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J. Vet. Med. A 48, 313±319 (2001) Ó 2001 Blackwell Wissenschafts-Verlag, Berlin ISSN 0931±184X Department of Animal Biology, Section of Physiology, Faculty of Veterinary Medicine, University of CoÂrdoba, Spain Skeletal Muscle Fibre Characteristics in Young and Old Bulls and Metabolic Response after a Bull®ght E. I. AGUÈERA1,4, A. MUNÄOZ2, F. M. CASTEJOÂN1 and B. ESSEÂN-GUSTAVSSON3 Addresses of authors: 1Department of Animal Biology (Physiology), Faculty of Veterinary Medicine, University of CoÂrdoba; 2University of Cardenal-Herrera, Valencia, Spain; 3Department of Large Animal Clinical Sciences, Swedish University of Agricultural Sciences, Uppsala, Sweden; 4 Corresponding author With 3 tables (Received for publication August 3, 2000) Summary Fibre type composition, activities of enzymes such as citrate synthase (CS), 3-hydroxyacyl coenzyme A dehydrogenase (HAD), lactate dehydrogenase (LDH), as well as glycogen, lactate and pH levels were analysed in muscle biopsies (m. gluteus medius) obtained after bull®ghting from 10 young and 10 old bulls. No changes were seen in ®bre type composition between groups, but the older bulls had higher HAD and LDH activities. Low glycogen concentrations and low pH values were found in both groups, but the lactate concentration after bull®ghting was higher in the older group of bulls. The histochemical stain for glycogen revealed that type IIB ®bres in both young and old bulls contained more glycogen than seen in type IIA and type I ®bres. These results show that young and old bulls have similar muscle ®bre type composition, but the metabolic capacity differs, with a higher glycolytic capacity and lactate production in older bulls. Furthermore, it seems that the physical and emotional stress in connection with a bull®ght causes a marked depletion of glycogen, especially of type I and IIA ®bres. Introduction Skeletal muscle ®bres can be histochemically classi®ed into different ®bre types based on differences in the pH dependence of the myosin adenosine triphosphatase (m-ATPase) activity (Brooke and Kaiser, 1970). At alkaline pH, slow-contracting, type I ®bres, have a low ATPase activity, whereas fast-contracting, type II ®bres, have a high ATPase activity (Burke and Edgerton, 1975). Type II ®bres can be further subdivided into type IIA and type IIB ®bres on the basis of their differences in m-ATPase under different acidic conditions (Brooke and Kaiser, 1970). With another histochemical staining technique [Periodic Acid Schiff (PAS)] it is possible to evaluate the glycogen content within the muscle ®bres (Kugelberg and EdstroÈm, 1968). Evaluation of PAS stains of repeated muscle biopsies from horses and humans have been used to indicate how muscle ®bres are recruited during work (Lindholm et al., 1974; Saltin and Gollnick, 1983; Valberg, 1986). A high degree of depletion after exercise indicates that glycogen has been one of the main substrates for energy release during ®bre recruitment. This technique has also been used in pigs and cattle to study if glycogenolysis differs among U. S. Copyright Clearance Center Code Statement: 0931±184X/2001/4805±0313 $15.00/0 www.blackwell.de/synergy 314 AGUÈERA et al. ®bre types in connection with different stress situations (Lacourt and Tarrant, 1985; Karlsson et al., 1994). Information about energy contribution, provided through aerobic metabolism (oxidative capacity) but also to a great extent through anaerobic metabolism (glycolytic capacity with lactate formation), can be obtained by analysing enzyme activities representing different metabolic pathways (Pete and Sparmer, 1986). Several studies concerning skeletal muscle characteristics and their relation to age and training have been performed in humans and animals such as horses and pigs (EsseÂn et al., 1984; EsseÂnGustavsson and Lindholm, 1984, 1985; LoÂpez-Rivero et al., 1993). Results from these studies show that physically active animals and trained animals have a higher oxidative capacity, a higher glycogen content and a higher type IIA/IIB ratio in muscle in comparison with animals that are less active. There are only a few studies describing muscle characteristics in bulls and cattle which must be considered as animals that are not often exposed to marked physical activity (Solomon et al., 1985; Zerouala and Stickland, 1991; Totland and Kryvi, 1991; KarlstroÈm et al., 1994; Picard et al., 1995). However, never before have the effects of the physical and emotional stress in connection with a bull®ght been studied. The aim of this study was to investigate the muscle characteristics in both young and old bulls raised for bull®ghting and the metabolic responses after a bull®ght. Materials and Methods Animals and experimental procedures Two groups of bulls were studied in connection with four different bull®ghts which lasted around 20 min. The young group consisted of 10 bulls which were 1 year old, weighing between 180 and 216 kg and were derived from two different farms. The old group consisted of 10 bulls which were 4±5 years old, weighing between 515 and 584 kg and were derived from two other farms. All these bulls had always been raised outdoors and were handled at each farm by the same person. The average duration of the bull®ghts was the