Supplementation of sows with L-Arginine during gestating and lactation affects muscle traits of offspring related with postnatal growth and meat quality: From conception to consumption.

This study investigated the effect of dietary inclusion of 25 g/day of L-Arginine (n = 7) or iso­nitrogenous amounts of alanine (n = 6) from d 30 of gestation to d 28 of lactation of sows on performance, muscle traits and meat quality in offspring. From each litter, heaviest and smallest littermate of both sexes were reared from d 28 and slaughtered at d 140 in accordance with a 23factorial design. A response to L-Arginine were obtained on small females where L-Arginine increased birth weight, however this effect disappeared at weaning. L-Arginine increased daily gain by 7% and increased the cross-sectional area of the M. semitendinosus in small females by 14%, suggesting an increased lean ratio. Mechanistic studies showed firstly, that small female littermates had increased number of muscle fibres (myogenesis) after L-Arginine treatment (11%) and secondly increased total DNA (12%) as a consequence of satellite cell proliferation. Traits describing tenderness seem to be affected by L-Arginine but further studies are needed.

In vitro differentiation of progenitor cells isolated from juvenile pig hearts - expression of relevant gene and protein markers.

OBJECTIVES: Heart failure is a significant cause of mortality worldwide, and most current therapies treat only its symptoms. The results of cardiac stem cell research suggest a promising treatment option for heart failure, but there is currently an unmet demand for better research models. We have therefore, for the first time, isolated, expanded and differentiated progenitor cells obtained from juvenile pig hearts to use as a platform for cardiac stem cell research. DESIGN: Progenitor cells were isolated from the left ventricles of porcine hearts using collagenase enzymatic digestion and Percoll®-gradient centrifugation. Cells were proliferated in Matrigel®-coated wells. Cell differentiation was initiated by applying 5-azacytidine and subsequently controlled by modifying the serum concentration. Western blotting and qPCR were used to determine protein and gene expression, respectively. RESULTS: Cardiac-specific genes, from the following proteins: troponin I-3, and myosin-heavy-chain 7 were stably expressed during proliferation and differentiation. Connexin-43 was upregulated and Actinin alfa 2 was downregulated during differentiation. The immature-cardiomyocyte marker GATA binding protein 4 was stably expressed but with a decrease in expression at day 4 of differentiation. Smooth muscle actin decreased in expression and Von Willebrand factor were stably expressed during differentiation. Smooth muscle protein expression was documented but no expression of cardiac-specific proteins after differentiation was found. CONCLUSION: The isolated progenitor cells had key cardiac-lineage gene expression characteristics but they did not express cardiac-specific proteins. Smooth muscle protein was expressed confirming commitment to the smooth muscle lineage.

Constitutive expression of cytochrome P450 in foetal and adult porcine livers-Effects of body weight.

The liver hosts a great number of enzymatically driven processes, including detoxification. The super-family of enzymes named cytochrome P450 (CYP) is the major participant in that process. The expression of CYPs is affected by several factors including life-stage (foetal vs. adult). In the present study we investigated the impact of birth-weight (high or low birth weight) and life-stage on constitutive expression of porcine hepatic CYP1A1, CYP1A2, CYP2A19, CYP2B22, CYP2C33, CYP2D25, CYP2E1 and CYP3A29, as well as the transcription factors controlling their expression; aryl hydrocarbon receptor, constitutive androstane receptor, pregnane X receptor, C/EBP and hepatocyte nuclear factors 1 and 4. Both RT-PCR and western blotting showed a marked increase in the expression of the adult pigs compared with prenatal pigs. Moreover, CYP2E1 mRNA expression was 7.5 fold higher in foetuses with low birth weight compared with foetuses with high birth weight. Gender did not affect the mRNA expression within the different life-stages. These results indicate a similarity to what is observed in humans and porcine foetuses may therefore be a model for humans when studying expression of CYPs.

Polyacetylenes from carrots (Daucus carota) improve glucose uptake in vitro in adipocytes and myotubes.

