Cellular patterns of post-mortem glycogenolysis in longissimus dorsi muscles of pigs at different live weights

Synopsis Serial frozen sections of longissimus dorsi muscles from twelve Canadian Yorkshire breed pigs at different live weights (13–86kg) were stained for glycogen by the periodic acid-Schiff (PAS) reaction and reacted for NAD tetrazolium reductase to determine the mitochondrial content of muscle fibres. Sections from muscles taken immediatelypost mortem and at 5 hrpost mortem were compared and the patterns of glycogenolysis in high, intermediate and low mitochondrial content fibres were assessed on the basis of the percentage of initially PAS-positive fibres which became PAS-negative 5 hrpost mortem. This form of assessment was necessary because not all fibres were PAS-positive immediatelypost mortem. In smaller pigs, only a few muscle fibres depleted their stainable glycogen by 5 hr and most of these fibres had a high mitochondrial content. In larger pigs, most or all initially PAS-positive fibres became PAS-negative by 5 hrpost mortem. Fibres with a low mitochondrial content accounted for most of the glycogenolysis detected histochemically in larger animals. The overall percentage of PAS-positive fibres was related to glycogen concentration (mg/g),r=0.52,P<0.025, when samples with all PAS-positive fibres were excluded.     

Relationships between mitochondrial content of muscle fibres and patterns of glyogendepletion postmortem.

Sternomandibularis muscles were removed from slaughtered adult cattle immediately after exsanguination. On the basis of the density of diformazan granules deposited by a reaction for NAD tetrazolium reductase, approximately equal numbers of muscle fibres with high and low mitochondrial content were identified in serial frozen sections. In samples taken immediately after exanguination both types of muscle fibres exhibited glycogen phosphorylase activity and were stained equally by the periodic acid-Schiff (PAS) reaction for glycogen. In unstimulated muscle samples 1 hr postmortem, no loss of PAS staining was detected. In electrically stimulated samples 1 hr postmortem, large numbers of muscle fibres with a low mitochondrial content but only some muscle fibres with a high mitochondrial content became PAS-negative. Stimulation-induced glycogen depletion was completely prevented by the interfaicular injection of magnesium sulphate solution. In unstimulated samples between 5 and 24 hr postmortem, some muscle fibres with a high mitochondrial content but only a few muscle fibres with a low mitochondrial content became PAS-negative.     

Pathological changes in the microstructure of longissimus lumborum muscle from five breeds of pigs

The aim of the study was to determine the extent of histopathological changes in m. longissimus lumborum of PL, PLW, Duroc, Pietrain, and Pu?awska pigs (N = 30 per breed) aged 210 days. Changes in fibre size (atrophy, hypertrophy - giant fibres), changes in fibre shape (angular fibres), degenerative lesions (necrosis with phagocytosis) and connective tissue hypertrophy were evaluated. The percentage of individual pathological changes in m. longissimus lumborum of the analysed pig breeds was relatively low. Significantly more normal fibres were found in the muscles of Pu?awska compared to Pietrain pigs. Muscle fibre atrophy was the most frequent and extensive histopathological change. The muscles of Pu?awska pigs had significantly fewer atrophic, giant and angular fibres, significantly less necrosis with phagocytosis, and less animals with connective tissue hypertrophy compared to the other pig breeds. On the other hand, Pietrain pigs were characterized by a greater number of animals with giant fibres and a significantly higher proportion of giant fibres compared to the other breeds. Also the diameter of giant fibres was the largest in Pietrain, intermediate in PL and PLW, and the smallest in Duroc and Pulawska pigs. Moreover, current findings indicate that giant fibres may arise from each muscle fibre type (I, IIA and IIB). It is concluded that selection of pigs for increased leanness contributes to the incidence of histopathological changes, which may decrease pork quality.     

