Indirect myosin immunocytochemistry for the identification of fibre types in equine skeletal muscle.

The histochemical ATPase method for muscle fibre typing was first described by Brooke and Kaiser in 1970. However, problems have been found with the subdivision of type II fibres using this technique. To determine whether indirect myosin immunocytochemistry using anti-slow (5-4D), anti-fast (1A10) and anti-fast red (5-2B) monoclonal antibodies with cross reactivity for type I, II and IIa fibres, respectively, in a number of species, could identify three fibre types in equine skeletal muscle, data on fibre type composition and fibre size obtained using the two different techniques were compared. Results indicate that different myosin heavy chains can coexist in single equine muscle fibres. Type I and type II fibres were identified by immunocytochemistry, but subdivision of type II fibres was not possible. Although the percentage of type I and type II fibres was not significantly different for the two techniques, a few fibres reacted with both the 1A10 and 5-4D antibodies.     

Fibre type proportions of the buccinator muscle in clinically normal adult dogs

In order to provide normal morphological data and reference values for fibre type proportions, against which changes due to facial nerve paralysis or primary facial muscle myopathies can be compared, a histological and histochemical study was carried out on the buccinator muscle of clinically normal adult dogs. Using myosin adenosine triphosphatase under acidic pre-incubation (pH 4.3) conditions, three histochemical fibre types--1, 2A and 2C were recognised. There was no significant difference in mean diameter between left- and right-sided buccinator muscle samples for type 1 versus 2. Mean diameters of fibre types were: type 1, 27.02 +/- 6.61 microns and type 2, 29.05 +/- 7.75 microns. The histographical distribution of fibre type diameters was unimodal. The buccinator muscle had a type 2 fibre predominance with a mean type 1 to type 2 ratio of 32:68. The number of fibres with internal nuclei was less than or equal to 1 per cent, atrophy-hypertrophy factors typically were less than or equal to 200, and variability coefficients approximated 250. Muscle spindles were not observed in any muscle sample.     

Effects of repeated biopsying on muscle tissue in horses

The collection of muscle samples in horses to estimate their performance potential, adaptations to training and diagnosis of exertional myopathies is of increasing interest. This study was designed to examine whether repeated biopsying of the gluteus medius muscle at 7 week intervals alters morphology and muscle fibre variables in the sampling area. The gluteus had been biopsied 3 times previously, each biopsy separated by a 7 week interval. Seven weeks after the last biopsy, this study was initiated. Percutaneous needle biopsies were taken from left and right muscles at 2 depths (2 and 6 cm) and at 2 sites for each depth (22 and 25 cm from the tuber coxae) of 7 mature Thoroughbreds. The 22 cm site was located close (2 cm caudal) to an area of the muscle that had been biopsied previously. The 25 cm site was 5 cm apart from this area. A total of 52 samples were available for histology, electron microscopy and immunohistochemistry. The latter was used to study percentages, sizes and capillaries of muscle fibre types. Most muscle samples collected (75%) had normal morphology, but the remaining 25% showed signs of muscle repair. In some circumstances, they showed regenerative signs of complete muscle repair, while in others ineffective muscle repair (scar formation) was evident. Nevertheless, sufficient normal tissue was always available for measuring routine muscle variables. Samples collected 3 cm laterally apart showed large differences with regard to muscle fibre type variables examined. These results show that repeated muscle samples in intervals of 7 weeks do not have effects that would impair evaluation of muscle fibre variables for diagnosis of effectivity of conditioning programmes. Furthermore, they emphasise that the only way of keeping variations through sample collection technique small is to standardise all biopsying techniques accurately.     

Muscle cellularity at cranial and caudal levels of the trunk musculature of commercial size sea bass, Dicentrarchus labrax (Linnaeus, 1758)

In eight specimens of Atlantic sea bass of commercial size (congruent with 350 g) muscle cellularity was studied at two selected sampling levels of the trunk axial musculature: caudal (anal opening) and cranial (fourth radius of the dorsal fin). The following parameters were quantified at both sampling levels: white muscle cross-sectional area, white muscle fibre diameter (900-1200 fibres), muscle fibre number and muscle fibre density. Results showed a higher total cross-sectional area at cranial than at caudal level (P < 0.05), what is related with their different gross morphology. However, the white muscle fibre size distribution, as well as the muscle fibre number and density did not show significant differences between them. This study contributes to typify muscle fibre sampling in sea bass of commercial size what is of great interest for morphometric studies where white muscle cellularity is commonly correlated with textural or organoleptic parameters.     