A dichloromethane (DCM) extract of carrot roots was found to stimulate insulin-dependent glucose uptake (GU) in adipocytes in a dose dependent manner. Bioassay-guided fractionation of the DCM extract resulted in the isolation of the polyacetylenes falcarinol and falcarindiol. Both polyacetylenes were able to significantly stimulate basal and/or insulin-dependent GU in 3T3-L1 adipocytes and porcine myotube cell cultures in a dose-dependent manner. Falcarindiol increased peroxisome proliferator-activated receptor (PPAR)γ-mediated transactivation significantly at concentrations of 3, 10 and 30 µM, while PPARγ-mediated transactivation by falcarinol was only observed at 10 µM. Docking studies accordingly indicated that falcarindiol binds to the ligand binding domain of PPARγ with higher affinity than falcarinol and that both polyacetylenes exhibit characteristics of PPARγ partial agonists. Falcarinol was shown to inhibit adipocyte differentiation as evident by gene expression studies and Oil Red O staining, whereas falcarindiol did not inhibit adipocyte differentiation, which indicates that these polyacetylenes have distinct modes of action. The results of the present study suggest that falcarinol and falcarindiol may represent scaffolds for novel partial PPARγ agonists with possible antidiabetic properties.

Screening for bioactive metabolites in plant extracts modulating glucose uptake and fat accumulation.

Dichloromethane and methanol extracts of seven different food and medicinal plants were tested in a screening platform for identification of extracts with potential bioactivity related to insulin-dependent glucose uptake and fat accumulation. The screening platform included a series of in vitro bioassays, peroxisome proliferator-activated receptor (PPAR) γ-mediated transactivation, adipocyte differentiation of 3T3-L1 cell cultures, and glucose uptake in both 3T3-L1 adipocytes and primary porcine myotubes, as well as one in vivo bioassay, fat accumulation in the nematode Caenorhabditis elegans. We found that dichloromethane extracts of aerial parts of golden root (Rhodiola rosea) and common elder (Sambucus nigra) as well as the dichloromethane extracts of thyme (Thymus vulgaris) and carrot (Daucus carota) were able to stimulate insulin-dependent glucose uptake in both adipocytes and myotubes while weekly activating PPARγ without promoting adipocyte differentiation. In addition, these extracts were able to decrease fat accumulation in C. elegans. Methanol extracts of summer savory (Satureja hortensis), common elder, and broccoli (Brassica oleracea) enhanced glucose uptake in myotubes but were not able to activate PPARγ, indicating a PPARγ-independent effect on glucose uptake.

Bioactive components from flowers of Sambucus nigra L. increase glucose uptake in primary porcine myotube cultures and reduce fat accumulation in Caenorhabditis elegans.

Obesity and insulin resistance in skeletal muscles are major features of type 2 diabetes. In the present study, we examined the potential of Sambucus nigra flower (elderflowers) extracts to stimulate glucose uptake (GU) in primary porcine myotubes and reduce fat accumulation (FAc) in Caenorhabditis elegans. Bioassay guided chromatographic fractionations of extracts and fractions resulted in the identification of naringenin and 5-O- caffeoylquinic acid exhibiting a significant increase in GU. In addition, phenolic compounds related to those found in elderflowers were also tested, and among these, kaempferol, ferulic acid, p-coumaric acid, and caffeic acid increased GU significantly. FAc was significantly reduced in C. elegans, when treated with elderflower extracts, their fractions and the metabolites naringenin, quercetin-3-O-rutinoside, quercetin-3-O-glucoside, quercetin-3-O-5″-acetylglycoside, kaempferol-3-O-rutinoside, isorhamnetin-3-O-rutinoside, and isorhamnetin-3-O-glucoside and the related phenolic compounds kaempferol and ferulic acid. The study indicates that elderflower extracts contain bioactive compounds capable of modulating glucose and lipid metabolism, suitable for nutraceutical and pharmaceutical applications.

Phytanic acid stimulates glucose uptake in a model of skeletal muscles, the primary porcine myotubes.

