Recruitment pattern of muscle fibre type during high intensity exercise (60 -- 100% VO2max) in Thoroughbred horsesIntroductionMaterials and methodsAnimalsMeasurement of VO2maxExercise protocolSamplingHistochemical and immunohistochemical analysisBiochemical analysisStatistical analysisResultsAnimal conditionsHistochemical analysisBiochemical analysisDiscussionAcknowledgementsReferencesRecruitment pattern of muscle fibre type during high intensityexercise (60–100% VO2max) in Thoroughbred horsesS. Yamanoa, D. Etob, A. Hiragab, H. Miyataa,*aDepartment of Biological Sciences, Faculty of Agriculture, Yamaguchi University, Yoshida 1677-1, Yamaguchi 753-8515, JapanbThe Equine Research Institute, Japan Racing Association, 321-4 Tokami-cho, Utsunomiya, Tochigi 320-0856, JapanAccepted 15 April 2005AbstractTo consider the optimal training programme for Thoroughbred horses, we examined the recruitment pattern of muscle fibresincluding hybrid muscle fibres in well-trained Thoroughbred horses. The horses performed exercise at three different intensitiesand durations; i.e., 100% VO2max for 4 min, 80% and 60% VO2max for 8 min on a treadmill with 10% incline. Muscle samples wereobtained from the middle gluteal muscle before, during (4 min at 80% and 60% VO2max), and after exercise. Four muscle fibre types(types I, IIA, IIA/IIX, and IIX) were immunohistochemically identified, and optical density of periodic acid Schiff staining (OD-PAS) in each fibre type, and the glycogen content of the muscle sample, were determined by quantitative histochemical andbiochemical procedures. The changes in OD-PAS showed that the recruitment of all fibre types were identical at the final time stageof each exercise bout, i.e., 4 min running at 100% VO2max, and 8 min running at 80% and 60% VO2max. The changes in OD-PAS oftype IIA/IIX fibre were very similar to those of type IIX fibre. The recruitment of these fibres were obviously more facilitated by4 min running at 100% VO2max than by 4 min running at 80% or 60% VO2max. Short duration with high intensity exercise, suchas 4 min running at 100% VO2max or 8 min running at 80% or 60% VO2max, is effective to stimulate type IIX fibre and IIA/IIXfibres that have the fastest speed of contraction. 2005 Elsevier Ltd. All rights reserved.Keywords: Thoroughbred; Muscle fibre; Recruitment pattern; Glycogen; PAS stain1. IntroductionIn high intensity exercise over the aerobic thresh-old, skeletal muscles mainly produce their active en-ergy by using glycogen. An indication of musclefibre recruitment is often obtained from histochemicalstaining intensity for glycogen in each muscle fibretype immediately after a certain exercise. Muscle fibrerecruitment patterns of human and other mammalshave been studied under various exercise intensitiesand durations. These studies have demonstrated thatregardless of the intensity or duration of exercise, anorder of muscle fibre recruitment occurred from typeI fibre with low thres hold through type IIA fibre withhigher threshold to type IIB fibre with the highestthreshold. For example, in humans, Vollestad et al.(1984) reported that initial glycogen depletion couldonly be detected in type I and IIA fibre, followedby IIAB fibre, and finally depletion of glycogen intype IIB fibr e during exhaustive bicycle exercise at75% VO2max. Green et al. (1990) demonstrated thatthe pattern of depletion in type I and IIA fibres wasparallel and obvious during the first 15 min of bicycleexercise at 59% VO2max, althoug h glycogen concen-tration in type IIB fibre was not significantly alteredthroughout the exercise. In Standard trotter horses,the middle gluteal muscle exhibited glycogen depletion0034-5288/$ - see front matter  2005 Elsevier Ltd. All rights reserved.doi:10.1016/j.rvsc.2005.04.006*Corresponding author. Tel.:/fax: +81 839 33 5866.E-mail address: [email protected] (H. Miyata).www.elsevier.com/locate/rvscResearch in Veterinary Science xxx (2005) xxx–xxxARTICLE IN PRESSin all fibre types with the greatest loss in type I andleast in type IIB after high speed trotting (Valberg,1986). In Thoroughbred horses, White and Snow(1987) showed that significant glycogen depletion wasfound in all fibre types in the middle gluteal muscleof horses that underwent an exercise test consistingof four gallops over a 620 m course with 5 minbetween the start of each gallop. However, Hodgsonet al. (1984) reported that no significant glycogendepletion in type I fibres was found despite significantdecreases in type IIA and IIB fibres after maximal gal-lop. Up to date, there have been no systematic studiesinvestigating the recruitment pattern of muscle fibretypes during high intensity exercise in Thoroughbredhorses. The results of the recruitment patterns duringhigh intensity exercise will be important in consider-ation of the training methods for Thoroughbredhorses, because the horses are usually subjected tohigh speed races of less than 3 min duration.In our previous studies, we suggested that a mixtureof type IIX and IIA fibres, which have a high speed ofcontraction and a high ability to resist fatigue, may bethe most suitable composition of muscle fibres forcompetition (Miyata et al., 1999; Yamano et al.,2002). In addition, we demonstrated a possibility thatconventional training programmes do not result inthe maximal development of anaerobic capacity ofThoroughbred horses (Eto et al., 2004). In the samestudy, we also suggested that continuously high inten-sity training may result in a high percentage of retire-ment from the training programme. Therefore, toconsider optimal training programmes for Thorough-bred horses, it is very important to know which inten-sity and duration is the most effective to induce typeIIX and IIA fibre recruitment in the main active mus-cles. In this study, we examined the recruitment patternof muscle fibres including hybrid muscle fibres, typeIIA/IIX, in the middle gluteal muscle of Thoroughbredhorses during three intensity exercises, 100%, 80% and60% VO2max. These intensities are approximately overthe aerobic threshold, and frequently applied for theconventional training programme. This is a first studyto investigate the recruitment patterns of hybrid musclefibre.2. Materials and methods2.1. AnimalsEight Thoroughbred horses (six males and two geld-ings; 3–9 years old) that weighed 459 ± 25 kg were usedin this study. Before the experiments, the horses hadacclimatized to exercise on a