Effects of parent Shamo cocks on the histochemical properties of M. iliotibialis lateralis and M. supracoracoideus on their crossbred broilers

1. Four Shamo (a Japanese game bird) cocks showing different characteristics in the histochemical properties of M. iliotibialis lateralis (ITL) were crossed with White Rock hens to produce male and female crossbred broilers of the 4 lines (90 d of age). Normal broilers (56 d) were used, for comparison. 2. Histochemical properties of ITL and M. supracoracoideus (SC) were compared among the crossbred lines and normal broilers. Myofibres were divided into Types II R, II I and II W showing high, moderate and low reduced nicotinamide adenine dinucleotide dehydrogenase (NADH-DH) activities, respectively. 3. In the ITL of the crossbred cockerels, the percentage of Type II R and II I fibres decreased and conversely Type II W increased in comparison to those in the Shamo. 4. Sex differences of the histochemical properties were recognised only in the ITL of the crossbred, in which the percentage of Type II R fibres was greater in the male. 5. The different characteristics of the parent Shamo cocks were reproduced only in the different fibre type composition of the ITL muscle in the crossbred cockerels. 6. The histochemical features of fibre type seemed to develop with bird age, particularly subsarcolemmal accumulation of formazan granules (indicating high NADH-DH activity) in Type IIR fibres. 7. Breed, line, sex and age differences in the histochemical properties were demonstrated clearly in ITL but not in SC.     

Muscle characteristics in young Norwegian-Swedish Coldblooded Trotters and associations with breeding index, body size and early training

Reasons for performing study: The trotting speed of Norwegian‐Swedish Coldblooded Trotters (NSCTs) is, although inferior to Standardbred speeds, gradually increasing. This development is mainly attributed to breeding progresses. Characteristics of the middle gluteal muscle have recently been described in a small number of these horses but the actual spread in muscle parameters within the population is unknown. Objectives: To describe the characteristics of the middle gluteal muscle and their naturally occurring variability in young NSCT horses and investigate possible association with body size, individual breeding index and early training. Methods: Biopsies of the middle gluteal muscle of 144 NSCT horses born in 2005 were collected at age 17–24 months. The horses were randomly selected by stratified sampling with gender and paternal progeny as stratification factors. The muscle samples were analysed for fibre type composition, fibre type areas, capillary supply and the activity of the key metabolic enzymes citrate synthase, 3‐OH‐acyl‐CoA‐dehydrogenase and lactate dehydrogenase. Correlation analysis including muscle parameters and body size, individual breeding index and early training was performed. Results: Large interindividual differences were identified in muscle characteristics. Male horses had higher percentages of type IIA fibres and lower percentages of type IIX fibres than age‐matched females. Fibre type areas followed the order type IIX>IIAX>IIA>I, while the capillary supply per fibre type area increased in the opposite order. No gender‐related differences were found in fibre type areas or capillary supply. Height was correlated to the relative area of type I fibres. Early training was associated with increased oxidative capacity and increased percentage of type IIA fibres. The lowest breeding index was found in untrained horses. Conclusions: Muscle characteristics differ among young NSCT horses, but the innate oxidative capacity seems to be lower than in Standardbreds of comparable age. Early training may influence on these parameters.     

Training and growth induced changes in the middle gluteal muscle of young Standardbred trotters

The middle gluteal muscle of five, two-year-old untrained trotters was investigated by repeated needle biopsy sampling over a training period of six months. A second group of five, three-year-old untrained horses was included to examine the effect of growth. After the training period increases were found in the relative distribution of slow twitch (ST) fibres from 18 per cent to 25 per cent and fast twitch (FTa) fibres from 36 per cent to 45 per cent, and a decrease in FTb fibres from 46 per cent to 30 per cent. A proportionally equal reduction (approximately 18 per cent) in the cross sectional area of all fibre types was observed after the first two months of training succeeded by an increase to approximately pretraining levels at the end of the period. The number of capillaries per fibre was enhanced from 1.7 to 2.4. Proliferation of capillaries occurred around fibres of all types. The metabolic adaptations showed increases in the activities of 3-hydroxy-acyl-CoA dehydrogenase (HAD) (50 per cent) and citrate synthase (31 per cent). Growth had no effect on the relative fibre type distribution nor the capillary per fibre ratio, but as the mean fibre area increased 36 per cent (primarily because of increases in FT fibres) the number of capillaries/mm2 was lower in the older untrained horses (350 capillaries/mm2) compared with the younger untrained ones (460 capillaries/mm2). Increase with growth was found in the activity of phosphorylase and HAD and a decrease was seen in the activity of hexokinase. It is concluded that the training programme exclusively induced alterations which improved the aerobic capacity of the muscle.     