BACKGROUND: Phytanic acid (PA) is a chlorophyll metabolite with potentials in regulating glucose metabolism, as it is a natural ligand of the peroxisome proliferator-activated receptor (PPAR) that is known to regulate hepatic glucose homeostasis. This study aimed to establish primary porcine myotubes as a model for measuring glucose uptake and glycogen synthesis, and to examine the impact of physiological amounts of PA on glucose uptake and glycogen synthesis either alone or in combination with insulin. METHODS: Porcine satellite cells were cultured into differentiated myotubes and tritiated 2-deoxyglucose (2-DOG) was used to measure glucose uptake, in relation to PA and 2-DOG exposure times and also in relation to PA and insulin concentrations. The MIXED procedure model of SAS was used for statistical analysis of data. RESULTS: PA increased glucose uptake by approximately 35%, and the presence of insulin further increased the uptake, but this further increase in uptake was non- additive and less pronounced at high insulin concentrations. There was no effect of PA alone on glycogen synthesis, while the insulin stimulation of glycogen was increased by 20% in the presence of PA. PA neither stimulated glucose uptake nor glycogen synthesis in insulin-resistant myotubes generated by excess glucose exposure. CONCLUSIONS: Primary porcine myotubes were established as a model of skeletal muscles for measuring glucose uptake and glycogen synthesis, and we showed that PA can play a role in stimulating glucose uptake at no or inadequate insulin concentrations.

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.

Gene expression profiling of porcine skeletal muscle in the early recovery phase following acute physical activity.

Acute physical activity elicits changes in gene expression in skeletal muscles to promote metabolic changes and to repair exercise-induced muscle injuries. In the present time-course study, pigs were submitted to an acute bout of treadmill running until near exhaustion to determine the impact of unaccustomed exercise on global transcriptional profiles in porcine skeletal muscles. Using a combined microarray and candidate gene approach, we identified a suite of genes that are differentially expressed in muscles during postexercise recovery. Several members of the heat shock protein family and proteins associated with proteolytic events, such as the muscle-specific E3 ubiquitin ligase atrogin-1, were significantly upregulated, suggesting that protein breakdown, prevention of protein aggregation and stabilization of unfolded proteins are important processes for restoration of cellular homeostasis. We also detected an upregulation of genes that are associated with muscle cell proliferation and differentiation, including MUSTN1, ASB5 and CSRP3, possibly reflecting activation, differentiation and fusion of satellite cells to facilitate repair of muscle damage. In addition, exercise increased expression of the orphan nuclear hormone receptor NR4A3, which regulates metabolic functions associated with lipid, carbohydrate and energy homeostasis. Finally, we observed an unanticipated induction of the long non-coding RNA transcript NEAT1, which has been implicated in RNA processing and nuclear retention of adenosine-to-inosine edited mRNAs in the ribonucleoprotein bodies called paraspeckles. These findings expand the complexity of pathways affected by acute contractile activity of skeletal muscle, contributing to a better understanding of the molecular processes that occur in muscle tissue in the recovery phase.

Improving meat quality of organic pork through post mortem handling of carcasses: an innovative approach.

This study was conducted to examine the best combination of post mortem chilling, suspension and ageing in order to optimize tenderness of organic pork at slaughter, which may be tougher than conventionally produced pork, because of lower daily gain. Combinations of stepwise chilling with a holding period of 6h at 10°C or traditional blast tunnel chilling, suspension in the pelvic bone or Achilles Tendon and ageing 2 or 4 days post mortem were tested. Stepwise chilling and ageing improved tenderness of the loin, and the effects were additive, whereas pelvic suspension was less effective in texture improvements, and non-additive to stepwise chilling. Stepwise chilling improved tenderness to a similar degree as can be obtained within 2-4 days of extended ageing, however, the minimum temperature during the holding period seems to be crucial in order to obtain a positive effect of stepwise chilling, and it should be above 7.5°C.

Metabolomics reveals relationship between plasma inositols and birth weight: possible markers for fetal programming of type 2 diabetes.

Epidemiological studies in man and with experimental animal models have shown that intrauterine growth restriction (IUGR) resulting in low birth weight is associated with higher risk of programming welfare diseases in later life. In the pig, severe IUGR occurs naturally and contribute substantially to a large intralitter variation in birth weight and may therefore be a good model for man. In the present paper the natural form of IUGR in pigs was studied close to term by nuclear magnetic resonance (NMR-)based metabolomics. The NMR-based investigations revealed different metabolic profiles of plasma samples from low-birth weight (LW) and high-birth weight (HW) piglets, respectively, and differences were assigned to levels of glucose and myo-inositol. Further studies by GC-MS revealed that LW piglets had a significant higher concentration of myoinositol and D-chiro-inositol in plasma compared to larger littermates. Myo-inositol and D-chiro-inositol have been coupled with glucose intolerance and insulin resistance in adults, and the present paper therefore suggests that IUGR is related to impaired glucose metabolism during fetal development, which may cause type 2 diabetes in adulthood.

