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Keywords:

  • horse;
  • fibre type;
  • training;
  • MCT1;
  • CD147

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conflicts of interest
  8. Manufacturers' addresses
  9. References

Reasons for performing study: Muscular changes caused by training are breed-specific and studies on the Norwegian-Swedish Coldblooded Trotter (NSCT) are limited. Knowledge about lactate-transporters in muscle in this light draught breed used for harness racing is lacking.

Objectives: To identify muscular changes associated with training in young NSCTs and investigate muscular distribution of the monocarboxylate transporter 1 (MCT1) and its ancillary protein CD147, which facilitate lactate transport across membranes.

Methods: Nine horses were followed from the start of their training period until the end of their 3-year-old season. A biopsy sample of the middle gluteal muscle was collected on 4 occasions. On the last 3 sampling occasions, individual VLa4-values (the speed corresponding to a blood lactate concentration of 4 mmol/l) were determined in an incremental exercise test on a high-speed treadmill. One horse was excluded due to lameness. Histochemical and immunohistochemical analyses were performed on all muscle samples to determine fibre types (I, IIA, IIAX, IIX), oxidative capacity (NADH) and the expression of MCT1 and CD147. The activity of selected metabolic enzymes in the muscle before and after training was determined.

Results: The percentage of type IIX fibres decreased with training while the percentage of type IIAX fibres increased. The activity of citrate synthase and 3-OH-acyl-CoA-dehydrogenase increased with training. The expression of MCT1 was lower in membranes and cytoplasm of type IIX fibres compared to all other fibre types both before and after training. The antibody against CD147 stained membranes and cytoplasm of all fibres. The first VLa4-value was lower than the last 2 in all horses.

Conclusions: Muscular changes with training of NSCTs were similar to those reported in Standardbreds, indicating fibre type transitions and increased oxidative capacity. Expression of MCT1 differed among fibre types and was related to the oxidative capacity of the fibres.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conflicts of interest
  8. Manufacturers' addresses
  9. References

Coldblooded Trotters are used for harness racing in Norway, Sweden and Finland. The Norwegian and Swedish horses belong to a breed called the Norwegian-Swedish Coldblooded Trotter (NSCT) that has evolved from a rural carriage and riding horse. The maximal trotting speed of NSCTs is lower than that of Standardbreds (Uhlin 2007; Karlström et al. 2009), which have been bred purely for trotting for centuries. Standardbreds and Coldblooded Trotters therefore compete in separate races, but under otherwise identical conditions.

The composition of fibre types within the middle gluteal muscle has been subject to numerous studies of equine exercise physiology. There is variation within, as well as between, breeds.

Horses used for endurance sports typically have higher percentages of oxidative, slow contracting type I fibres than sprinters, which have high percentages of the faster contracting but less oxidative type IIA and IIX fibres, of which type IIX is the fastest (Snow and Guy 1980). Muscle fibres possess the ability to change their own metabolic and contracting properties by converting to another fibre type as a result of training impulses (Pette 1998). It has been demonstrated that muscular responses to a standardised training programme vary between breeds (Rivero et al. 1995). Muscular adaptations to training must therefore be determined in different breeds independently.

The use of the activity of myosin ATPase (mATPase) to classify muscle fibres (Brooke and Kaiser 1970) has been replaced by a more sensitive immunohistochemical method (Rivero et al. 1996; Karlström and Essén-Gustavsson 2002). With this method, myosin heavy chains (MHC) characteristic for the main fibre types I, IIA and IIX are stained (Rivero et al. 1996). Since the introduction of immunohistochemical methods hybrid IIAX fibres have also been identified in equine muscles (Rivero et al. 1996; Karlström and Essén-Gustavsson 2002; Karlström et al. 2009).

