Patrick Ecolan

New Insights into Muscle Fiber Types in the Pig

The accurate classification of skeletal muscle fiber types according to myosin heavy chain (MyHC) polymorphism remains a difficult task in the pig. Combined myofibrillar ATPase and metabolic enzyme histochemistry, in situ hybridization, and immunocytochemistry were performed on serial transverse sections of pig longissimus (L) and rhomboideus (R) muscles at 100 kg body weight to give a new insight into muscle fiber typing in the pig. Several monoclonal antibodies (MAbs) either specific for a single MyHC (I, IIa, or IIb) or of multiple MyHCs (IIa + IIx or I + IIx + IIb) were used. No monospecific IIx antibody was available for the pig. All three adult Type II isoforms were expressed in the white L muscle, whereas no IIb was observed in the red R muscle, which was confirmed using RNase protection analysis. In most fibers, the distribution of the transcripts closely matched that of the corresponding proteins. When observed, co-expression of MyHCs mostly occured for IIx and IIb in L muscle, and was more common at the protein (11.5%) than at the mRNA (2.2%) level. A minor proportion of myofibers showed a mismatch between MyHC mRNA and protein. According to the type grouping distribution of myofibers encountered in pig muscle, MyHC isoform expression followed the rank order of I→Iia→Iix→IIb from the center to the periphery of the islets, concomitantly with a decrease in oxidative metabolism and an increase in fiber size. The developmental origin and functional significance of the type grouping distribution are discussed.

Interactive effect of food deprivation and agonistic behavior on blood parameters and muscle glycogen in pigs

Agonistic behavior, neuroendocrine and plasma metabolite changes, and muscle glycogen content were studied in 16 fed and 16 24 h-fasted domestic Large White pigs (100 +/- 5 kg) submitted to dyadic encounters (30 min) in a novel environment. Comparisons were made with corresponding control pigs (eight fed and eight 24 h-fasted animals) kept under resting conditions. At rest, fasting resulted in a significant decrease in plasma insulin, increase in plasma-free fatty acids, and decrease in glycogen content in the predominantly red Semispinalis muscle. Fasted pigs displayed significantly more submissive acts than fed ones. In response to dyadic encounters, fed and fasted pigs showed similar rise in plasma levels of cortisol, catecholamines, and lactate, but stress-induced hyperglycemia was suppressed in food-deprived animals. Fasting enhanced stress-induced glycogen depletion in the predominantly white Longissimus muscle but this effect was significant only in fast-twitch glycolytic fibres (alpha W). In the Semispinalis of fasted pigs, however, dyadic encounters did not induce further glycogen depletion. The present findings suggest that in response to dyadic encounters, fasting-induced changes in glucose metabolism lead to a higher dependence on endogenous energy reserves, i.e., glycogen, in working muscles.

Consequences of divergent selection for residual feed intake in pigs on muscle energy metabolism and meat quality

Selection to decrease Residual Feed Intake (RFI) is a relevant way to improve feed efficiency in growing pigs. However, RFI criterion is correlated with body composition and muscle characteristics. Present study evaluated adaptive responses to divergent selection on RFI on muscle metabolism and homeostasis through AMP-activated protein kinase pathway. Consequences on technological and sensory meat quality were also analyzed in two lines of Large White pigs after six generations of divergent selection on RFI. RFI(-) pigs (n=60) exhibited similar growth rate but lower feed intake and conversion ratio, and were leaner than RFI(+) pigs (n=57). Despite higher glycogen content, metabolic enzyme capacities involved in glycolytic, fatty acid oxidation pathway and energy balance were reduced in the Longissimus muscle of the RFI(-) pigs. Reduced muscle homeostasis in the RFI(-) line influenced post-mortem metabolism and impaired technological quality traits of loin and ham but had only slight effects on meat eating quality.

Glycogen depletion according to muscle and fibre types in response to dyadic encounters in pigs (Sus scrofa domesticus)-relationships with plasma epinephrine and aggressive behaviour

Changes in glycogen content according to fibre type were assessed in a predominantly white (Longissimus) and a predominantly red (Semispinalis) pig muscle, in response to dyadic encounters involving aggressive interactions. Tested animals showed significantly lower glycogen levels than the control in the Semispinalis, but not in the Longissimus muscle. Histological treatment of muscle serial cuts followed by computerized image analysis showed that the observed decrease in muscle Semispinalis glycogen level occurred only in fast-twitch fibres. Total glycogen and glycogen contents in fast-twitch fibres of the Semispinalis muscle were closely and negatively related to aggressive behaviour, but not with plasma epinephrine levels during and at the end of the encounters. The present results provide indirect evidences suggesting a major influence of fighting-induced physical activity on muscle glycogen depletion in response to aggressive interactions in pigs.