NMR-based metabonomic investigation of heat stress in myotubes reveals a time-dependent change in the metabolites.

NMR-based metabonomics was applied to elucidate the time-dependent stress responses in mouse myotubes after heat exposure of either 42 or 45 degrees C for 1 h. Principal component analysis (PCA) revealed that the gradual time-dependent changes in metabolites contributing to the clustering and separation of the control samples from the different time points after heat stress primarily are in the metabolites glucose, leucine, lysine, phenylalanine, creatine, glutamine, and acetate. In addition, PC scores revealed a maximum change in metabolite composition 4 h after the stress exposure; thereafter, samples returned toward control samples, however, without reaching the control samples even 10 h after stress. The results also indicate that the myotubes efficiently regulate the pH level by release of lactate to the culture medium at a heat stress level of 42 degrees C, which is a temperature level reached in muscles of pigs during exposure to slaughter stress.

Creatine-induced activation of antioxidative defence in myotube cultures revealed by explorative NMR-based metabonomics and proteomics.

BACKGROUND: Creatine is a key intermediate in energy metabolism and supplementation of creatine has been used for increasing muscle mass, strength and endurance. Creatine supplementation has also been reported to trigger the skeletal muscle expression of insulin like growth factor I, to increase the fat-free mass and improve cognition in elderly, and more explorative approaches like transcriptomics has revealed additional information. The aim of the present study was to reveal additional insight into the biochemical effects of creatine supplementation at the protein and metabolite level by integrating the explorative techniques, proteomics and NMR metabonomics, in a systems biology approach. METHODS: Differentiated mouse myotube cultures (C2C12) were exposed to 5 mM creatine monohydrate (CMH) for 24 hours. For proteomics studies, lysed myotubes were analyzed in single 2-DGE gels where the first dimension of protein separation was pI 5-8 and second dimension was a 12.5% Criterion gel. Differentially expressed protein spots of significance were excised from the gel, desalted and identified by peptide mass fingerprinting using MALDI-TOF MS. For NMR metabonomic studies, chloroform/methanol extractions of the myotubes were subjected to one-dimensional 1H NMR spectroscopy and the intracellular oxidative status of myotubes was assessed by intracellular DCFH2 oxidation after 24 h pre-incubation with CMH. RESULTS: The identified differentially expressed proteins included vimentin, malate dehydrogenase, peroxiredoxin, thioredoxin dependent peroxide reductase, and 75 kDa and 78 kDa glucose regulated protein precursors. After CMH exposure, up-regulated proteomic spots correlated positively with the NMR signals from creatine, while down-regulated proteomic spots were negatively correlated with these NMR signals. The identified differentially regulated proteins were related to energy metabolism, glucose regulated stress, cellular structure and the antioxidative defence system. The suggested improvement of the antioxidative defence was confirmed by a reduced intracellular DCFH2 oxidation with increasing concentrations of CMH in the 24 h pre-incubation medium. CONCLUSIONS: The explorative approach of this study combined with the determination of a decreased intracellular DCFH2 oxidation revealed an additional stimulation of cellular antioxidative mechanisms when myotubes were exposed to CMH. This may contribute to an increased exercise performance mediated by increased ability to cope with training-induced increases in oxidative stress.

Oxidative stress-induced metabolic changes in mouse C2C12 myotubes studied with high-resolution 13C, 1H, and 31P NMR spectroscopy.