Monocarboxylate transporters (MCT), that facilitate the transport of lactate and other monocarboxylates together with protons across membranes, have been suggested to be the most important regulators of muscle pH during exercise (Messonnier et al. 2007; Juel 2008). Two MCT isoforms, MCT1 and MCT4, are expressed in the muscles of horses and other species (Halestrap and Price 1999; Koho et al. 2006). MCT1 is thought to contribute to lactate influx into oxidative muscle fibres, where lactate may serve as a substrate for ATP regeneration. MCT4 is expressed especially in cells with a high glycolytic rate, suggesting a role in lactic acid efflux (Halestrap and Price 1999; Juel 2008). Both isoforms need an ancillary protein, CD147 (EMMPRIN, basigin, neurothelin), in order to form a functional unit on the membranes (Wilson et al. 2002). Although the presence of MCT4 in equine muscle cell membranes has been confirmed in Western blots (Koho et al. 2006), the same antibody has failed to work in immunohistochemistry (Mykkänen et al. 2010).

In rats, the expression of MCT1 correlates highly with the oxidative capacity of muscles (McCullagh et al. 1996), but in man only minor fibre type variation in the expression of MCT1 is seen (Pilegaard et al. 1999a). In Standardbred horses MCT1 is expressed in all fibre types and the expression correlates with the oxidative capacity (Mykkänen et al. 2010). The expression of MCT1 increases with training in man and rodents (Bonen et al. 1998; Pilegaard et al. 1999b), but in horses the amount of MCT1 has been found to be similar in moderately trained and older, race-fit individuals (Koho et al. 2006).

To our knowledge, longitudinal studies of the fibre type distribution of MCT1 in muscles of horses in training have not been performed. The aim of the present study was to identify muscular changes associated with training in young NSCTs, and to investigate changes of MCT1 and CD147 in different fibres.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conflicts of interest
  8. Manufacturers' addresses
  9. References

Horses

Nine NSCT horses (5 colts, 4 fillies) born in Norway in 2005 were followed from the start of their training period until the end of their 3 year season. All horses were privately owned, and trained by 6 different trainers at 5 different training facilities. All horses were intended for early racing. One filly became lame early in the study period and was excluded. One colt had lameness problems towards the end of the study period, but was still included in the complete study. The project was approved by The National Animal Research Authority.

Experimental protocol

Percutaneous biopsy samples of the left middle gluteal muscle were collected on 4 occasions; O1 (15.12.06–09.05.07), O2 (November 2007), O3 (May 2008) and O4 (December 2008).

According to the trainers all horses were trained for 45–60 min 4–5 times a week throughout the study period. Before O1, training consisted of slow trotting and walking. By the time of O2, training against high resistance had been introduced for all horses. At O3, intervals of faster trotting had become a gradually increasing part of the training programme. At O4, one horse had started racing and 2 started shortly after.

At O1 the biopsy sample was obtained at the individual horse's training yard. At O2, O3 and O4, the sample collection took place at the Norwegian School of Veterinary Science, and was combined with determination of individual VLa4-values (treadmill speed corresponding to a blood lactate concentration of 4 mmol/l) in an incremental exercise test on a high speed treadmill1.

Muscle biopsies

The biopsy samples were collected from the midpoint of a line from the tuber coxae to the root of the tail according to the procedure described by Lindholm and Piehl (1974) after mild sedation of the horses. The biopsy needle was directed perpendicular to the skin and inserted to a fixed depth of 4 cm that had previously been marked on the needle. All biopsies were collected by the same person to avoid bias.

Two muscle samples were collected from each horse. One sample, intended for histological procedures, was rolled in talcum powder prior to freezing. Both samples were frozen in liquid nitrogen within 3–5 min after sampling. All samples were stored at −80°C until analysed.

Exercise test

Day 1: The horse was exercised at a moderate intensity on the treadmill (2000–2500 m). Subsequently, a biopsy sample of the left middle gluteal muscle was collected.

Day 2: All horses were weighed before the exercise test. The treadmill incline was set to 3°. After 4 min warm-up at 2 m/s, the speed was increased to 4 m/s. Thereafter, the horses ran 2 min at the speed steps 4, 5, 6, 7 and 8 m/s, respectively, while blood samples were drawn from an indwelling catheter in the left jugular vein during the last 15 s of each speed step. The blood was collected in Lithium-heparin blood collection tubes, stored on ice. The test was ended at lower speed steps if the horse showed signs of fatigue or poor trotting technique.