Fibre composition and enzyme activities in five different muscles from the Svalbard reindeer

beta R fibres (type I) constitute less than 10% of the semimembranosus and longissimus dorsi muscles and about twice as much of the gluteobiceps and flexor hallucis. Except for longissimus dorsi, 50% or more consist of alpha W (type IIB) fibres--in semimembranous, as much as 70%. Despite the comparatively large content of alpha W fibres, both the oxidative capacity and the capacity to metabolize fatty acids is high. Furthermore, unexpectedly small differences in oxidative capacity between the three fibre types beta R, alpha R and alpha W (I, IIA and IIB) are revealed by histochemical staining. These results indicate a tendency to bring the three fibre types closer together as regards metabolic activities, as an adaptation to the relatively tranquil life of this animal. However, the large content of alpha W fibres does not accord well with this way of life, as they guarantee quick movements. The comparatively high oxidative capacity of the alpha W fibres in the Svalbard reindeer and the fact that during starvation it is primarily alpha W fibres that contribute to the energy supply by protein degradation may nevertheless account for their abundant occurrence.     

Parallel-fibered muscles transplanted with neurovascular repair into bipennate muscle sites in rabbits.

Experiments were performed on 20 New Zealand White male rabbits. Our hypotheses were that (1) latissimus dorsi (LTD) muscles transplanted into the site of a bipennate rectus femoris (RFM) muscle with neurovascular repair would retain their parallel-fibered structure and (2) the parallel-fibered structure of latissimus dorsi grafts would reduce their total fiber cross-sectional area and adversely affect force development relative to that of bipennate rectus femoris grafts and muscles. Compared with their respective donor muscles, 120 to 150 days after grafting, latissimus dorsi and rectus femoris grafts showed no change in the number of fibers and a decrease in the mean single-fiber cross-sectional area to approximately 70 percent. The latissimus dorsi grafts, which remained parallel-fibered, developed maximum forces 34 and 23 percent of the values for fully activated rectus femoris grafts and muscles, respectively. The deficit in the maximum force of the latissimus dorsi grafts resulted primarily from the smaller total-fiber cross-sectional area as a result of the parallel-fibered structure.     

Muscle weights and succinic dehydrogenase distribution in the hind limb musculature of two rodents (Thryonomys gregorianus and Pedetes capensis) with different locomotory habits

The hind limb muscles of the spring hare (Pedetes capensis) were found to be relatively heavier than the hind limb muscles of the cane rat (Thryonomys gregorianus). The distribution of succine dehydrogenase activity was investigated in four of these muscles (m. gluteus superficialis, m.semimembranosus, m. biceps femoris and m.rectus femoris) from both animals. It was found that the spring hare had a higher proportion of low-activity fibres in all four muscles than the cane rat. All muscle fibre types were also smaller in diameter in the spring hare than the cane rat. These results are discussed in relation to the different locomotry habits of the two animals.     

Congenital muscle fibre type disproportion: clinical, morphological and biochemical findings in children

Muscle biopsies from quadriceps femoris muscle of normal subjects and subjects with symptoms of congenital myopathy, aged 1-3 years, were examined for morphological and biochemical differences. Four patients showed clinical signs of Congenital Fibre Type Disproportion (CFTD) as described originally by Brooke (1973), while the other cases showed only signs of hypotonia and diffuse weakness as described by Clancy et al. (1980). No morphological differences between patients with different clinical signs were found in any biopsy sample from the quadriceps femoris muscle, as regards fibre size; type I fibres were always smaller than type II fibres. The difference in fibre size was more than 15% in all cases, and the variability coefficient of the larger type II fibres was less than 250. Nevertheless, as regards fibre occurrence, two patients showed more type I fibres than type II fibres, four patients showed more type II fibres than type I fibres and one patient had a marked type II fibre predominance. Enzyme activities assayed in the crude mitochondrial fraction showed no abnormalities between normal subjects and patients. An increase in the activity of lactate dehydrogenase in the crude extract was found in all cases.     

Transitional stages in the histochemical development of muscle fibres during post-natal growth

Synopsis Serial frozen sections of longissimus dorsi muscles from seven pigs at different live weights (13 to 127 kg) were reacted for ATPase by the calcium method at an alkaline pH and for NADH oxidative activity. One hundred muscle fibres from each animal were identified individually in serial sections and their staining intensity was measured with a microscope photometer at 600 nm. For each section, staining intensity of fibres (% tranmission) was measured and converted to the nearest one-tenth unit of the range from the darkest to the lightest staining fibres. Frequency of occurrence of fibre types was plotted on a 10×10 grid using the range co-ordinates for NADH oxidative activity (vertical) and ATPase activity (horizontal). The commonly recognized histochemical fibre types in this muscle appeared as crowded areas in the grid but, in many cases, these areas were part of a continuous L shaped distribution. In fibres having an ATPase staining intensity of 1.0 and 0.9 units of the range, a continuous but skewed distribution with regard to NADH oxidative activity was detected. In fibres with NADH oxidative activity of 0.6 to 1.0 units of the range, a continuous but irregular distribution with regard to ATPase activity was detected. Within this range, there was some evidence of a growth-related shift towards weaker ATPase activity.     