In this study, stress in relation to slaughter was investigated in a model system by the use of (13)C, (1)H, and (31)P nuclear magnetic resonance (NMR) spectroscopy for elucidating changes in the metabolites in C2C12 myotubes exposed to H(2)O(2)-induced stress. Oxidative stress resulted in lower levels of several metabolites, mainly amino acids; however, higher levels of alanine were apparent in the (13)C spectra after incubation with [(13)C(1)]glucose. In the (13)C spectra [(13)C(3)]lactate tended to increase after exposure to increasing concentrations of H(2)O(2); conversely, a tendency to lower levels of the unlabeled ((12)C) lactate were identified in the (1)H spectra after stress exposure. These data indicate an increase in de novo synthesis of alanine, concomitant with a release of lactate from the myotubes to the medium at oxidative stress conditions. The changes in the metabolite levels could possibly be useful as markers for meat quality traits.

Metabolic profiling of heat or anoxic stress in mouse C2C12 myotubes using multinuclear magnetic resonance spectroscopy.

In the present study, the metabolic effects of heat and anoxic stress in myotubes from the mouse cell line C2C12 were investigated by using a combination of (13)C, (1)H, and (31)P nuclear magnetic resonance (NMR) spectroscopy and enrichment with [(13)C]-glucose. Both the (13)C and the (1)H NMR spectra showed reduced levels of the amino acids alanine, glutamate, and aspartate after heat or anoxic stress. The decreases were smallest at 42 degrees C, larger at 45 degrees C, and most pronounced after anoxic conditions. In addition, in both the (1)H and the (31)P NMR spectra, decreases in the high-energy phosphate compounds adenosine triphosphate and phosphocreatine with increasing severity of stress were identified. At anoxic conditions, an increase in (13)C-labeled lactate and appearance of glycerol-3-phosphate were observed. Accumulation of lactate and glycerol-3-phosphate is in agreement with a shift to anaerobic metabolism due to inhibition of the aerobic pathway in the mitochondria. Conversely, lower levels of unlabeled ((12)C) lactate were apparent at increasing severity of stress, which indicate that lactate is released from the myotubes to the medium. In conclusion, the metabolites identified in the present study may be useful markers for identifying severity of stress in muscles.

Compensatory growth response as a strategy to enhance tenderness in entire male and female pork M. longissimus thoracis.

The effects of restrictive feeding strategies aimed at promoting a compensatory growth response were investigated with respect to tenderness improvement in pork M. longissimus thoracis. Compensatory growth response is defined by increased weight in pigs fed ad libitum after a period with restricted feeding compared to pigs fed ad libitum the entire fattening period. Specifically, the aim was to study the sensory textural characteristics after female and entire male pigs have been exposed to restrictions in either energy, protein or both in specific growth periods. It was found that textural differences were caused by feeding strategies and not related to variation in intramuscular fat, which did not vary significantly between genders. The female pigs demonstrated compensatory growth response and the texture was significantly (P<0.001) improved by low dietary level of energy from day 50 to 90 and normal dietary level of protein during the entire feeding period. For the entire male pigs, low level of protein in the late feeding period significantly (P<0.05) improved the texture. Also, low level of protein and normal level of energy in the early feeding period resulted in improved tenderness. However, these texture improvements were deemed not to be an effect of compensatory growth since the entire male pigs did not compensate for the feeding restriction in the early feeding period. The tenderness enhancement for the entire male pigs compromised the production results in terms of 7-15% lower carcass weight.

The polyacetylenes falcarinol and falcarindiol affect stress responses in myotube cultures in a biphasic manner.

The effects of the bioactive polyacetylenes, falcarinol and falcarindiol, present in carrots, celery, celeriac and other umbelliferous vegetables, on the stress responses in primary myotube cultures, were studied. Biphasic responses on cellular stress responses in myotube cultures were investigated by exposing them to various concentrations of falcarinol and falcarindiol for 24 h before testing effects of 100 microM H(2)O(2) on the intracellular formation of reactive oxygen species (ROS), transcription of the antioxidative enzyme cytosolic glutathione peroxidase (cGPx), and the heat shock proteins (HSP) HSP70 and HO1. At low concentrations (1.6 to 25 microM) polyacetylenes caused a slightly accelerated intra-cellular ROS formation, increased cGPx transcription and decreased HSP70 and HO1 transcription. The increased cGPx transcription may be interpreted as an adaptive response to the increased ROS formation and may have caused a reduced demand for the protective functions of the HSPs. ROS formation, however, was substantially decreased after pre-incubation with both polyacetylenes at 50 and 100 microM, the cGPx transcription was reduced and the HSP70 and HO1 transcription increased, indicating a need for the protective and repairing functions of the HSPs. In conclusion, pre-incubation with low concentrations of both polyacetylenes prior to H(2)O(2) exposure induced a cytoprotective effect whereas higher concentrations had adverse effects.