same for the two groups, i.e. both the old and the young bulls were involved in the ®ght for 20 min. Every bull charged the matador around 40 times with a distance of 5 m during each charge. The average speed of movement was dif®cult to determine. Muscle biopsies Muscle biopsies were obtained as soon as possible after death (within 5 min) from the gluteus medius muscle. The samples were obtained with the percutaneous biopsy technique (Lindholm and Piehl, 1974). All muscle biopsies were taken by the same person and always in the same area in the middle of the muscle at a depth of 5 cm. The samples were quick-frozen in liquid nitrogen and then kept at ±80°C until analysed. Histochemical analyses The muscle samples for histochemical analyses were mounted on a metal chuck with an 1 embedding medium (Tissue-Teck ll; Tissue Teck, Nussloch, Germany). Serial sections (10 lm) were cut in a cryostat microtome at ± 20°C and stained for myo®brillar ATPase after both alkaline (pH 10.3) and acid (pH 4.3 and 4.6) pre-incubation (Brooke and Kaiser, 1970). The ®bres were identi®ed as type I, IIA and IIB. At least 150±200 ®bres were identi®ed on each section. An evaluation of the glycogen content in the muscle ®bres was obtained from cross-sections (20 lm) by staining for PAS (Pearse, 1961). The ®bres were subjectively classi®ed as `high', `medium' or `low' according to the staining intensity and then compared with the ATPase stain to identify the ®bre type. Skeletal Muscle in Bulls after a Bull®ght 315 Enzyme analyses Muscle tissue for enzyme analyses was freeze dried, dissected free of blood, fat and connective tissue, weighed and homogenized in ice-chilled 0.1 M potassium phosphate buffer (pH 7.3) in an ultrasound disintegrator. The activities of citrate synthase (CS), 3-hydroxyacyl coenzyme A dehydrogenase (HAD), and lactate dehydrogenase (LDH) were determined at 25°C by ¯uorometry (EsseÂn et al., 1980, 1984). Glycogen and lactate Muscle tissue was freeze dried, dissected free of blood, fat and connective tissue and weighed. After boiling 0.1±0.5 mg of the sample for 2 h in 1 ml of 1 M hydrochloric acid to hydrolyse the glycogen, the glucose concentration was determined (Lowry and Passonneau, 1973). Lactate was analysed in perchloric acid extracts (Lowry and Passonneau, 1973). pH The pH was determined after homogenization a piece of muscle in potassium chloride solution containing iodoacetate to inhibit further glycolysis (Harris et al., 1989). Statistical analysis Data are presented as means standard deviation (SD). The Mann±Whitney U-test was applied to test differences between the groups. Statistical signi®cance was accepted if P < 0.05. Table 1. Mean values for ®bre types (I, IIA and IIB), activities of citrate synthase (CS), 3-hydroxyacyl coenzyme A dehydrogenase (HAD) and lactate dehydrogenase (LDH) in gluteus medius muscle of young and old bulls Enzyme activities (mmol/(kg dry matter h) Fibre type composition (%) Group Young group (n = 10) Old group (n = 10) I IIA IIB CS HAD LDH 39 7 39 13 37 9 32 7 24 8 29 12 21 6 18 6 14 4* 20 5 1668 333* 2204 797 n = number of bulls. * P £ 0.05 (signi®cant differences between young and old bulls). Table 2. Mean values ( standard deviation) for concentrations of glycogen and lactate and pH values in gluteus medius muscle of young and old bulls Group Young group (n = 10) Old group (n = 10) Glycogen (mmol/kg dry matter) Lactate (mmol/kg dry matter) pH 67 31 55 27 150 23* 256 56 6.00 0.2 5.89 0.1 n = number of bulls. * P £ 0.05 (signi®cant differences between young and old bulls). 316 AGUÈERA et al. Results Fibre type composition and activities of CS, HAD, and LDH from young and old bulls are presented in Table 1. No differences were seen in ®bre type composition between groups, but the older bulls had higher HAD and LDH activities. Muscle lactate and glycogen concentration and pH from young and old bulls are presented in Table 2. The lactate concentration after a bull®ght was higher in the older bulls than in the younger group of bulls. Low glycogen concentrations and low pH values were found in both groups. The PAS stain could only be performed in eight of the samples from the young group and from ®ve of the samples from the old group. Fibres were subjectively rated as high, medium or low and the results are shown in Table 3. All type IIB ®bres in both young and old bulls revealed a high staining intensity for glycogen, whereas most of the type I and IIA ®bres showed a low or medium staining intensity. Discussion The results for ®bre type composition and enzyme activities in gluteus muscle agree with ®ndings from an earlier study in three 1.5-year-old steers (KarlstroÈm et al., 1994). Other studies in bulls or cattle have usually investigated muscles like longissimus dorsi or semitendinosus, as these muscles are often used in studies investigating relationships with meat quality (Solomon et al., 1985; Zerouala and Stickland, 1991; Picard et al., 1995). The gluteus medius muscle was chosen for this study as it is a muscle considered to be important for locomotion. Muscle ®bre type composition in an animal is partly determined by genetic factors, but factors such as growth, training and physical ®tness may also in¯uence muscle characteristics. Changes in the type I/II ratio are seldom seen in