Histochemical patterns in normal and splaylegged piglet muscle fibers

Summary The histochemical pattern of muscle fiber types of the longissimus dorsi and biceps femoris muscles was investigated in normal and splaylegged piglets at birth and seven days later. Only slight differences between the muscle fibers at birth were found using histochemical reactions for alkaline adenosine triphosphatase (ATPase), succinate dehydrogenase (SDH), phosphorylase (PH) activities, and for the periodic acid-Schiff (PAS) reaction. With the method for acid-preincubated ATPase activity, high activity was observed in Type I muscle fibers and low activity in Type II muscle fibers in animals of both groups investigated. However, a higher number of Type I fibers was found in muscles of normal piglets, suggesting a faster and more advanced process of transformation of Type II into Type I muscle fibers in unaffected animals. Thus the histochemical conversion appears to be retarded in muscles of splaylegged animals, which have a histochemical pattern similar to that of normal prenatal animals. Cholinesterase activity in motor endplates was well developed; its staining revealed smaller sized and irregularly arranged endplates in muscles of affected piglets. Fiber type differentiation in muscles of animals which recovered from splayleg becomes fully developed and comparable to normal piglets seven days after birth. The number of fibers which became converted from Type II to Type I was increased; the fiber types were differentiated with regard to the PAS reaction and to their ATPase, SDH and PH activities. Morphological features of motor endplates in muscles of normal and surviving splaylegged piglets are similar.Histochemical investigation of the fiber type differentiation thus suggests that full recovery occurs within the first week of postnatal life in muscles affected by pathological changes accompanying splayleg.     

Histochemical patterns in normal and splaylegged piglet muscle fibers

The histochemical pattern of muscle fiber types of the longissimus dorsi and biceps femoris muscles was investigated in normal and splaylegged piglets at birth and seven days later. Only slight differences between the muscle fibers at birth were found using histochemical reactions for alkaline adenosine triphosphatase (ATPase), succinate dehydrogenase (SDH), phosphorylase (PH) activities, and for the periodic acid-Schiff (PAS) reaction. With the method for acid-preincubated ATPase activity, high activity was observed in Type I muscle fibers and low activity in Type II muscle fibers in animals of both groups investigated. However, a higher number of Type I fibers was found in muscles of normal piglets, suggesting a faster and more advanced process of transformation of Type II into Type I muscle fibers in unaffected animals. Thus the histochemical conversion appears to be retarded in muscles of splaylegged animals, which have a histochemical pattern similar to that of normal prenatal animals. Cholinesterase activity in motor endplates was well developed; its staining revealed smaller sized and irregularly arranged endplates in muscles of affected piglets. Fiber type differentiation in muscles of animals which recovered from splayleg becomes fully developed and comparable to normal piglets seven days after birth. The number of fibers which became converted from Type II to Type I was increased; the fiber types were differentiated with regard to the PAS reaction and to their ATPase, SDH and PH activities. Morphological features of motor endplates in muscles of normal and surviving splaylegged piglets are similar. Histochemical investigation of the fiber type differentiation thus suggests that full recovery occurs within the first week of postnatal life in muscles affected by pathological changes accompanying splayleg.     