Ca2+-activated myosin-ATPases, creatine and adenylate kinases regulate mitochondrial function according to myofibre type in rabbit

Mitochondrial respiration rates and their regulation by ADP, AMP and creatine, were studied at different free Ca2+ concentrations (0.1 versus 0.4 μm) on permeabilized fibre bundles of rabbit skeletal muscles differing in their myosin heavy chain profiles. Four fibre bundle types were obtained: pure types I and IIx, and mixed types IIax (approximately 50% IIa and 50% IIx fibres) and IIb+ (60% IIb fibres, plus IIx and IIa). At rest, pure type I fibres displayed a much higher apparent Km for ADP (212 μm) than IIx fibres (8 μm). Within the IIax and IIb+ mixed fibre bundle types, two KADPm values were observed (70 μm and 5 μm). Comparison between pure IIx and mixed types indicates that the intermediate Km of 70 μm most probably corresponds to the mitochondrial affinity for ADP in IIa fibres, the lowest Km for ADP (5 μm) corresponding to IIx and IIb types. Activation of mitochondrial creatine and adenylate kinase reactions stimulated mitochondrial respiration only in type I and IIax fibre bundles, indicating an efficient coupling between both kinases and ADP rephosphorylation in type I and, likely, IIa fibres, since no effect was observed in pure IIx fibres. Following Ca2+‐induced activation of myosin‐ATPase, an increase in mitochondrial sensitivity to ADP of 45% and 250% was observed in type IIax and I bundles, respectively, an effect mostly prevented by addition of vanadate, an inhibitor of myosin‐ATPase. Ca2+‐induced activation of myosin‐ATPase also prevented the stimulation of respiration rates by creatine and AMP in I and IIax bundles. In addition to differential regulation of mitochondrial respiration and energy transfer systems at rest in I and IIa versus IIx and IIb muscle fibres, our results indicate a regulation of phosphotransfer systems by Ca2+ via the stimulation of myosin‐ATPases in type I and IIa fibres of rabbit muscles.

Effect of muscle type and food deprivation for 24 hours on the composition of the lipid fraction in muscles of large white pigs

The present study was designed to examine the lipid composition of a predominantly glycolytic (M. longissimus lumborum, LL) and a predominantly oxidative (M. semispinalis capitis, SC) muscle sampled at slaughter from Large White pigs fed or deprived of food for 24 h. The intramuscular lipid content was almost four-fold higher in the red SC than in the white LL. When expressed on a muscle tissue basis, the results indicated that the LL had significantly lower amount of tri-, di- and monoglycerides, free fatty acids, cholesterol and phospholipids than the SC. In both muscles, phosphatidylcholine represented the major constituent of the phospholipid fraction. The difference in phospholipid content between the LL and the SC was significant only for cardiolipin, phosphatidylethanolamine and phosphatidylinositol. When expressed on total lipid basis, the results showed that the SC contained significantly lower amount of phospholipids and higher amount of triglycerides than the LL, thus suggesting that the influence of muscle type on the content of intramuscular lipid was primarily due to a difference in the amount of triglycerides. Food deprivation for 24 h resulted in a significantly higher content of free fatty acids, as expressed on a muscle tissue basis, in both muscles. Relative to total lipid, food deprivation resulted in increased levels of free fatty acids and monoglycerides only in the LL. These results suggest that food deprivation for 24 h induced lipolysis in muscle, as evidenced by a modification in the relative composition of the neutral lipid fraction, but that this effect was muscle-dependent.

Influence of early postnatal cold exposure on myofiber maturation in pig skeletal muscle

Early after birth, piglets rely almost exclusively on muscular shivering thermogenesis to produce heat in the cold and this can possibly modulate skeletal muscle development. An experiment involving 10 individually housed piglets was conducted to determine the influence of cold (24–15°C, D5C group) vs. thermoneutrality (34–30°C, D5TN group) between birth and 5 days on myosin heavy chain (MyHC) polymorphism and metabolic characteristics of longissimus lumborum (LL) and rhomboideus (RH) muscles. Five additional piglets were sacrificed at birth. Piglets exposed to cold received 43% more artificial milk on a liveweight basis in order to achieve similar growth rates. D5C piglets produced 93% more heat and exhibited intense shivering during the whole experiment. Contractile and metabolic characteristics of muscles were determined by immunocytochemistry, electrophoresis and enzyme activities. At least eight MyHC isoforms were detected, including atypical expressions of the α-cardiac and extraocular isoforms. Dramatic changes in MyHC composition, myofiber cross-sectional area (CSA) and energy metabolism occurred between birth and 5 days. Cold exposure did not affect either the total number of fibers or the CSA, but it did influence muscle maturation. In particular, it increased the expression of α-cardiac and type I MyHC, and decreased that of fetal MyHC, confirming an acceleration in the rate of postnatal maturation. An increase in oxidative enzyme activities was observed in both muscles in the cold, whereas the activity of a glycolytic enzyme, lactate dehydrogenase, remained unchanged. Cold exposure also induced an increase in T3 plasma levels. The extent to which these changes are the result of sustained shivering or are due to the action of hormonal factors, such as thyroid hormones, are discussed.

