occupied by skeletal muscle are of great value in estimating the athletic potential of elite competitive horses, in both longdistance events and short-distance races.13 Our study showed an increase in the cross-sectional area of the longissimus dorsi muscle in all experimental groups during the course of training. Training induces this enhancement, characterizing an improved body composition of animals by increasing fat-free mass. Training led to increased muscle cross-sectional area and a decreased layer of fat, which was not influenced by creatine. The lack of effect of creatine was also observed by other authors.12,14 A well-accepted theory is that creatine-mediated water retention increases cell size by modifying the concentration gradient.15,16 This could explain the weight increase seen in humans after the ingestion of dietary creatine.5 However, no evidence was seen of significant weight gain in animals in this study during the course of creatine supplementation, although a horse would have to show considerable weight gain to demonstrate a statistically significant increase.8 The ultrasound measurement of the layer of fat of the longissimus dorsi muscle is generally used to determine the meat tenderness of animals destined for consumption. However, this study proposed the use of this technique to evaluate training, to examine the correlation between cross-sectional area and thickness of the layer of fat in an established anatomic part, using a standard aerobic training program. Cross-sectional area increased and fat layer thickness decreased after the training period, whereas body weight and body conditioning score of the animals did not change (data shown elsewhere12). Thus, during the monthly evaluations, a diminution in adipose tissue and Volume 27, Number 3
an increase in muscle mass were noted as a result of the intensity of aerobic exercise used. This effect is supported by other works13,17 that found a negative correlation between the amount of body fat and the athletic performance of horses in races and in long distance events. Another research group18 studied the influence of fat mass in endurance horses using Arabian and part-Arabian horses. The percentage of body fat in horses competing in a 150-mile race was approximately 7.8%. When performance during the competition was compared with the percentage obtained for this variable, horses that were more successful in the course had lower body fat (6.5%) in relation to animals that did not finish the test (11%). Although body composition is considered an important factor for the healthy animal, few findings have been published referring to this evaluation in horses, particularly athletic horses during sport competitions.13 Some of these procedures are expensive in terms of personnel and equipment and are not used in the field because of their difficult operation, making the technique impractical for veterinary examinations in routine training and during sport competitions. The ultrasound technique was carried out easily and safely in the athletic horses used in these experiments, meanwhile not affecting their daily schedule, mainly training, a very important aspect for the horse in any type of sport. Because of the advantages of ultrasound experienced in this study, this technique can be recommended for routine use during training. Analysis of the data showed that, in the first month of training, the cross-sectional area of the longissimus dorsi muscle increased considerably and the thickness of the layer of fat diminished. ACKNOWLEDGMENTS Supported by Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP), Proc. 01/02207-4, Sa˜o Paulo, SP. The authors thank Dr. A. Leyva for his assistance in the preparation of the manuscript. REFERENCES 1. Evans DL. Training and fitness in athletic horses: rural industries research and development corporation. 2000; Available at: http:// www.rirdc.gov.au/reports/Index.htm. Accessed June 21, 2001. 2. Brannon TA, Adams GR, Conniff CL, Baldwin KM. Effects of creatine loading and training on running performance and biochemical properties of rat skeletal muscle. Med Sci Sport Exer 1996;29: 489–495. 3. Casey A, Constantin-Teodosiu D, Howell S, Hultman E, Greenhaff PL. Creatine ingestion favorably affects performance and muscle metabolism during maximal exercise in humans. Am J Physiol 1996;271:E31–E37. 4. Vandenberghe K, Goris M, Van Hecke P, Van Leemputte M, Vangerven L, Hespel P. Long term creatine intake is beneficial to muscle performance during resistance training. J Appl Physiol 1997; 83:2055–2063.
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