Within 30 min after sampling, the plasma lactate concentration ([lactateplasma]) was determined using a blood gas analyser equipped with a lactate analysing electrode (ABL 800 Flex)2. Plasma lactate was converted to blood lactate using the equation [lactateblood]= 0.4 + 0.61 *[lactateplasma] (Lindner 1997). The individual VLa4 from each exercise test was then calculated using the equation ln [lactateblood]=β01* (V [m/s]).

The values for β0 and β1 were determined from the data set of ln transformed [lactateblood] from each horse, and the equation was solved for [lactateblood]= 4 to give an individual VLa4-value.

Immunohistochemistry and histochemistry

The muscle samples were mounted on blocks using OCT embedding medium, and oriented so that myofibres could be cut transversely in a cryostat (Reichert-Jung)3 at −20°C. Serial sections were collected for immunohistochemistry and histochemistry.

Identification of myosin isoforms was performed in all samples with the commercially available monoclonal antibodies (Mab) N2-2614 (MHC I + IIA) and A4-744 (MHC IIA) (Karlström and Essén-Gustavsson 2002; Karlström et al. 2009). The sections used for MHC isoform staining were fixed in methanol for 5 min at +4°C, after which they were washed in PBS and blocked with 5% rabbit serum5. The sections were then incubated with primary MHC antibodies for 2 h at room temperature, after which they were stained with the Peroxidase-AntiPeroxidase (PAP) method5.

Horse MCT1 (GenBank accession No. AY457175.1), MCT4 (GenBank accession No. EF564279.2) and horse CD147 (GenBank accession No. EF564280.1) have been sequenced (Reeben et al. 2006). This information was used to synthesise C-terminal peptides of MCT1, MCT4 and CD147 used to immunise rabbits. Antibodies were subsequently harvested and purified with affinity chromatography. Peptide synthesis, immunisation and purification were done by Sigma Genosys6. The antibodies were tested and gave a single band in Western blots. Preincubation of the antibody with the peptide used to immunise the rabbits blocked the staining. The MCT4 antibody does not work in immunohistochemistry (Mykkänen et al. 2010) and was, therefore, not used in the present study.

Due to differences in the quality of the muscle samples, samples from O1 in 2 horses and from O2 in 6 horses were used for analysis of MCT1 and CD147 before training. Samples from O4 were used for analysis of MCT1 and CD147 after training. The sections used for MCT1 and CD147 staining were blocked with 5% goat serum5. The sections were then incubated with the primary antibody for 35 min at room temperature, after which they were stained with DakoCytomation EnVision+ System-HRP (DAP)-kit5. All immunohistochemical slides included a negative control for the primary antibody. The stained slides were dehydrated and mounted in DPX.

The same samples were analysed for oxidative capacity of individual fibres using the NADH tetrazolium reductase method (Novikoff et al. 1961). Additional sections from all samples were stained for the demonstration of mATPase activity after acid (pH 4.6) preincubation (Brooke and Kaiser 1970). The histochemically stained sections were mounted in Kaiser's Glycerol gelatine.

Photomicrographs were made of all stained sections7.

Measurements of muscle fibre characteristics

Since no commercial antibody against MHCIIX is available, fibres were classified as I, IIA, IIAX or IIX based on a combination of immunohistochemistry and mATPase stainings. Fibres staining as IIX in the mATPase staining that also showed antibody staining for MHCIIA were classified as hybrid IIAX fibres. Fibres staining as type I in the mATPase staining that also showed antibody staining for MHCIIA were classified as hybrid I-IIA fibres. Morphometric analyses were performed on the mATPase pictures. Approximately 200 adjacent cross-sectioned fibres from each biopsy sample were typed, counted and measured in representative areas of the sections without artifacts using a standard morphometric computer program (Leica QWin Pro V 3.5.1)8 modified for muscle analyses.

The amount of MCT1 and CD147 antibody in membranes of the different fibre types was analysed using AIDA image analyser9. As the system was not always able to separate membranes from adjacent fibres, measurements were done in areas where 2 adjacent fibres were of the same type. The measurements, therefore, represented the combined staining intensity of 2 membranes. Only membrane sections that were not close to capillaries were measured. Five to 10 membrane sections from each fibre type were analysed in each slide. The average staining intensity of membranes in type IIX fibres was set to be one, and used as baseline for each slide. The average staining intensity for MCT1 or CD147 in membranes of the fibre types I, IIA and IIAX were accordingly expressed as relative figures. Artifacts from the staining or sectioning processes, or lack of adjacent fibres caused some missing values.

