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An in vitro assay was developed to study protease activity during the maturation of beef postmortem. Myofibrils were purified from the semitendinosus and used as a sentinel for assessing the activity of endogenous proteases in longissimus thoracis et lumborum (LTL) and the extensor carpi radialis (ER) over time postmortem in beef carcasses. Samples were collected from each muscle at 0, 1, 2, 7, and 14 d of aging and snap frozen. Samples were powdered and added to an in vitro proteolysis assay containing buffer and purified myofibrils. Aliquots were collected at 0, 2, 120, 480, and 1440 min of incubation, and intact desmin and troponin-T were quantified. Digestions at 0 and 1 d using either muscle had little desmin degradation during the entire digestion period. In contrast, LTL muscle collected at 2, 7, and 14 d had the greatest proteolytic capacity as indicated by disappearance of intact desmin by 480 and 1440 min incubation. Though degradation ensued using powdered ER muscle, disappearance of intact proteins was limited. Degradation in vitro paralleled that observed in intact muscle. Addition of ethylene glycol tetra-acetic acid (EGTA), a cysteine protease inhibitor, and calpastatin inhibited proteolysis and suggest proteolytic activity observed in muscles and detected in our proteolysis assays is due to an active calpain protease. Collectively, our data show an active protease is minimal in bovine muscle until 48 h postmortem in the LTL muscle and suggest an in vitro assay containing purified myofibrils is a potential tool for studying temporal changes in proteolysis during the maturation and tenderization of beef across muscles., Competing Interests: Declaration of competing interest This work is supported in part by A1364 Novel Foods and Innovative Manufacturing Technology [grant no. 2020-67017-31269] from the USDA National Institute of Food and Agriculture., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Reduction in muscle glycogen triggered by adverse antemortem handling events alters postmortem energy metabolism and results in a high ultimate pH and dark, firm and dry beef, often referred to as 'dark-cutting'. However, the relationship between atypical dark (AT) beef, postmortem energy metabolism and underlying tissue characteristics remains somewhat unclear. Cattle harvested in the US and Canada representing normal (pH < 5.6), AT dark (pH 5.6-5.8) and dark cutting (DC; pH > 5.8) beef were analyzed for tissue characteristics related to energy metabolism. Results show AT dark beef is more oxidative but similar to normal beef in glycolytic potential and nucleotide abundance. Mitochondria DNA content (P < 0.05, Canada; P < 0.005, US) and oxidative enzymes for DC and AT dark beef were greater (P < 0.01; Canada and US) compared to normal beef. Myoglobin tracked (P < 0.01) with color classification. These findings show both DC and AT beef are inherently more oxidative and raise the possibility that more oxidative muscle may be more prone to develop dark beef., Competing Interests: Declaration of Competing Interest None., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The objective of this study was to determine if ractopamine (RAC) impacts postmortem muscle metabolism and subsequent pork quality in Halothane (HAL) and Rendement Napole (RN) mutant pigs. All RAC fed pigs had increased (P < 0.04) L* values. HAL and RN mutants muscle had lower (P < 0.01) pH values but RAC feeding had no effect. RN mutants had higher and lower (P < 0.05) muscle pH and temperatures, respectfully at 15 min and RN mutant pigs had greater (P < 0.0001) glycogen initially but lactate levels similar to wild type (WT) pigs at 24 h. RAC lowered (P < 0.05) glycogen in RN mutants but not in HAL mutated or WT pig muscle. These data show RAC feeding changes postmortem energy metabolism but does not change pH and pork quality hallmark of two major pig gene mutations and supports our contention that ultimate meat quality traits and their biochemical drivers may be more complex than originally reasoned., (Published by Elsevier Ltd.)

A kinetic model structure was developed to describe the major variations in energy metabolism and to gain further understanding of pH changes in postmortem muscle experimentally observed with an in vitro glycolytic system. Comparison with experiments showed that the model could describe the kinetics of major metabolites and pH under varied conditions. Optimized model parameters definitively and consistently showed the observed effects of mitochondria, indicating a desirable level of model complexity. Simulation and analysis of pH variations based on the model suggested that phosphofructokinase activity has the strongest impact on the rate and extent of postmortem pH decline. Postmortem pH is also influenced by rates of ATP hydrolysis and glycolysis, and to a much lesser extent, pH buffering capacity. Other reactions, including those mediated by creatine kinase, adenylate kinase, and AMP deaminase, have minimal effects on postmortem pH decline., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Insufficient acidification results in dark, firm, and dry beef. While this defect is often indicative of a stress event antemortem, muscle tissue may change in response to feeding regime. Longissimus dorsi muscle samples from 10 grain-fed and 10 grass-fed market weight, angus-crossbred beef cattle were collected postmortem. Lower (P < .05) L* and a* values were recorded for steaks from grass-fed cattle. Higher (P < .05) ultimate pH values were noted in lean of grass-fed cattle compared to grain-fed cattle, yet differences in lactate, glycogen and glucose were not detected. Further, increased (P < .05) ultimate pH values and lower (P < .05) lactate accumulations were noted when samples from grass-fed cattle were subjected to an in vitro glycolysis system. Muscle from grass-fed beef possessed nearly two-fold more (P < .05) succinate dehydrogenase and (P < .001) myoglobin than that of grain-fed cattle. These data show lean from grass-fed beef has greater enzymes reflective of oxidative metabolism and suggest dark lean from grass-fed cattle may be a function of more oxidative metabolism rather than a stress-related event antemortem., (Copyright © 2019. Published by Elsevier Ltd.)