Establishment and conditions for growth and differentiation of a myoblast cell line derived from the semimembranosus muscle of newborn piglets.

To establish an adequate model to study the proliferation and differentiation of porcine skeletal muscle in response to bioactive compounds, a pool of satellite cells was derived from the semimembranosus muscle (SM) of newborn piglets using a Percoll gradient centrifugation. The final yield amounted to 4.1 x 10(6) cells/g muscle tissue. The percentage of muscle satellite cells has been determined by immunostaining for desmin and subsequent fluorescence analysis by flow cytometry, which revealed 95% of desmin-positive cells. For proliferation studies, satellite cell born myoblasts were seeded in gelatin-coated 96-well microplates at about 5 x 10(3) cells per well. Cells were grown for 1 day in MEMalpha plus 10% fetal bovine serum (FBS) and 10% horse serum (HS), followed by 2 d cultivation in serum-free growth medium. For differentiation studies, myoblasts were cultured in matrigel-coated 24-well plates for 4 d with growth medium containing 10% FBS and 10% HS. At 80% confluence, cells were grown for 24 h in medium plus 10% FBS and 1 microM insulin to initiate differentiation. Subsequently, the cells were cultured in serum-free differentiation medium (SFDM) for 3 d to form myotubes. Cultures reached a maximum fusion rate of approximately 20% after 96 h. By establishing this culture system, we provide an advanced and appropriate in vitro model to study porcine skeletal muscle cell growth and differentiation including the responses to various bioactive compounds.

Role of phospholipase A2 in the induction of drip loss in porcine muscle.

The role of phospholipase A2 in the induction of drip loss from pig muscle has been investigated. In samples from porcine M. longissimus dorsi, total PLA2 activity as well as mRNA and protein levels of the group VIA iPLA2 (iPLA2-VIA) increased during the initial 4 h post-mortem period. Morphological studies of porcine muscle showed that at 4 h post-mortem, gaps had formed between muscle fibers and that the sarcolemma membrane borders appeared blurred. At the same time iPLA2-VIA protein levels were increased inside muscle fibers and at the sarcolemma. iPLA2-VIA mRNA abundance in samples from different breeds of pigs with variations in drip loss revealed no clear correlation between drip loss level and iPLA2-VIA expression. Together, these data indicate that during the post-mortem period, iPLA2-VIA expression and activity is increased at the muscle fiber membranes. PLA2 activity may affect membrane permeability and consequently the progression of drip formation in porcine muscle.

Post-mortem activity of the glycogen debranching enzyme and change in the glycogen pools in porcine M. longissimus dorsi from carriers and non-carriers of the RN(-) gene.

Glycogen debranching enzyme (GDE) is together with glycogen phosphorylase responsible for the degradation of glycogen. The present study compares the post-mortem activity of GDE and breakdown of the glycogen pools in M. longissimus dorsi of RN(-) carrier pigs and in wild type animals. The activity of GDE (n=14) and pH (n=20) was measured 0.5, 3, 5, 24 and 48h post-mortem. The change in pro-glycogen and in macro-glycogen content (n=20) was followed until 216h post-mortem and the transcription level of GDE, glycogenin and glycogen synthase m-RNA (n=19) were measured 0.5h post-mortem. Both the activity of GDE and the transcription level of GDE were found to be similar in RN(-) carriers and wild type animals shortly after slaughter. However, the activity declined faster in wild type animals compared with RN(-) carriers with increasing time post-mortem. The contents of both pro-glycogen and macro-glycogen were higher in RN(-) carriers compared with wild type animals, and further, the proportion of macro-glycogen was higher in RN(-) carriers compared with wild type animals. During the post-mortem period, only degradation of pro-glycogen was observed in both genotypes. The decrease in pH was faster and the ultimate pH lower in RN(-) carriers than in wild type animals. It was suggested that the higher GDE activity in the late phase of the post-mortem period in muscles from RN(-) carriers renders the extended pH decrease in these muscles.