Body composition, muscle fibre characteristics and postnatal growth capacity of pigs born from sows supplemented with L-carnitine

Recently, it has been shown that supplementation of sows with L-carnitine increases their plasma concentrations of insulin-like growth factor (IGF)-I, and it has been hypothesized that this may stimulate fetal myogenesis. This study was performed to investigate whether piglets of sows supplemented with L-carnitine differ in muscle fibre characteristics, chemical body composition and postnatal growth capability from pigs of control sows. Muscle fibre characteristics and chemical body composition were determined at weaning in 21 piglets of control sows and 21 piglets of sows treated with L-carnitine with similar body weights; postnatal growth capability was determined from weaning until slaughter at a body weight of 118 kg in 80 pigs of control sows and 80 pigs of sows treated with L-camitine which had also similar body weights at weaning. Piglets of sows supplemented with L-carnitine did not differ in number, area, diameter and type (percentages of slow twitch oxidative + fast twitch oxidative fibres, and fast twitch glycolytic fibres) of muscle fibres in m. longissimus dorsi and m. semitendinosus and in chermical body composition (concentrations of dry matter, crude protein, crude fat) from piglets of control sows. Postnatal growth capability (body weight gains, feed conversion ratio) from weaning to slaughter as well as carcass composition (carcass yield, meat thickness, fat thickness) was also not different between pigs of sows treated with L-carnitine and pigs of control sows. In conclusion, data of this study do not support the hypothesis that L-carnitine supplementation of sows during pregnancy enhances fetal muscle fibre development and increases postnatal growth capability of the offspring.     

Cytophotometry of post mortem glycogenolysis in different histochemical types of muscle fibres of the pig.

Synopsis Serial transverse sections of porcine longissimus dorsi muscle (18 pigs, 50 to 178 kg live weight) were reacted for NAD tetrazolium reductase and ATPase at pH9.4, and for glycogen with the periodic acid-Schiff (PAS) reaction. Three histochemical types of muscle fibre were identified; (1) strong ATPase and weak NADH oxidative activity; (2) strong ATPase and intermediate NADH oxidative activity; and (3) weak ATPase and strong NADH oxidative activity. Immediatepost mortem samples from one side of each animal were compared with a laterpost mortem sample from the other side by measuring the absorbance of PAS-stained glycogen at 570 nm with a microscope photometer. Laterpost mortem absorbance was expressed as a percentage of immediatepost mortem absorbance in each category of muscle fibre in order to compensate for distributional error and different starting levels of glycogen. Muscle fibres with weak ATPase and strong NADH oxidative activity showed a progressive decrease in absorbance of PAS-stained glycogenpost mortem. In some animals, fibres with strong ATPase and intermediate or weak NADH oxidative activity showed an initialpost mortem increase in absorbance of PAS-stained glycogen which was then followed by a progressive decrease. The maximum rates of decrease in absorbance in the three fibre types did not differ to any great extent.     

Proteome analysis of early post-mortem changes in two bovine muscle types: M. longissimus dorsi and M. semitendinosis

To study early post-mortem changes in muscle tissues from bull calves, cytosole proteins from two muscles: M. longissimus dorsi (LD) and M. semitendinosis (ST) at 0 and 24 h after slaughter were analysed by 2-DE. Principal component analysis (PCA) and rotation testing were used to analyse the protein patterns in the two muscles in order to select protein spots that were significantly different at the two time-points. Selected proteins were identified by MALDI-TOF/TOF. Five proteins, namely cofilin, lactoylglutathione lyase, substrate protein of mitochondrial ATP-dependent proteinase SP-22, HSP 27 and HSP20, were changed in both LD and ST muscles during post-mortem storage. Fifteen additional protein changes were observed in either LD or ST muscles, and some of these changes have not previously been observed to change during post-mortem storage of bovine muscles. Further studies will reveal the relevance of these biomarkers for meat quality.     

Ultrastructural transformation of fast chicken muscle fibres induced by nerve cross-union

Summary The fast posterior latissimus dorsi (PLD) muscle of newly hatched chickens was transposed and cross-innervated by the slow-type nerve originally innervating the anterior latissimus dorsi (ALD) muscle. The innervation and the ultrastructure of the cross-innervated posterior latissimus dorsi (PLD-X) muscle was investigated from one week up to 18 months after the operation and compared with that of the control fast (PLD-C) and control slow (ALD-C) muscles. All nerve terminals in the PLD-X muscle were of the slow type. Yet the degree of ultrastructural transformation differed from fibre to fibre. Only about 30% of PLD-X fibres had transformed ultrastructure closely resembling the control slow fibres. In this group of maximally altered fibres, the myofibrils had large diameters, wide Z lines and indistinct M lines as the control slow fibres. The amount of mitochondria was increased to levels found in control slow fibres. The mean percentage of triads was also comparable to that of control slow fibres, being approximately by two thirds lower than in control fast fibres.The differences in the degree of ultrastructural transformation are presumably due to different plasticity of muscle cells at the time of cross-innervation. In the transposed PLD-X muscles large areas undergo degeneration and regeneration. It is suggested that an almost complete changeover of the fibre type is only brought about after cross-innervation of newly differentiating muscle cells, whereas partial alteration occurs after reinnervation of young myofibres.The skillful technical assistance of Dr. Z. Liková, Mrs. M. Sobotková, Ing. M. Doubek and Mr. H. Kunz is gratefully acknowledged.     