Muscle satellite cells (SC) are resident stem-like cells that play an integral role in skeletal muscle growth and repair. Understanding how SC maintain their identities and dynamic properties is critical to animal growth. However, the genetic and environmental factors governing SC behaviors and the underpinning mechanisms remain unknown. To explore whether genetic selection influences SC behaviors, we used 2 lines of chickens selected for over 50 generations with over a 10-fold difference in body weight at 56 d of age-the Virginia high weight selection (HWS) and low weight selection (LWS) lines. To study these 2 lines, we performed both in vivo and in vitro experiments. In vivo, we studied the abundance of SC in normal physiological settings and tested their functional roles in muscle regeneration using a muscle injury model. In vitro, we isolated SC from chicken skeletal muscle and assayed their ability to proliferate and differentiate under cultured conditions. Immunohistochemical staining of breast muscle (pectoralis major) revealed that muscle fibers from HWS chickens possessed more SC than those from LWS. Further analysis showed that the SC pool from HWS muscles contained a higher percentage of activated SC compared to that of LWS. When isolated SC from HWS and LWS muscles were cultured, HWS SC exhibited greater abilities to proliferate and differentiate than those SC from LWS. To test whether the observed in vitro differences in SC properties could be confirmed in vivo, we subjected chicken breast muscle to barium chloride to induce muscle injury and regeneration. Consistent with in vitro data, breast muscle in HWS chicken experienced a faster and more robust recovery than that of LWS, as evidenced by quicker regeneration and larger muscle fiber size. Taken together, these findings suggest divergent selection for body weight not only results in correlated responses in SC number, but also changes SC growth kinetics. Further dissection of the molecular mechanism will aid the identification of the target molecules for growth intervention in chickens.

Postmortem energy metabolism drives hydrogen accumulation in muscle and results in a fairly constant ultimate pH. Extended glycolysis results in adverse pork quality and may be possible with greater adenonucleotide availability postmortem. We hypothesized that slowing adenonucleotide removal by reducing AMP deaminase activity would extend glycolysis and lower the ultimate pH of muscle. Longissimus muscle samples were incorporated into an in vitro system that mimics postmortem glycolysis with or without pentostatin, an AMP deaminase inhibitor. Pentostatin lowered ultimate pH and increased lactate and glucose 6-phosphate with time. Based on these results and that AMPK γ3(R200Q) mutated pigs (RN⁻) produce low ultimate pH pork, we hypothesized AMP deaminase abundance and activity would be lower in RN⁻ muscle than wild-type. RN⁻ muscle contained lower AMP deaminase abundance and activity. These data show that altering adenonucleotide availability postmortem can extend postmortem pH decline and suggest that AMP deaminase activity may, in part, contribute to the low ultimate pH observed in RN⁻ pork., (Published by Elsevier Ltd.)

Meat quality development, or the transformation of muscle to meat, involves a myriad of biochemical pathways that are largely well-studied in living muscle tissue. However, these pathways are less predictable when homeostatic ranges are violated. In addition, there is far less known about how various management or environmental stimuli impact these pathways, either by substrate load or altered cellular environment. Likewise, it is largely accepted that oxygen plays little to no role in the conversion of muscle to meat, as anaerobic metabolism predominates in the muscle tissue. Even so, the oxygen tension within the tissues does not fall precipitously at exsanguination. Therefore, transition to an anaerobic environment may impact energy metabolism postmortem. Antemortem handling, on the other hand, clearly impacts meat quality development, yet the exact mechanisms remain a mystery. In this paper, we will attempt to review those factors known to affect postmortem energy metabolism in muscle and explore those areas where additional work may be fruitful., (Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Muscles in most domestic animals differ in function and growth potential based largely on muscle fiber type composition. Though much is known about satellite cells (SC), information is limited regarding how populations of SC differ with muscle fiber type, especially in pigs. Therefore, the objective of this study was to isolate and culture SC from red (RST) and white (WST) portions of the semitendinosus muscle of neonatal and adult pigs and determine their capacity to proliferate, differentiate, and express various myosin heavy chain (MyHC) isoforms in vitro. Porcine satellite cells were isolated from RST and WST muscles of 6-wk-old and adult (>6-mo-old) pigs and cultured under standard conditions. Muscle from neonatal pigs yielded nearly 10 times more (P < 0.001) presumptive satellite cells as those from adult pigs, with fusion percentages close to 60% for the former. The RST yielded more (P < 0.001) SC per gram muscle compared to WST, 8.1 ± 0.2 × 10(4) cells versus 6.7 ± 0.1 × 10(4) cells/gram muscle in young pigs, and 9.7 ± 0.4 × 10(3) cells versus 5.5 ± 0.4 × 10(3) cells/gram muscle in adult pigs, respectively. Likewise, satellite cells from RST proliferated faster (P < 0.001) than those from WST across both ages, as indicated by a shorter cell doubling time, 18.6 ± 0.8 h versus 21.3 ± 0.9 h in young pigs, and 23.2 ± 0.7 h versus 26.7 ± 0.9 h in adult pigs, respectively. As a result of shorter times to confluence, satellite cells from RST also formed myotubes earlier than those SC originating from WST. Once induced, however, SC from WST differentiated and fused faster (P < 0.05) as evidenced by fusion percentage within the first 24 h, 41.6% versus 34.3%, respectively; but reached similar ultimate fusion percentages similar to WST by 48 h. Over 90% of MyHC expressed in maximally fused SC cultures from both RST and WST was restricted to the embryonic isoform. Type IIX MyHC mRNA was not detected in any culture. Myotube cultures from RST expressed more (P < 0.01) Type I MyHC isoform mRNA than those from WST, whereas those cultures from WST expressed more (P < 0.05) Type II (including Types IIA and IIB) MyHC transcripts. These data show SC cultures from porcine fast and slow muscles express MyHC profiles largely reflective of their muscle of origin and suggest satellite cells are partially restricted to a particular muscle phenotype in which they are juxtapositioned. Understanding the molecular nature of these intrinsic control mechanisms may lead to improved strategies for augmenting meat animal growth or muscle regeneration.