muscle, but changes can be seen in the type IIA/IIB ratio after several months or years of exposure to physical activity (EsseÂn et al., 1980; RoneÂus et al., 1992). In contrast, rapid changes can sometimes be observed in enzyme activities due to training (EsseÂn-Gustavsson et al., 1989). Even though the older bulls had been exposed to several years of outdoor life they had a similar ®bre type composition as the younger bulls. The only increased demand on the ®bres with time will be due to growth and, thus, the increase that occurs in body weight. The enzyme activities showed some differences between the young and the old group, but the results also show that bulls are not athletic animals like racehorses. Racehorses have been bred for speed and endurance and this type of physical activity requires a high capacity for aerobic metabolism (EsseÂn-Gustavsson et al., 1984). In racehorses for instance, values for CS and HAD can be two or three times higher than seen in bulls (EsseÂn-Gustavsson and Lindholm, 1985; Valberg, 1987). Glycolytic capacity and LDH activity is high in both racehorses and bulls which indicates that for both these animals glycogenolysis with lactate production is an important metabolic pathway for energy release. Muscle lactate levels in standardbred trotters and thoroughbreds after racing can be as high as 150±200 mmol/kg (Valberg, 1987). Similar high muscle lactate levels were also found in this study in the young bulls after the bull®ght. In the older group even higher lactate values were found and they also had a higher capacity for lactate production, as indicated by the higher LDH activity. Perhaps this could suggest that muscle oxidative potential in this species is lower than in equine athletes. In this way, ®ghting bulls would not have to facilitate the use of glucose, free fatty acids and triglycerides. Also, it could be associated with less respiratory, cardiovascular and haematological functionality. In horses, pH values may reach values around 6.4 after intensive exercise (Harris et al., 1989). Even though the samples in this study were obtained within 5 min after death, it cannot be excluded that part of the high lactate production and the low pH values were related to the glycogenolysis that is observed in muscle after death. However, the low glycogen levels and the high lactate concentrations and low pH values found in n = number of bulls. Young group (n = 8) Old group (n = 5) ± 6 (0±33) High 15 (13±25) 30 (7±71) Medium Type I 85 (75±100) 64 (29±93) Low ± 7 (0±20) High 55 (25±100) 73 (55±100) Medium Type IIA 45 (8±75) 20 (10±45) Low 100 100 High ± ± Medium Type IIB Table 3. Mean percentage (and range) of ®bres within a ®bre type stained as either high, medium or low with the Periodic Acid Schiff (PAS) stain ± ± Low Skeletal Muscle in Bulls after a Bull®ght 317 318 AGUÈERA et al. this study suggest that glycogenolysis is a most important pathway for energy release during the stressful events that occur in connection with a bull®ght. The bulls have been selected only for ®ghting and were raised outdoors without a high level of physical activity and no contact with people other than the farmer. The day before the bull®ght, the bulls were transported to the bull ring and this together with the actual bull®ght must be regarded as extremely physical and emotionally stressful events. Unfortunately, nothing is known about resting glycogen levels of these bulls, but based on other studies, resting muscle glycogen levels are around 400±500 mmol/kg dry matter (EsseÂn-Gustavsson and Lindholm, 1990). The low glycogen levels found in this study may thus indicate that a high rate of glycogenolysis occurs during a bull®ght. Interestingly, glycogen was selectively lost from type I and IIA ®bres, as all of the type IIB ®bres still contained glycogen. In horses and humans, ®bres are recruited in the following order: type I, type IIA and type IIB if the physical activity increases (Lindholm et al., 1974; Hodgson et al., 1983; Saltin and Gollnick, 1983; Valberg, 1986). It is therefore likely that type I ®bres of the bulls in this study are preferentially recruited at low intensity of exercise such as posture and walking and with an increase in the workload there is an activation of type II ®bres. During a bull®ght, type IIB ®bres may be recruited when the bull attacks. However, fatigue may quickly develop in these ®bres due to their low oxidative capacity, causing rapid lactate production. The emotional stress will result in sympathetic stimulation and release of catecholamines which stimulate glycogenolysis. When familiar Fresian bulls were mixed with unfamiliar bulls and also when adrenaline was injected, there was a marked glycogen depletion in the ®bres (Lacourt and Tarrant, 1985). Adrenaline caused a more marked glycogen depletion in the type I ®bres than was seen for type IIA and IIB ®bres. The greatest demand before and during the bull®ght will thus be on type I and IIA ®bres which may be supported by the low glycogen content found in these ®bres. In conclusion, muscle ®bre type composition does not differ between young and old bulls, but metabolic capacities differ with a higher glycolytic capacity and lactate production in older than younger bulls. 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