31P NMR study of postmortem metabolism in porcine and bovine muscles

31P NMR spectroscopy was used to evaluate interspecies differences in muscle fibre types and related postmortem metabolism. M. longissimus thoracis (MLT) and m. pectoralis superficialis (MPS) of bulls and MLT of pigs were investigated. In perchloric acid extracts NMR resonances for sugar phosphates (SP), inorganic phosphate (Pi), glycerophosphorylcholine (GPC), phosphocreatine (PCr), adenosine triposphate (ATP), adenosine diphosphate (ADP) as well as for NAD+/NADH could be distinguished. Also, glycogen and lactate contents and pH were determined. The relative contents of phosphorus compounds in bovine muscles of similar participation of muscle fibre are similar. Bovine muscles contain a relatively large proportion of PCr (48% of all phosphates 15 minutes post-mortem in MPS) whereas porcine MLT show lower PCr content (11% 15 minutes post-mortem). On the other hand, the ATP content is relatively higher in porcine MLT when compared with bovine muscles in the early phases of the postmortem processes. No NMR-detectable levels of GPC were measured in porcine MLT in contrast to bovine muscles. This suggests that the GPC content does not depend solely on the fibre participation but is also animal species determined. The 24 hour postmortem metabolism patterns of bovine and porcine muscles have many common traits. CP disappeared first followed by ATP. Simultaneously, the Pi concentrations increased. However, the content of SP remained relatively constant in porcine, but not in bovine muscles where it increased only gradually. The significantly higher concentrations of SP and lactate as well as the lower values of glycogen and pH measured for porcine as compared with bovine muscles suggest an enhanced glycolysis during the early phases of postmortem processes in porcine muscles.     

Comparative aspects of muscle fibre size and succinic dehydrogenase distribution in the longissimus dorsi muscle of several species of East African mammals

The distribution of succinic dehydrogenase enzyme activity was investigated in frozen sections of longissimus dorsi muscle taken from several species of East African game animal (giraffe, hartebeest, wildebeest, oryx, gerenuk and dik-dik) as well as local zebu cattle. Muscle fibres were classified as red (high succinic dehydrogenase activity), white (low activity) or intermediate. The mean diameter and percentage distribution of each fibre type were noted as well as the overall mean muscle fibre diameter (MFD) for each species. The diameters of red muscle fibres were found to be between 54 and 62% of the diameters of the white muscle fibres for all species with MFD differences between species being up to over 100%. The variation in MFD was found to be significantly and positively correlated with live weight, when zebu values were omitted. It was found that the variation in MFD was significantly related to the diameter of muscle fibre types and not to the percent distribution of these fibre types.     

Effect of training on fibre composition and phosphate metabolites in rest measured in vitro in muscles of young pigs.

1. Fibre type composition and phosphate metabolites were studied in m. longissimus thoracis (MLT), m. rectus femoris (MRF) and m. triceps brachii (MTB) in trained (N = 6) and sedentary (N = 6) pigs. 2. Samples were analyzed histochemically and by means of in vitro 31P NMR spectroscopy. 3. Training (duration 11 weeks) consisted of treadmill running at a speed of 1.1 m/sec. The daily exercise time of trained animals gradually increased from 10 min during the very first days to 60 min at the end of the 4th week. 4. During the final 7 weeks exercise time remained unchanged. Sedentary animals were not subjected to training. 5. A higher proportion of type beta R fibres in MRF, MTB and MLT and a lower proportion of type alpha W fibres were found in the trained group of animals compared to the control group. 6. In MLT no significant differences in the proportion of type alpha W were observed between both groups. 7. No significant differences in average fibre diameter of muscle fibres were found between groups. 8. No differences in concentration of phosphate compounds were observed between trained and sedentary groups. 9. Muscles with a higher proportion of type IIb fibres in both groups of pigs contained higher amounts of phosphocreatine (PCr) and were also characterized by a higher ratio of PCr to inorganic phosphate (Pi).     