Rate and extent of postmortem metabolism control pork quality development. Our objective was to evaluate the role of the phosphagen system (phosphocreatine, PCr; and creatine, Cr) on metabolism and pork quality. Muscle PCr and Cr were manipulated by feeding pigs the creatine analogue, β-guanidinopropionic acid (β-GPA). In experiment 1, pigs received standard (control) diet or β-GPA supplemented (2%) diet (1 wk or 2 wk). Supplementation with β-GPA (2 wk) decreased total Cr (PCr+Cr; P=0.02) and improved pork color (decreased reflectance, P=0.003); however, β-GPA supplementation reduced growth performance (P=0.007). To separate effects of phosphagen system and growth, a second experiment was conducted with control, pair-fed, and 2 wk β-GPA (1%) supplementation; pigs were also offered a control or β-GPA supplemented flavored beverage. Neither treatment influenced pork quality. Immediately postmortem, ATP/ADP was higher in control compared to pair-fed (P<0.05); subsequently, ATP/ADP was similar among all groups. Loss of the phosphagen system may lead to adaptive changes that promote conservation of cellular ATP., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The calpain protease system, in particular, μ-calpain is involved in the disassembly of specific myofibrillar proteins, resulting in tenderization of meat postmortem. Given the size, complexity, and integral nature of titin to the structure of the sarcomere, it is plausible that the length of a sarcomere may alter the susceptibility of various domains of titin to cleavage by the calpains. Therefore, we hypothesized titin degradation differs in a sarcomere-length-dependent manner in beef. After slaughter, beef carcasses were split and sides were either suspended by the Achilles tendon (normal suspension, NS) or by the aitchbone (hip suspension, HS). Immediately after suspension, samples were dissected from the LM, psoas major (PM), and semitendinosus (STN) muscles to serve as 0-d controls. After 24 h, 4 steaks were removed from each muscle and randomly assigned to 1-, 4-, 7-, or 10-d aging treatments. After the assigned aging period, myofibrils were purified for determination of sarcomere length. Warner-Bratzler shear force analysis was also performed to evaluate differences in tenderness. Muscle proteins were solubilized and subjected to SDS-VAGE (vertical agarose gel electrophoresis) to evaluate titin degradation. Sarcomere lengths differed (P < 0.0001) between contralateral muscles of NS and HS carcasses. Quantification of SDS-VAGE gels revealed less (P < 0.05) intact titin in the PM muscle of NS carcasses at each aging period compared with the PM of HS carcasses. No significant differences (P > 0.05) were detected in the disappearance of intact titin among suspension methods in the LM or STN. These data demonstrate that suspension method alters proteolysis of titin and suggest an increase in sarcomere length may contribute to the susceptibility of titin to postmortem proteolysis in beef.