In vitro characterization of proliferation and differentiation of pig satellite cells

Skeletal muscle contains various muscle fiber types exhibiting different contractile properties based on the myosin heavy chain (MyHC) isoform profile. Muscle fiber type composition is highly variable and influences growth performance and meat quality, but underlying mechanisms regulating fiber type composition remain poorly understood. The aim of the present work was to develop a model based on muscle satellite cell culture to further investigate the regulation of adult MyHC isoforms expression in pig skeletal muscle. Satellite cells were harvested from the mostly fast-twitch glycolytic longissimus (LM) and predominantly slow-twitch oxidative rhomboideus (RM) muscles of 6-week-old piglets. Satellite cells were allowed to proliferate up to 80% confluence, reached after 7 day of proliferation (D7), and then induced to differentiate. Kinetics of proliferation and differentiation were similar between muscles and more than 95% of the cells were myogenic (desmin positive) at D7 with a fusion index reaching 65 ± 9% after 4 day of differentiation. One-dimensional SDS polyacrylamide gel electrophoresis revealed that satellite cells from both muscles only expressed the embryonic and fetal MyHC isoforms in culture, without any of the adult MyHC isoforms that were expressed in vivo. Interestingly, triiodothyronine (T3) induced de novo expression of adult fast and α-cardiac MyHC in vitro making our culture system a valuable tool to study de novo expression of adult MyHC isoforms and its regulation by intrinsic and/or extrinsic factors.

Fiber type composition and enzyme activities of muscles in two models of obese rats

1. In oxidative (soleus) and glycolytic (extensor digitorum longus) muscles of obese Zucker rats, a significant decrease in the percentage of relative area occupied by glycolytic fibers was observed. 2. The activity of citrate synthase and beta-hydroxy-acyl-CoA-dehydrogenase was significantly higher in muscles of obese than of lean Zucker rats. 3. In rats, 6 weeks after lesion of the ventromedial hypothalamus, no changes were observed. 4. This indicates that neither the proportion of oxidative fibers, nor the oxidative capacities are decreased in skeletal muscles of obese rats suggesting that insulin resistance cannot be ascribed to a higher glycolytic-oxidative fiber ratio.

Influence of production system in local and conventional pig breeds on stress indicators at slaughter, muscle and meat traits and pork eating quality

Sensory quality of pork is a complex phenotype determined by interactions between genetic and environmental factors. This study aimed at describing the respective influences of breed and production system on the development of pork quality. Plasma stress indicators and Longissimus muscle (LM) composition, physicochemical and sensory quality traits were determined in two contrasted breeds - the conventional Large White (LW, n=40) and the French local Basque (B, n=60). Pigs were reared in either a conventional (C; n=20 per breed), alternative (A; sawdust bedding and outdoor area, n=20 per breed) or extensive system (E; free-range, n=20 B). All the pigs from A and C systems were slaughtered at the same slaughterhouse, whereas B pigs from the E system were slaughtered at a local commercial abattoir. Major breed differences were found for almost all traits under study. LM from B pigs exhibited higher lipid, lower water and collagen concentrations, as well as lower collagen thermal solubility (P0.05) influence plasma stress indicators, LM chemical composition and physicochemical or sensory traits of pork. In contrast, within the B pigs, the E system affected the meat quality more. Lower plasma cortisol levels (P<0.05), but higher plasma lactate, creatine kinase and lactate dehydrogenase activities, and more skin lesions (P<0.05), indicating higher muscular activity during pre-slaughter handling, were found in pigs produced in the E compared with the C system. E pigs exhibited higher meat pH1 and pHu values and shear force (P<0.01) and exhibited lower lightness, hue angle and drip and thawing losses (P<0.01) compared with the C pigs, whereas LM lipid, protein or collagen concentrations were not affected. Regarding sensory traits, the E system produced redder meat, but did not impact the eating quality of pork. Altogether, this study demonstrates that differences in meat quality between B and LW breeds can be modulated by extensive pig production system.