The staining intensity for MCT1 and CD147 in cytoplasm was determined by measuring the intensity of the light that penetrated the stained fibres. These measurements were performed with Olympus Cell^P imaging system10. Five to 10 fibres of each type were examined in each slide. For clarity, measured light intensities were mathematically converted to staining intensities. The average staining intensity in type IIX fibres was set to one, and intensities measured in other fibre types in the same slide were expressed as relative figures.

The same technique was used to evaluate the staining intensity in NADH tetrazolium reductase stainings.

Enzyme activities

Freeze-dried muscle samples from O1 and O4 were dissected free of fat, connective tissue and blood under a dissection microscope and weighed. Each sample was then homogenised in ice cooled 0.1 M phosphate buffer at pH 7.3. The activities of citrate synthase (CS) as a marker for oxidative capacity, 3-OH-acyl-CoA-dehydrogenase (HAD) as a marker for lipid oxidation, hexokinase (HK) as a marker for blood glucose utilisation and lactate dehydrogenase (LDH) as a marker for glycolytic capacity in the muscle were analysed at room temperature (Essén et al. 1980; Essén-Gustavsson et al. 1983).

Statistics

The results are expressed as mean with 95% confidence interval. Analysis of variance (ANOVA) with repeated measurements was used to study the development of muscle characteristics throughout the study period. For comparison of groups, ANOVA was performed. Differences were considered significant at P≤0.05.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conflicts of interest
  8. Manufacturers' addresses
  9. References

The mean bodyweight with 95% confidence interval was 467 [442–491] kg at O2, 461 [429–493] kg at O3 and 484 [448–521] kg at O4.

VLa4

One of the horses was extremely excited during exercise tests on the treadmill. The work intensity of this particular horse could not be controlled and determination of a reliable VLa4 failed. Hence, VLa4 values from this horse are missing. In another horse, the exercise test at O4 was not performed due to lameness. Mean values with 95% confidence intervals for VLa4 were 6.2 [5.8–6.6] m/s at O2, 7.1 [6.8–7.4] m/s at O3 and 6.8 [6.6–6.9] m/s at O4. All horses had higher VLa4-values at O3 and O4 than at O2.

Muscle fibre composition and fibre type area

Three samples from O1, one sample from O2 and one from O3 contained high amounts of type I fibres, far beyond what has previously been reported in this breed (Karlström et al. 2009) as well as in Standardbreds (Ronéus 1993). They were also not in line with other observations from the same horse and, therefore, excluded. A thorough description of this phenomenon will be presented in a separate paper (Ihler et al. unpublished data).

No hybrid I-IIA fibres were found. The percentage of type IIX fibres decreased significantly with time, while the percentage of type IIAX fibres increased. There was also a tendency for increasing type I fibre percentages (P = 0.09). The percentage of type IIA fibres did not change significantly during the study period (Table 1).

Table 1. Mean (95% confidence interval) fibre type composition and fibre type area in the middle gluteal muscle of young Norwegian-Swedish Coldblooded Trotters in training at 4 sampling occasions
Sampling occasion1234
N (age in months)5 (18–22)6 (28–31)7 (34–37)8 (41–44)
  1. N, number of observations.

Fibre types (%)    
 I11 (4–17)13 (7–19)17 (11–22)15 (9–21)
 IIA39 (28–50)37 (30–44)45 (41–49)40 (34–46)
 IIAX8 (4–13)10 (7–14)9 (7–11)14 (4–23)
 IIX42 (29–56)40 (31–49)29 (23–36)31 (28–34)
Fibre type area (µm2)    
 I1947 (1313–2580)1646 (1204–2089)2045 (1507–2583)2652 (2056–3248)
 IIA2073 (1128–3018)2612 (2079–3173)2732 (2278–3187)3076 (2631–3521)
 IIAX2535 (1887–3183)3851 (3037–4667)3513 (2699–4327)3731 (2519–4942)
 IIX3908 (2055–5761)4647 (3318–5976)4515 (3096–5934)5578 (4069–7086)
Mean area (µm2)3026 (1949–4104)3571 (2678–4465)3291 (2530–4053)3885 (3379–4390)

There was a tendency for increasing cross-sectional areas of the type IIA fibres (P = 0.09), while no significant change was seen in the areas of the fibre types I, IIAX and IIX (Table 1).