The objective of this study was to investigate the potential of a novel optical reflectance imaging method to predict beef tenderness. Two-dimensional (2D) optical reflectance in beef muscles induced by a point incident light was acquired. A set of five parameters were extracted from each reflectance image to describe quantitatively the reflectance profiles. Two parameters, q and B, were derived by numerically fitting the equi-intensity contours of the reflectance pattern. Two spatial gradients were calculated along the directions parallel and perpendicular to muscle fibers and total scattering intensity was obtained by excluding the specular reflectance. This method was applied to analyze 2D images of optical diffuse reflectance in 336 beef samples obtained from 14 steers in which large variations in tenderness were generated by altering animal genetics, suspension method and aging time as well as utilizing muscles varying in their inherent tenderness. Tenderness was evaluated using Warner-Bratzler shear force (WBSF). The effects of animal breed, muscle, types of suspension, and aging were investigated and results indicate that the scattering intensity measured at 1-d was correlated (R(2)=0.50 at lambda=720 nm) with 10-d WBSF in M. longissimus dorsi muscles; and the q parameters measured at 1-d was correlated (R(2)=0.46 at lambda=720 nm) with 10-d WBSF in M. psoas major muscles. These results show analyzing 2D reflectance images of meat surfaces provides valuable information regarding the physical characteristics of meat that are responsible for beef tenderness., (Copyright 2009 Elsevier Ltd. All rights reserved.)

The calmodulin/Ca2+-dependent serine/threonine phophatase, calcineurin (CaN), has been implicated in controlling muscle fiber phenotype. However, little information is available concerning the expression of CaN in porcine skeletal muscle. Therefore, the porcine CaN alpha (CaN-A) was cloned by reverse transcription-PCR and its expression characterized in selected porcine skeletal muscles. We successfully cloned porcine CaN gene using semitendinosus muscle (GenBank accession number AF193515). Sequence analysis showed both the full length and a 30-bp deletion splice variant in coding region of the gene reported in other species. The deduced AA sequence showed 99.4% homology with the rat CaN-A delta isoform gene. Real-time PCR analysis showed CaN is present in all tissues. However, using primers targeting the region containing the 30-bp deletion, the full length sequence is only found in skeletal muscle and brain tissues. Using a CaN-A monoclonal antibody, we localized CaN-A in porcine LM and soleus muscle and the red and white portions of the semitendinosus muscle. The CaN-A protein was abundant in fast fibers and primarily localized in the cytoplasm, whereas slow fibers expressed reduced abundance of CaN-A. Further studies are required to understand the functions of CaN-A isoform in skeletal muscle.

The purpose of this study was to determine the effect of 5'-AMP-activated protein kinase (AMPK) on energy metabolism and myosin heavy chain (MyHC) isoform expression in growing pigs using chronic treatment with 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR) as a model. Four-week-old pigs were given daily injections of AICAR or 0.9% saline for 10 d. Treatment with AICAR increased (P < 0.05) AMPK activity in semitendinosus muscles (STM). Expression of skeletal muscle specific glucose transporter 4 (GLUT4) was also enhanced (P < 0.05) by AICAR treatment. Using real-time PCR, electrophoresis, and Western blot analyses, we confirmed that AICAR treatment caused a decrease (P < 0.05) in type IIa MyHC isoform mRNA and protein levels and a concomitant increase (P < 0.05) in type IIx MyHC containing fibers. Consistent with a MyHC isoform shift from IIa to IIx, muscles from pigs treated with AICAR had greater (P < 0.05) lactate dehydrogenase (LDH) activity. Moreover, muscle of treated pigs expressed greater (P < 0.05) message for LDH. Administration of AICAR, however, did not alter expression of PPAR-gamma coactivator-1alpha, fatty acid translocase, citrate synthase, or the activity of cytochrome c oxidase. Overall, these results indicate that activation of AMPK by AICAR causes muscle to assume a faster-contracting, more glycolytic nature. These data are in direct contrast to documented effects in rodent models, but these effects may be dependent on the time of administration and the overall growth status of the animal.

Muscle shortening and postmortem proteolysis both influence beef tenderness, but their interacting effects on tenderness are relatively unknown. Inherent myofibril structure and the extent of overlap between myosin and actin filaments are hypothesized to affect the availability of substrates for degradation by calpains. The objective of this study was to determine the influence of sarcomere length on the extent of calpain-induced proteolysis of bovine myofibrils in vitro. Bovine semitendinosus muscles were excised within 20 min postmortem and dissected into strips, which were stretched and attached to applicator sticks or allowed slack to generate samples with different sarcomere lengths upon rigor completion. Samples were allowed to undergo rigor in a neutral pH buffer containing a protease inhibitor. Myofibrils were isolated and incubated at room temperature with excess exogenous mu-calpain at a ratio of 1:800 (wt/wt; enzyme:myofibrillar protein) at pH 6.8 for 0, 2, 60, 1,440, or 2,880 min. Purified troponin was subjected to the same digestion conditions. Proteolysis of troponin T (TnT) was monitored using SDS-PAGE and Western blotting. Sarcomere length was greater (P < 0.0001) in stretched versus shortened samples (2.99 microm +/- 0.03 vs. 2.12 +/- 0.03 microm, respectively, means +/- SE). Western blots for both stretched and shortened samples exhibited bands corresponding to intact TnT and TnT fragments. The abundance of intact TnT decreased (P < 0.0001) with incubation time across both treatments. At 1,440 and 2,880 min, less (P < 0.05) intact TnT was detected in samples with long sarcomeres. These data indicate proteolysis of TnT occurs to a greater extent in samples with longer sarcomeres, possibly due to easier access of proteases to their targeted substrates. Degradation patterns of TnT were qualitatively similar between myofibrils and purified troponin after incubation with mu-calpain. Therefore, it is unlikely that the mechanism by which proteolysis is limited in short sarcomeres involves an actomyosin-mediated interference of TnT.