Low temperature activation of post mortem glycogenolysis in bovine skeletal muscle fibres.

Synopsis Trimmed strips of stermomandibularis muscles taken from freshlyslaughtered cattle were placed in an isotonic myograph and cooled to 1°C. Spontaneous activity due to neuromuscular irritability was minimized by keeping muscle surfaces moist and anaerobic and was monitored by electromyography. Muscle strips were removed and frozen for histochemical analysis after they had completed their initial phase of cold-induced shortening (several hours). Control strips maintained for an equal time at 24°C rarely depleted the stainable glycogen in any of their muscle fibres so as to become PAS-negative. In chilled muscle strips, however, glycogenolysis was activated in some muscle fibres and they became PAS-negative. In serial sections, most of the PAS-negative fibres exhibited strong ATPase and weak succinate dehydrogenase activity. Fibres with weak ATPase and strong succinate dehydrogenase activity rarely became PAS-negative. These results are in agreement with biochemical reports of a cold-induced (<5°C) activation of glycolysis in skeletal musclepost mortem. Investigations on untrimmed lengths of excised sternoman dibularis muscle indicated that longitudinal muscle damage caused in cutting muscle strips for the myograph and/or their more rapid rate of initial cooling had facilitated the depletion of stainable glycogen.     

Association of 3 polymorphisms in porcine troponin I genes (TNNI1 andTNNI2) with meat quality traits

The contractile protein troponin I (TnI), a constituent protein of the troponin complex located on the thin filaments of striated muscle, is involved in inhibition of calcium-induced myosin AT Pase activity (and thus contraction). TnI-slow (slow-twitch skeletal muscle isoform, named TNNI1) and TnI-fast (fast-twitch skeletal muscle isoform, named TNNI2) are muscle-fiber-type-specific proteins, and expression of their genes may affect the composition of muscle fiber, thereby influencing the meat quality traits. Thus, the TnI genes are potential candidate genes for traits related to meat quality in animals. Association of 2 SNPs (EU743939:g.5174T>C in intron 4, and EU743939:g.8350C>A in intron 7) of theTNNI1 gene and a SNP (EU696779:g.1167C>T in intron 3) of theTNNI2 gene with 11 meat quality traits were studied on 334 Large White × Meishan F₂ pigs. In theTNNI1 gene, g.5174T>C and g.8350C>A were found to be significantly associated with intramuscular fat content and meat color value of biceps femoris. The g.5174T>C also showed significant effects on meat color value and marbling score of longissimus dorsi, as well as pH of longissimus dorsi and semispinalis capitis. The g.1167C>T polymorphism in theTNNI2 gene affected significantly the pH of longissimus dorsi, meat color value of longissimus dorsi and semispinalis capitis, marbling score of longissimus dorsi, and intramuscular fat.     

Comparison of red and white muscles by cytophotometry of their muscle fibre populations

Synopsis Samples from two red muscles (vastus intermedius and vastus medialis) and two white muscles (biceps femoris and gluteus medius) were taken from four pigs. Serial transverse sections were reacted for ATPase and NADH oxidative activity. Sections were mapped with a projection microscope so that the staining intensity of individual fibres for the two reactions could be measured with a simple microscope photometer. Transmission values at 600 nm were converted to units of 0–10 for the range from darkest to lightest staining fibres on each section to cancel variation in staining intensity between sections. The aim of the study was to use simple cytophotometry instead of subjective judgement in the categorization of different histochemical types of muscle fibres. Cytophotometry enabled clear resolution of the major fibre types (types I and II using the ATPase reaction), partial resolution of more variable characteristics (NADH oxidative activity in type I and II fibres) and no resolution of subtle subtypes (IA and IB with the NADH oxidative reaction). However, between the major fibre types, cytophotometry revealed variable numbers of fibres with transitional characteristics. There were more of these fibres in red muscles. With sections reacted for ATPase, transmission values for low magnification fields containing 100 to 200 fibres were correlated (r=–0.91) with the ratio of type I:II fibres.