Monocarboxylate transporter 1, CD147 and NADH dehydrogenase

Differences between fibre types: Overall, the expression of MCT1 in membranes was significantly higher in the fibre types I, IIA and IIAX than the reference value from IIX fibres. In the latter, the membranes were minimally stained or not stained at all (Fig 1). The expression of membrane MCT1 was also significantly higher in the fibre types I and IIA than in IIAX fibres.

image

Figure 1. Relative intensities of staining for MCT1 and CD147 in membranes of the fibre types I, IIA and IIAX before and after training. The dotted line represents the average staining intensity in IIX fibres, which was used as reference in each slide. Error bars represent 2 s.e. B=before training, A=after training.

Download figure to PowerPoint

CD147 was expressed in the membranes of all fibre types. Still, the expression was significantly higher than the reference in IIA and IIAX fibres, with a tendency for higher expression also in type I fibres (P = 0.06; Fig 1).

The cytoplasmic contents of both MCT1 and CD147 were significantly higher than the reference from IIX fibres in the fibre types I, IIA and IIAX (Fig 2). The cytoplasmic contents of MCT1 were significantly higher in type I fibres than in IIA and IIAX fibres, while no difference was seen between IIA and IIAX fibres. There was a tendency for higher expression of CD147 in type I fibres than in IIA fibres (P = 0.06), but there was no difference between IIAX fibres and the fibre types I and IIA.

image

Figure 2. Relative intensities of staining for MCT1 and CD147 in cytoplasm of fibre types I, IIA and IIAX before and after training. The dotted line represents the average staining intensity in IIX fibres, which was used as reference in each slide. Error bars represent 2 s.e. B=before training, A=after training. *=significant change in paired observations.

Download figure to PowerPoint

The intensity of the NADH tetrazolium reductase staining was significantly higher than the reference in the fibre types I (mean [confidence interval]: 1.35x [1.31x–1.40x]), IIA (1.18x [1.15x–1.21x]) and IIAX (1.14x [1.11x–1.17x]). The differences between the latter 3 fibre types were also significant.

Changes with training: Paired observations were analysed for changes with training. No significant changes were identified in the relative distribution of MCT1 and CD147 in membranes of different muscle fibre types. All observations (including nonpaired) before and after training are presented in Figure 1.

The relative cytoplasmic contents of both MCT1 and CD147 seemed to increase with training in the fibre types I, IIA and IIAX. In this low number of observations, these changes were only significant for MCT1 in IIAX fibres and for CD147 in IIA fibres. All observations (including nonpaired) before and after training are presented in Figure 2.

The relative distribution of the NADH tetrazolium reductase staining did not change with training.

Muscle enzymes

Due to the exclusion of some biopsies as described above, muscle enzyme activities before and after training could only be compared in 5 horses. Significant increases were seen in the activities of CS and HAD (Table 2).

Table 2. Mean [95% confidence interval] activity of the enzymes Citrate-synthase (CS), 3-OH-acyl-CoA-dehydrogenase (HAD), Hexokinase (HK) and Lactate-dehydrogenase (LDH) in muscles of 5 Norwegian-Swedish Coldblooded Trotters before and after training
EnzymeBefore trainingAfter trainingP value (paired t test)
  1. Enzyme activity is given in mmol/kg d.w./min. P values from paired t tests are also given.

CS28.1 [24.6–31.6]31.6 [28.1–35.1]0.03
HAD33.2 [29.5–36.9]35.4 [31.6–39.1]0.03
HK3.0 [1.9–4.1]4.1 [3.0–5.2]0.16
LDH1703 [1348–2058]1975 [1620–2330]0.26

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conflicts of interest
  8. Manufacturers' addresses
  9. References

The present study identifies changes in muscle fibre composition of young NSCT horses that are in agreement with the theory of fibre type transitions in a graded and orderly sequential manner from fast to slow with training (Pette 1998). Similar observations have been reported in a previous study of the same breed (Karlström et al. 2009), as well as in studies of Standardbreds (Ronéus et al. 1992; Ronéus 1993). According to the fibre type dependent distribution of the NADH-staining, these changes reflect an overall increase in the oxidative capacity, further supported by the increased activity of CS and HAD.