Genetic selection for improved growth and overall meatiness has resulted in the occurrence of 2 major mutations in pigs, the Rendement Napole (RN) and Halothane (Hal) gene mutations. At the tissue level, these mutations influence energy metabolism in skeletal muscle and muscle fiber type composition, yet also influence total body composition. The RN mutation affects the adenosine monophosphate-activated protein kinase gamma subunit and results in increased glycogen deposition in the muscle, whereas the Hal mutation alters sarcoplasmic calcium release mechanisms and results in altered energy metabolism. From a meat quality standpoint, these mutations independently influence the extent and rate of muscle energy metabolism postmortem, respectively. Even though these mutations alter overall muscle energy metabolism and histochemically derived muscle fiber type independently, their effects have not been yet fully elucidated in respect to myosin heavy chain (MyHC) isoform content and those enzymes responsible for defining energetics of the tissue. Therefore, the objective of this study was to determine the collective effects of the RN and Hal genes on genes and gene products associated with different muscle fiber types in pig skeletal muscle. To overcome potential pitfalls associated with traditional muscle fiber typing, real-time PCR, gel electrophoresis, and Western blotting were used to evaluate MyHC composition and several energy-related gene expressions in muscles from wild-type, RN, Hal, and Hal-RN mutant pigs. The MyHC mRNA levels displayed sequential transitions from IIb to IIx and IIa in pigs bearing the RN mutation. In addition, our results showed MyHC protein isoform abundance is correlated with mRNA level supporting the hypothesis that MyHC genes are transcriptionally controlled. However, transcript abundance of genes involved in energy metabolism, including lactate dehydrogenase, citrate synthase, glycogen synthase, and peroxisome proliferator-activated receptor alpha, was not different between genotypes. These data show that the RN and Hal gene mutations alter muscle fiber type composition and suggest that muscle fiber energy metabolism and speed of contraction, the 2 determinants of muscle fiber type, can be uncoupled.

Unlabelled: The effects of increased dietary protein on resistance training (RT)-induced changes in body composition and skeletal muscle fiber size are uncertain in older people., Objectives: We hypothesized that the ingestion of more animal-based foods, especially eggs, to achieve a higher protein intake would enhance RT-induced changes in body composition., Setting: West Lafayette, IN., Participants: 36 older people (age 61 +/- 1 y; mean +/- SEM)., Intervention: Subjects completed RT three d/wk for 12 weeks, and consumed omnivorous diets that contained either 0.9 +/- 0.1 (lower protein) or 1.2 +/- 0.0 (higher protein) g protein x kg(-1) x d(-1) (12 +/- 3 and 17 +/- 5% of energy intakes, respectively), with the higher protein intake achieved by consuming more eggs, meats, and dairy foods. The lower and higher protein diets contained 213 +/- 21 and 610 +/- 105 mg cholesterol/d, respectively., Measurements: Strength, body composition, serum lipid-lipoprotein profile, urinary creatinine, skeletal muscle fiber type and size., Results: Among all subjects, over time (i.e. with RT) body weight was unchanged, lean mass (1.1 +/- 0.2 kg) increased, and fat mass (-1.4 +/- 0.2 kg) decreased (all changes P < 0.05). Regional (i.e. trunk, legs, arms) lean mass increased and fat mass decreased. Whole body muscle mass (24-h urinary creatinine excretion) increased, but skeletal muscle (vastus lateralis) type 1, type 2a, and type 2x fiber cross-sectional areas did not change from baseline. Serum total and LDL cholesterol decreased (P < 0.05) and HDL cholesterol and triacylglycerol were unchanged. Dietary protein and cholesterol intakes did not influence these responses to RT., Conclusion: Consumption of diets that contained moderately higher protein and variable amounts of cholesterol did not differentially affect body composition, skeletal muscle fiber size, or serum lipid-lipoprotein profile responses to resistance training in older people.

Muscle contraction stimulates glucose transport independent of insulin. Glucose uptake into muscle cells is positively related to skeletal muscle-specific glucose transporter (GLUT-4) expression. Therefore, our objective was to determine the effects of the contraction-mediated signals, calcium and AMP-activated protein kinase (AMPK), on glucose uptake and GLUT-4 expression under acute and chronic conditions. To accomplish this, we used pharmacological agents, cell culture, and pigs possessing genetic mutations for increased cytosolic calcium and constitutively active AMPK. In C2C12 myotubes, caffeine, a sarcoplasmic reticulum calcium-releasing agent, had a biphasic effect on GLUT-4 expression and glucose uptake. Low-concentration (1.25 to 2 mM) or short-term (4 h) caffeine treatment together with the AMPK activator, 5-aminoimidazole-4-carboxamide-1-beta-D-ribonucleoside (AICAR), had an additive effect on GLUT-4 expression. However, high-concentration (2.5 to 5 mM) or long-term (4 to 30 h) caffeine treatment decreased AMPK-induced GLUT-4 expression without affecting cell viability. The negative effect of caffeine on AICAR-induced GLUT-4 expression was reduced by dantrolene, which desensitizes the ryanodine receptor. Consistent with cell culture data, increases in GLUT-4 mRNA and protein expression induced by AMPK were blunted in pigs possessing genetic mutations for both increased cytosolic calcium and constitutively active AMPK. Altogether, these data suggest that chronic exposure to elevated cytosolic calcium concentration blocks AMPK-induced GLUT-4 expression in skeletal muscle.