The Post-larval Development of Lateral Musculature in Gilthead Sea Bream Sparus aurata (L.) and Sea Bass Dicentrarchus labrax (L.)

Fibre-type differentiation of lateral musculature has been studied in gilthead sea bream Sparus aurata (L.) and sea bass Dicentrarchus labrax (L.) during post-larval development using ultrastructural, histochemical and morphometric techniques. The study showed three muscle layers: red, intermediate (or pink) and white. Initially, most of the red muscle showed low myosin ATPase (m-ATPase) activity fibres, whereas near the transverse septum some small high m-ATPase activity fibres appeared and later acquired a rosette aspect. Afterwards, during adult growth the red muscle showed a histochemical mosaic appearance. The pink muscle in sea bass was observed at the beginning of juvenile development by the oxidative technique (NADH-RT reaction) whereas in gilthead sea bream it was also observed at the end of larval development. The pink layer consists of high m-ATPase activity fibres. However, along the muscle development other low and moderate m-ATPase activity fibres were observed close to the red and white muscles, respectively. The white muscle of juvenile fish showed a histochemical mosaic appearance near the pink muscle. In adult specimens the mosaic white muscle spread out occupying the whole of the myotome. Morphometric analysis shows a significant increase in mean fibre diameter during post-larval development, as shown by the Student's t-test (hypertrophic growth). Skewness and kurtosis values of fibre diameters point to the generation of a new fibres from the myosatellite cells (hyperplastic growth).     

Evaluation of functional muscle anatomy scalability in the canine hind limb

In contrast to other mammals, the large variation in dog sizes is not accompanied by any significant genetic re-organization. In order to study the relationship between body mass, limb length and the functional anatomical muscle parameters of the canine hind limb, a large dataset comprising of muscle masses, optimal muscle fibre lengths and physiological cross-sectional area's (PCSA) were acquired for twenty-five muscles in ten dogs of sizes varying between 20 kg and 52 kg. The potential of body mass and limb length for reliably scaling individual muscle masses, optimal muscle fibre lengths and PCSA's were examined. For the majority of the muscles of the canine hind limb, neither body mass nor limb length were reliable scaling parameter for either muscle masses, PCSA's and optimal fibre length. These results indicate the need of a breed-specific approach to musculoskeletal modelling in future canine musculoskeletal research.     

Enzyme histochemistry on muscle biopsies as an aid in the diagnosis of diseases of the equine neuromuscular system: a study of six cases

Muscle biopsies from six horses with clinical histories of muscle atrophy, muscle tremors, myopathic symptoms, unsteadiness of pelvic limbs and progressive ataxia were examined. Muscle biopsies were studied with enzyme histochemical techniques to evaluate the diagnostic values of these methods in cases suspected of suffering from neuromuscular disorders. Hypertrophy, atrophy, fibre splitting, waxy degeneration, phagocytosis and necrosis were seen in haematoxylin eosin stained sections of the different cases. Fibre type predominance and fibre type grouping were seen in the calcium ion stimulated myosine ATP-ase (Ca-ATP-ase) stained sections of some cases. 'Moth-eaten fibres' were demonstrated in three cases by staining with NADH: nitro blue tetrazolium oxidoreductase (NADH-TR), succinate dehydrogenase (SDH), NADH dependent malate dehydrogenase, cytochrome c oxidase and by lactate dehydrogenase. The catabolic enzymes, acid phosphatase (ACP) and 5'-nucleotidase were active in cases with fibre phagocytosis. The oxidative part of the pentose phosphate pathway in myopathic tissue seemed to be important in three cases, demonstrated by the increased activity of glucose-6-phosphate dehydrogenase (GPDH) and 6-phosphogluconate dehydrogenase (PGDH). The important feature of diseased horse muscle was that the pathohistochemical changes were exactly the same as in diseased skeletal muscles of humans. The application of tissue saving enzyme histochemical techniques can be recommended in the study of muscle tissue from horses suffering from suspected neuromuscular disorders.     