The results of the present study also indicate that the expression of MCT1 is related to the oxidative capacity of the fibres, with the highest expression in the most oxidative fibre types. This is in agreement with earlier findings in man, rat and Standardbred horses (Fishbein et al. 2002; Hashimoto et al. 2005; Mykkänen et al. 2010). The latter study also identified CD147 in the membranes of type IIX fibres that hardly expressed membrane MCT1 in agreement with the results of the present study. This finding is probably related to the fact that CD147 acts as a chaperone for both MCT1 and MCT4 (Kirk et al. 2000; Wilson et al. 2002).

In man and rat, training is associated with increased expression of MCT1 (Juel 2008) and it has been shown that training can activate the MCT1-gene (Zoll et al. 2006).

No training effects were detected on the relative distribution of membrane MCT1 or membrane CD147 between the different fibre types in the studied horses. The method that was used only allows for comparison of staining intensities between different fibre types within the same slide. Therefore, if a training-induced increase in staining intensity was equal in all fibre types, no changes in the relative staining intensity would be observed. To study this, a quantitative analysis of MCT1 and CD147 needs to be combined with the immunohistochemical analysis. Although the oxidative capacity can increase even in IIX fibres with training (Snow and Valberg 1994; Karlström et al. 2009), a previous study using Western blots did not reveal differences in the muscular contents of membrane MCT1 between moderately trained and older, race-fit horses (Koho et al. 2006).

In contrast, there was a tendency for the relative cytoplasm contents of MCT1 and CD147 to increase with training in the fibre types I, IIA and IIAX compared to the reference value from IIX fibres. The mitochondrial volume density in muscle fibres increases with training (Tyler et al. 1998) and the presence of MCT1 and CD147 in mitochondrial membranes has been demonstrated (Benton et al. 2004; Butz et al. 2004; Hashimoto et al. 2006). Mitochondrial MCT1 and CD147 seems to represent the most likely explanation for the staining of the cytoplasm as also indicated by previous results in young Standardbreds (Mykkänen et al. 2010).

The percentage of type IIX fibres, the fibre type with the lowest expression of MCT1 and the lowest oxidative capacity, decreased with training, while the percentages of other fibre types expressing more MCT1 and with higher oxidative capacities increased. This indicates that not only the oxidative capacity, but also the overall expression of MCT1 in the middle gluteal muscle of NSCT horses increases with training. Future studies including Western blot analysis are needed to confirm this theory.

Since lactate may serve as a substrate for ATP regeneration in muscle fibres with a high oxidative capacity and a high expression of MCT1, an increased proportion of such fibres would increase the capacity of the muscle to oxidise lactate. Accordingly, more substrate would be available for oxidative regeneration of ATP, and less lactate would be released into the blood, causing a delay in the onset of blood-lactate accumulation. This theory agrees with the higher VLa4-values observed after a period of training.

The volume and intensity of the training could not be standardised or quantified in this study, since the horses were privately owned and trained by 6 different trainers. However, all horses performed both resistance and interval training. Type IIX fibres are known to be recruited only at high exercise intensities (Snow and Valberg 1994). Since the percentage of IIX fibres decreased and the percentage of IIAX fibres increased after the training period, it appears plausible that adaptations in recruited IIX fibres include transformation to IIAX fibres with concomitant increase in oxidative capacity and expression of MCT1.

The samples were taken after moderate intensity exercise. This may have influenced the results, since a recent report shows that even a single bout of exercise may increase the amount of MCT1 without activation of MCT1-gene (Bickham et al. 2006).

The topographic heterogeneity of the equine middle gluteal muscle has been well documented; with higher contents of type I fibres in deep parts of the muscle (Bruce and Turek 1985; Rivero et al. 1993; Karlström et al. 1994). It is, therefore, essential to standardise the sampling point thoroughly. We marked all biopsy needles at 4 cm to make sure that all samples were collected from the same depth.