The interaction between sarcomere length and postmortem proteolysis as related to meat tenderness is not clear. The extent of thick and thin filament overlap alters actomyosin binding and may alter substrate availability during aging-induced tenderization. The objective of this study was to determine the influence of sarcomere length on proteolytic degradation in beef. Strips from bovine semitendinosus were either stretched 40% and restrained or allowed to shorten unrestrained in an ice bath. After rigor completion, 0.6-cm cross sections were fabricated and were randomly assigned to 2, 4, 7, or 10 d of aging treatments. Myofibrils were isolated for sarcomere length determination. Samples were collected and frozen for shear force analysis, and muscle proteins were extracted for SDS-PAGE and Western blotting analyses to determine troponin T (TnT) proteolysis. Sarcomere length was greater (P < 0.01) in stretched muscle samples compared with shortened samples (2.57 vs. 1.43 microm, respectively). Correspondingly, shear force values were greater (P < 0.05) in shortened samples than stretched samples. Western blots revealed the presence of 3 major intact TnT bands that diminished with time postmortem and 4 bands (TnT degradation products) that accumulated during postmortem storage. Quantification of intact TnT showed increased (P < 0.05) proteolysis at 4 and 7 d postmortem in samples with long sarcomeres. By 10 d, only traces of the greatest molecular weight intact TnT band were evident in both shortened and stretched samples, suggesting this TnT band may be more susceptible to proteolysis than other intact TnT bands. Degradation products of TnT appeared earlier postmortem in samples with long sarcomeres. The 30-kDa TnT fragment appeared after 7 d of postmortem storage in samples with long sarcomeres but not until 10 d in muscle containing short sarcomeres. Collectively, these data show that postmortem TnT proteolysis is sarcomere length-dependent and suggest that thick and thin filament overlap may influence the postmortem aging process in beef.

Ractopamine (RAC) improves growth by increasing lean accretion and decreasing fat deposition through repartitioning nutrients from adipose tissue to skeletal muscle. Although the process is not completely understood, RAC alters the proportion of muscle fiber type composition toward a faster-contracting phenotype. Because one of the primary determinants of contractile speed is the relative abundance of myosin heavy chain (MyHC) isoforms and because the genes encoding these isoforms are transcriptionally regulated, RAC likely alters MyHC gene expression. Using real-time PCR, the relative abundance of transcripts of individual type I, IIA, IIX, and IIB, and total MyHC, as well as glycogen synthase, citrate synthase, lactate dehydrogenase, peroxisome proliferator activated receptor alpha, beta1-adrenergic receptor (AR), and beta2-AR were determined in the LM of 44 pigs fed RAC (20 mg/kg) for 0, 1, 2, or 4 wk. In addition, MyHC isoform expression was determined in the LM and red semitendinosus and white semitendinosus muscles of 48 pigs fed RAC (20 mg/kg) for shorter periods of 12, 24, 48, or 96 h. Type I MyHC expression was unaffected (P > 0.73) by RAC administration. Type IIA MyHC expression decreased (P < 0.0001) by 96 h, was lower (P < 0.0001) by 1 wk, and returned to normal by 4 wk. Type IIX MyHC mRNA decreased (P < 0.001) by 2 wk and continued to decrease (P < 0.0001) by 4 wk. Most interesting was an increase (P < 0.0001) in type IIB MyHC by 12 h, which was maintained at an elevated level throughout the 4-wk feeding period. Abundance of glycogen synthase transcript was increased (P < 0.05) by 12 h, but was not different from controls at 2 wk, and was lower (P < 0.01) at 4 wk. Gene expression of beta1-AR was not affected by feeding RAC, whereas beta2-AR gene expression was decreased (P < 0.05) by 2 wk. These data show MyHC genes are differentially regulated by RAC and suggest that the beta adrenergic agonist-induced repartitioning effect is, in part, mediated by changing muscle fiber type-specific gene expression, perhaps through the beta2-AR.