Insulin sensitivity and muscle characteristics in calves at different levels of physical activity

This study was undertaken to investigate insulin sensitivity and muscle characteristics in calves at different levels of physical activity. Ten male calves of the Swedish Holstein breed were allocated to either an exercise or a non-exercise group. The exercise group performed light work on a treadmill, 5-15 min at 10-12 occasions during 4-5 weeks. The treadmill speed varied from 0.6 to 1.5 m/s. At the beginning and end of the experimental period, the hyperinsulinemic euglycemic clamp technique was used to assess insulin sensitivity and gluteus medius muscle biopsies specimens were obtained for histochemical and biochemical analyses. There were no significant differences in insulin sensitivity, muscle fibre types, enzyme activities or glycogen content between the groups before or after the experimental period. The areas of type I, IIA, IIB fibres and mean muscle fibre area increased significantly in both groups during the experimental period. Muscle fibre characteristics and insulin sensitivity varied among the calves. Insulin sensitivity correlated positively to the percentage of type I fibres at the start of the experiment, and negatively to the mean muscle fibre area at the end of the experimental period. The results indicate that insulin sensitivity in young calves is associated with the change seen in muscle fibre areas with growth, but not by a short period of light physical activity.     

Oxidative capacity of skeletal muscle fibres in racehorses: histochemical versus biochemical analysis

The oxidative capacity of skeletal muscle fibre types was evaluated histochemically using the nicotinamide dinucleotide diaphorase (NADH-D) staining, and biochemically by measuring the activity of citrate synthase (CS) in both whole muscle samples and in pools of fibres of identified type. Duplicate determinations of the NADH-D staining pattern resulted in standard deviations (sd) between duplicates of 6 and 11 per cent for two observers. The NADH-D pattern was found to differ between observers. Duplicate determinations of CS activity in the same fibre pools resulted in an sd value of 2.9 mumol/g/min. Measurements of whole muscle CS activity did not provide information about the distribution of oxidative capacity among fibre types. The NADH-D stain and CS activity in fibre pools both showed that, in general, type I and IIA fibres had a higher oxidative capacity than type IIB fibres. Biochemical techniques also showed, however, that the CS activity in type I and IIA fibres of different horses could vary as much as twofold, whereas the NADH-D rating showed a high intensity staining for most type I and IIA fibres in all horses. Furthermore, type IIB fibres received a lower NADH-D rating than the other fibre types even when the CS activities were quite similar. For purposes of research, biochemical measurement of oxidative capacity in individual muscle fibre types provides valuable quantitative and comparative information. The ease of histochemical NADH-D staining in comparison to fibre dissections makes this technique more practical for routine estimates of the distribution of oxidative capacity among muscle fibres.(ABSTRACT TRUNCATED AT 250 WORDS)     

Morphometry of pectoral development in turkey breeding stock

1. Musculo-skeletal development was measured in males and females of two genetic lines of turkey breeding stock: one line had been selected mainly for egg production (E-line) while the other had been selected for meat production as well as egg production (M-line). 2. The M-line had a more rapid growth of body weight and breast muscle weight than the E-line but breast muscle yield (breast muscle weight/body weight) did not differ between lines. 3. From an early age, breast length and keel length were longer in the M-line than in the E-line. 4. In older birds, keel depth was greater in the M-line than in the E-line. 5. No differences between lines were detected for depth of the pectoralis muscle, number of muscle fibres in the depth of the pectoralis, or muscle fibre cross sectional areas.     