In growing horses, the absolute depth of 4 cm will gradually become more superficial. The increase in bodyweight was so small that it was considered not to influence the relative sampling depth. A superficial sampling site was chosen, as the largest breed-related differences in muscle fibre composition have been found in these parts of the muscle (Rivero et al. 1995). Comparable sampling depths have been used in most other studies of horses, which is of importance when comparisons between breeds are done.

In conclusion, muscular adaptations to training in young NSCT horses are similar to those seen in Standardbreds. The results indicate that MCT1 and CD147 are expressed in the fibres in the order type I>IIA>IIAX>IIX, which also relates to the oxidative capacity seen among the fibre types. Furthermore, training may increase the expression of MCT1 and CD147 in muscle fibre cytoplasm, but this has to be confirmed with a larger number of horses.

Manufacturers' addresses

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conflicts of interest
  8. Manufacturers' addresses
  9. References

1 Säto AB, Knivsta, Sweden.

2 Radiometer Medical ApS, Brønshøj, Denmark.

3 Cambridge Instruments GmbH, Nussloch, Germany.

4 Alexis Biochemicals, Lausen, Switzerland.

5 Dako, Glostrup, Denmark.

6 Sigma Genosys, Cambridge, UK.

7 Nikon Coolpix Microscope system, Japan.

8 Leica Microsystems, Cambridge, UK.

9 Raytest Isotopenmeßgeräte GmbH, Straubenhardt, Germany.

10 Olympus Biosystems GmbH, München, Germany.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conflicts of interest
  8. Manufacturers' addresses
  9. References
  • Benton, C.R., Campbell, S.E., Tonouchi, M., Hatta, H. and Bonen, A. (2004) Monocarboxylate transporters in subsarcolemmal and intermyofibrillar mitochondria. Biochem. Biophys. Res. Commun. 323, 249-253.
  • Bickham, D.C., Bentley, D.J., Le Rossignol, P.F. and Cameron-Smith, D. (2006) The effects of short-term sprint training on MCT expression in moderately endurance-trained runners. Eur. J. appl. Physiol. 96, 636-643.
  • Bonen, A., McCullagh, K.J., Putman, C.T., Hultman, E., Jones, N.L. and Heigenhauser, G.J. (1998) Short-term training increases human muscle MCT1 and femoral venous lactate in relation to muscle lactate. Am. J. Physiol. 274, E102-E107.
  • Brooke, M.H. and Kaiser, K.K. (1970) Muscle fiber types: how many and what kind? Arch. Neurol. 23, 369-379.
  • Bruce, V. and Turek, R.J. (1985) Muscle fibre variation in the gluteus medius of the horse. Equine vet. J. 17, 317-321.
  • Butz, C.E., McClelland, G.B. and Brooks, G.A. (2004) MCT1 confirmed in rat striated muscle mitochondria. J. appl. Physiol. 97, 1059-1066.
  • Essén, B., Lindholm, A. and Thornton, J. (1980) Histochemical properties of muscle fibres types and enzyme activities in skeletal muscles of Standardbred trotters of different ages. Equine vet. J. 12, 175-180.
  • Essén-Gustavsson, B., Lindholm, A., McMiken, D., Persson, S. and Thornton, J. (1983) Skeletal muscle characteristics of young Standardbreds in relation to growth and early training. In: Equine Exercise Physiology, Eds: D.H.Snow, S.Persson and R.J.Rose, Granta Editions, Cambridge. pp 200-210.
  • Fishbein, W.N., Merezhinskaya, N. and Foellmer, J.W. (2002) Relative distribution of three major lactate transporters in frozen human tissues and their localization in unfixed skeletal muscle. Muscle Nerve 26, 101-112.
  • Halestrap, A.P. and Price, N.T. (1999) The proton-linked monocarboxylate transporter (MCT) family: structure, function and regulation. Biochem. J. 343, 281-299.
  • Hashimoto, T., Hussien, R. and Brooks, G.A. (2006) Colocalization of MCT1, CD147, and LDH in mitochondrial inner membrane of L6 muscle cells: evidence of a mitochondrial lactate oxidation complex. Am. J. Physiol. Endocrinol. Metab. 290, E1237-E1244.
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