Muscle fiber types are classified based on contractile speed and type of metabolism. Fast-contracting fibers involve mainly glycolytic-based metabolism, whereas slow-contracting fibers involve a more oxidative type of energy metabolism. The relationship between expression of the genes controlling these functional characteristics and their relative protein abundance in porcine muscle is unknown. The objective of this study was to determine the expression of adult myosin heavy-chain (MyHC) genes and their corresponding protein content in various porcine muscles. Moreover, changes in expression of 2 genes involved in energy metabolism (glycogen synthase and citrate synthase) were determined on muscles varying in MyHC. Using real-time PCR, the relative transcript abundance was determined for the adult MyHC isoforms (types I, IIA, IIX, and IIB), glycogen synthase, and citrate synthase in the masseter (MAS), diaphragm, longissimus, cutaneous trunci, and red and white semitendinosus muscles of 7 pigs. Each muscle was subjected to SDS-PAGE analyses to determine the relative abundance of each MyHC. The relative transcript abundance of type IIB MyHC was greatest (P < 0.05) in the longissimus, white semitendinosus, and cutaneous trunci muscles, whereas type I MyHC expression was greatest (P < 0.05) in the MAS, diaphragm, and red semitendinosus muscles. Glycogen synthase gene expression was least in the MAS (P < 0.01) but exhibited a pattern similar to MyHC IIB expression across muscles. Citrate synthase transcript abundance, however, varied (P < 0.05) independently of MyHC gene expression. Expression of types I and IIB MyHC was correlated with their tissue protein content (R2 = 0.76 and 0.78, respectively), whereas type IIA and X MyHC expression did not correlate with the SDS-PAGE-determined protein content. These data show differences in MyHC gene expression across various porcine muscles and suggest that expression of these genes is reflective of the type of myosin contained within the muscle. Moreover, these data show that expression of energy-specific genes differs greatly across porcine muscles with different functions.

Skeletal muscle atrophy results from an imbalance in protein degradation and protein synthesis and occurs in response to injury, various disease states, disuse, and normal aging. Current treatments for this debilitating condition are inadequate. More information about mechanisms involved in the onset and progression of muscle atrophy is necessary for development of more effective therapies. Here we show that expression of the mouse ether-a-go-go related gene (Merg1a) K+ channel is up-regulated in skeletal muscle of mice experiencing atrophy as a result of both malignant tumor expression and disuse. Further, ectopic expression of Merg1a in vivo induces atrophy in healthy wt-bearing mice, while expression of a dysfunctional Merg1a mutant suppresses atrophy in hindlimb-suspended mice. Treatment of hindlimb-suspended mice with astemizole, a known Merg1a channel blocker, inhibits atrophy in these animals. Importantly, in vivo expression of Merg1a in mouse skeletal muscle activates the ubiquitin proteasome pathway that is responsible for the majority of protein degradation that causes muscle atrophy, yet expression of a dysfunctional Merg1a mutant decreases levels of ubiquitin-proteasome proteolysis. Thus, expression of Merg1a likely initiates atrophy by activating ubiquitin-proteasome proteolysis. This gene and its product are potential targets for prevention and treatment of muscle atrophy.

The loss of normal weight-bearing activity, which occurs during bed rest, limb immobilization, and spaceflight, stimulates a catabolic response within the musculoskeletal system, which results in a loss of skeletal muscle mass and bone mineral. The mechanism by which loading of muscle and bone is sensed and translated into signals controlling tissue formation remains a major question in the field of musculoskeletal research. In this investigation, we have examined the ability of two potentially anti-atrophic proteins, IGF-I and Shh, to inhibit disuse atrophy within muscle and bone, when electroporated into skeletal muscle. We have found that electroporation and ectopic expression of IGF-I and/or Shh within the gastrocnemius/soleus muscle significantly stimulated muscle fiber hypertrophy and increases in muscle size. In addition, we report that electroporation and ectopic expression of IGF-I and/or Shh within the gastrocnemius/soleus muscle attenuated the lost of muscle fiber area, muscle mass, and muscle mass density that normally occurs during disuse muscle atrophy. Finally, we found that ectopic expression of IGF-I and Shh within the gastrocnemius/soleus muscle inhibits parameters of osteopenia within the tibia and fibula associated with hindlimb unloading. These results support the theory that skeletal muscle can regulate bone maintenance and could offer potentially novel and efficient therapeutic options for attenuating muscle and bone atrophy during aging, illness and spaceflight.

To determine the effects of deer antler velvet on maximal aerobic performance and the trainability of muscular strength and endurance, 38 active males were randomly assigned in a double-blind fashion to either deer antler velvet extract (n = 12), powder (n = 13), or placebo groups (n = 13). Subjects were tested prior to beginning supplementation and a 10-week strength program, and immediately post-training. All subjects were measured for circulating levels of testosterone, insulin-like growth factor, erythropoietin, red cell mass, plasma volume, and total blood volume. Additionally, muscular strength, endurance, and VO2max were determined. All groups improved 6 RM strength equivalently (41 +/- 26%, p < .001), but there was a greater increase in isokinetic knee extensor strength (30 +/- 21% vs. 13 +/- 15%, p = .04) and endurance (21 +/- 19% vs. 7 +/- 12%, p = .02) in the powder compared to placebo group. There were no endocrine, red cell mass or VO2max changes in any group. These findings do not support an erythropoetic or aerobic ergogenic effect of deer antler velvet. Further, the inconsistent findings regarding the effects of deer antler velvet powder supplementation on the development of strength suggests that further work is required to test the robustness of the observation that this supplement enhances the strength training response and to ensure this observation is not a type I error.