Neuroanatomy of the equine dorsal cricoarytenoid muscle: surgical implications

Reason for performing study: Studies are required to define more accurately and completely the neuroanatomy of the equine dorsal cricoarytenoid muscle as a prerequisite for developing a neuroprosthesis for recurrent laryngeal neuropathy. Objective: To describe the anatomy, innervation, fibre types and function of the equine dorsal cricoarytenoid muscle. Methods: Thirty‐one larynges were collected at necropsy from horses with no history of upper airway disease and 25 subjected to gross dissection. Thereafter, the following preparations were made on a subset of larynges: histochemical staining (n = 5), Sihler's and acetylcholinesterase staining for motor endplates (n = 2). An additional 6 larynges were collected and used for a muscle stimulation study. Results: Two neuromuscular compartments (NMC), each innervated by a primary nerve branch of the recurrent laryngeal nerve, were identified in all larynges. Stimulation of the lateral NMC produced more lateral displacement of the arytenoid cartilage than the medial NMC (P<0.05). The medial NMC tended to rotate the arytenoid cartilage dorsally. Motor endplates were identified at the junction of the middle and caudal thirds of each NMC. If fibre type grouping was present it was always present in both NMCs. Conclusions: The equine dorsal cricoarytenoid muscle has 2 distinct muscle NMCs with discrete innervation and lines of action. The lateral NMC appears to have a larger role in increasing cross‐sectional area of the rima glottidis. Potential relevance: This information should assist in planning surgical reinnervation procedures and development of a neuroprosthesis for recurrent laryngeal neuropathy.     

The effect of testosterone on gastrocnemius muscle fibres in growing and adult male and female rats: a histochemical,morphometric and ultrastructural study

In this study, the effect of testosterone on gastrocnemius muscle fibres in growing and adult rats (male and female) was examined using histochemical, morphometric and ultrastructural techniques. After physiological saline (PS), olive oil (OvO) or olive oil + testosterone (OvOT) injections on 72 rats (growing and mature, 36 male and 36 female), the sample tissues of fibre types of the gastrocnemius muscle taken were examined by histochemical [alkaline adenosine triphosphatase (alk-ATPase), acid ATPase (ac-ATPase)], morphometric and ultrastructural techniques. In PS-injected control groups, the gastrocnemius muscle of both sexes contained all the fibre types studied [slow-oxidative muscle fibres (type I), fast-oxidative glycolytic muscle fibres (type IIA) and fast-glycolytic muscle fibres (type IIB)]. The type I fibres had the smallest diameter, type IIA had a medium diameter and type IIB fibres had the largest diameter. In OvO-injected groups, it was observed that the OvO had little effect on the gastrocnemius muscles of either sex, although there was significant enlargement of type IIB fibres. After the injection of OvOT, hypertrophy of muscle fibres was determined by morphometric study. The biggest increase in diameter was on type I fibres. In addition, degenerations on some mitochondria, accumulation of lipid droplets on type I and type II fibres, an increase in glycogen particles, bifurcation of myofibrils, an increase in the number and diameter of units resembling T tubules and an increase in ribosomal content were also observed in the same group by transmission electron microscope. Consequently, it was determined that testosterone can induce protein synthesis in gastrocnemius muscle fibres, and induces changes in shape and size, and also can change the appearance and the number of fibres.     

Preliminary Study on Age- and Sex-Dependent Alterations in the Composition of Skeletal Muscle Fibers of Brasileiro de Hipismo Horses

The aim of this work was to characterize the distribution of myofibers in the gluteus medius muscle of inactive horses of the Brasileiro de Hipismo (BH) breed at different ages by means of histochemical analyses, according to sex and depth of the biopsy. A total of 78 inactive horses (9 castrated males, 35 stallions, and 34 females) of the BH breed, aged 1 to 4 years, were used. A percutaneous muscle biopsy was obtained with a 6.0-mm Bergström-type needle, which allowed the removal of muscle fragments at depths of 20 and 60 mm. Myofiber types were determined based on myofibrillar adenosine triphosphatase (mATPase) and nicotinamide dinucleotide tetrazolium reductase (NADH-TR) techniques. Morphometry of the fibers was determined based on cross-sectional area (CSA), mean frequency (F), and relative cross-sectional area (RCSA). The current study demonstrated that BH horses 3 and 4 years of age show a greater percentage of, and area occupied by, type IIA fibers and lower percentage of type IIX fibers in the gluteus medius muscle compared with horses 1 and 2 years of age. No difference was found between sexes in the frequency of and area occupied by the different fiber types at any of the depths and ages studied. In this study, females showed a greater CSA for all fibers in comparison with males, at 1 year of age. The results of the current study indicate that the gluteus medius muscle of inactive BH horses shows modifications in its structural and biochemical composition during the growth of the animals, leading to a better oxidative capacity.     

Different growth rates of male chicken skeletal muscles related to their histochemical properties

1. Early, M. pubo‐ischio‐femoralis pars medialis (PIF muscle) and late, M. iliotibialis lateralis pars postacetabularis (ITL muscle), maturing muscles were studied. These two muscles contained different populations of histo‐chemical fibre types.

2. The profile measurements of the muscles showed diphasic allometric growth relative to the weights. In the early stage of growth (up to 15 weeks after hatching), the muscle length, width and depth all increased, while in the later stage (from 15 to 35 weeks) muscle growth resulted mainly from the marked increase in the depth and to a lesser degree, from an increase in the width.

3. Type I fibres, observed predominantly in PIF muscle matured earlier than the Type II fibres of ITL muscle.

4. From these results, the detailed process of skeletal muscle growth in the chicken was discussed.     

Maternal dietary linoleic acid supplementation promotes muscle fibre type transformation in suckling piglets

As meat quality is basically dependent on muscle fibre characteristics, it is important to know how muscle fibres are regulated and transformed. This study aimed to investigate the effect of maternal dietary supplementation on muscle fibre types using 3% saturated fatty acid (palmitic acid, PA) or 3% unsaturated fatty acid (linoleic acid, LA) from 80 days of gestation to the weaning of offspring (25 days post‐natal). The results indicated that higher mRNA levels of MyHCI type genes were found in the soleus muscles of piglets that suckled from LA‐supplemented sows than from PA‐supplemented sows. In addition, LA treatment increased the gene expression of the type I muscle fibre marker troponin I (p < 0.01), suggesting that LA promoted muscle fibre type transformation to type I fibres. Moreover, PGC‐1α (p < 0.01) and MEF2c (p < 0.05) mRNA levels were higher in the piglets from the LA treatment group than in those from the PA treatment group. Furthermore, LA supplementation also significantly increased AMP‐activated protein kinase (AMPK) mRNA levels (p < 0.05), which is an upstream regulator of PGC‐1α. Collectively, these findings demonstrated that maternal dietary LA supplementation promoted muscle fibre transformation to type I fibre and that this process may be mediated through an AMPK‐dependent pathway.     

Effect of controlled exercise on middle gluteal muscle fibre composition in Thoroughbred foals

REASONS FOR PERFORMING STUDY: Most racehorses are trained regularly from about age 18 months; therefore, little information is available on the effect of training in Thoroughbred foals. HYPOTHESIS: Well-controlled exercise could improve muscle potential ability for endurance running. METHODS: Thoroughbred foals at age 2 months were separated into control and training (treadmill exercise) groups and samples obtained from the middle gluteal muscle at 2 and 12 months post partum. Muscle fibre compositions were determined by histochemical and electrophoretical techniques and succinic dehydrogenase (SDH) activity was analysed in each fibre type. RESULTS: All fibre types were hypertrophied with growth and type I and IIA fibres were significantly larger in the training than the control group at age 12 months. A significant increase of SDH activity was found in type IIX muscle fibres in the training group. CONCLUSIONS: Training in young Thoroughbred horses can facilitate muscle fibre hypertrophy and increase the oxidative capacity of type IIX fibres, which could potentially enhance stamina at high speeds. POTENTIAL RELEVANCE: To apply this result to practical training, further studies are needed to determine more effective and safe intensities of controlled exercise.     

Muscle glycogen depletion patterns during draught work in Standardbred horses

Muscle fibre recruitment was investigated during draught loaded exercise by studying glycogen depletion patterns from histochemical stains of muscle biopsies from the gluteus and semitendinosus muscles. Three Standardbred trotters performed several intervals of draught loaded exercise on a treadmill with 34 kp at a trot (7 m/sec) and with 34 and 80 kp, respectively at a walk (2m/sec). Exercise was continued until the horses were unwilling to continue. Glycogen depletion was seen in all three fibre types when trotting with 34 kp for 5 or 10 mins. When an equal weight resistance was pulled at a walk, glycogen depletion was first seen in type I fibres only, then followed by a small percentage of type IIA fibres after at least 1 h. When 80 kp was pulled at a walk both type I and IIA fibres showed glycogen depletion, and after at least 30 mins exercise a small percentage of type IIB fibres was also depleted. These results indicate that the muscle fibres are depleted, in order, from type I through IIA to IIB as the intensity or duration of draught work increases.