Feeding beta-adrenergic agonists promotes muscle growth. Early histological techniques failed to show precisely how feeding ractopamine-HCl (Paylean) alters muscle growth in pigs. To understand these effects, an indirect enzyme-linked immunosorbent assay (ELISA) was used to determine the abundance of each adult skeletal muscle myosin heavy chain isoform, one means of assigning muscle fiber type, in fast and slow muscles of pigs fed Paylean. Sixty growing pigs (-85 kg) were randomly assigned to three Paylean doses (0, 20, or 60 ppm). At 3, 7, 14, 28, and 42 d of treatment, four pigs per dose were harvested and white (WST) and red (RST) semitendinosus and longissimus (LM) muscles were removed and processed, and myosin heavy chain was quantified by ELISA. Feeding Paylean enhanced (P < 0.05) pigs' average daily gain. Muscle myosin heavy chain (slow, 2A, 2AX, and 2B) composition differed (P < 0.05) across muscles. Compared with LM, RST contained approximately five times more (P < 0.0001) slow and type 2A myosin heavy chain and three times more 2AX myosin heavy chain but nearly undetectable amounts of 2B myosin heavy chain. Myosin heavy chain composition of the WST closely resembled that of the LM (i.e., greater 2AX and 2B and less slow and 2A). After 42d of 60 ppm Paylean, the amount of slow, 2A, and 2AX myosin heavy chain decreased (P < 0.05) across the three muscles whereas the amount of 2B myosin heavy chain increased (P < 0.05). In contrast, relative amounts of 2A and 2AX myosin heavy chain increased (P < 0.05) in muscle of control pigs at 42d. Changes associated with the 20-ppm dose were intermediate to and different from (P < 0.05) control and 60 ppm treatments. Correlations (P < 0.05) among various myosin heavy chain within muscles suggest that slow, type 2A, and 2X decrease with increases in 2B myosin heavy chain. These data show that administration of Paylean affects myosin heavy chain isoform composition in a time- and dose-dependent manner and provides a mechanism of action for Paylean altering animal growth.

Objectives were to determine 1) effects on traits measured from birth to slaughter in F2 cross calves from sire breeds that differ in potential for lean tissue growth but have similar mature BW and 2) the gene action of the mutant Piedmontese myostatin allele. Hereford (normal muscling, H), Limousin (moderate increase in muscling, L), and Piedmontese (muscular hypertrophy, P) sires (20 to 25 per breed) were bred at random to crossbred cows to produce F1 calves that were inter se-mated within sire breed to produce F2 calves that were grown out, finished, and slaughtered. Piedmontese-cross calves were genotyped for the G-A transition mutation at the myostatin locus characteristic of P (msP). Genotypes were classified on the basis of having zero (P0), one (P1), or two (P2) copies of msP (H, n = 227; L, n = 207; P0, n = 40; P1, n = 107; and P2, n = 37). Limousin-cross F2 calves had heavier birth (but dystocia was not affected) and weaning weights, gained faster, had more muscle, less fat, larger pelvic area, and more efficient feed conversion than Hereford-cross F2 calves. Normal-muscled Piedmontese-cross F2 calves (P0) were similar to Hereford-cross F2 calves except that they required less assistance at birth in heifer dams, had less fat, gained slower, were less efficient, and had larger pelvic area. Addition of msP alleles (P1 and P2) consistently increased muscle through hyperplasia, decreased fat, and increased adjusted efficiency, but many of those changes were not linear. Residual variances for breed were heterogeneous for most traits related to muscularity. This heterogeneity was caused by increased variances for L and P and(or) lower variances for H. Accounting for the msP alleles decreased the variance for P in most traits, but heterogeneity remained for most traits among the five genotypes because L remained high, H was low, and(or) P2 was low. We conclude that differences in muscularity affect most traits, and when differences in muscularity include the msP allele, there is an incremental, but not equal, change in most traits with the addition of each copy of the msP allele. Advantages of L could be captured through normal crossbreeding and selection schemes but with some caution because of potential problems from increased variability. Advantages of P could be best captured through more complex breeding and selection programs that would lessen potential negative impacts and through marketing systems that do not penalize for very low fat.

Direct DNA injection into porcine skeletal muscle was investigated as an approach for studying roles of locally produced IGF-I on IGF-binding protein (IGFBP) production. To determine parameters for maximal reporter gene expression, and to investigate the effects of dose, time and weaning on exogenous DNA expression, plasmid DNA encoding firefly luciferase under control of a constitutive promoter and enhancer was injected in skeletal muscle of pigs. Results indicate that injected DNA does not migrate beyond 9 mm from injection sites and that 100 microg DNA injections resulted in optimal luciferase activity. Maximum amounts of recombinant protein were observed 3 days after injection, and were reduced by weaning. Using these data, a second DNA injection study was performed using plasmid DNA containing a cDNA insert for epitope-tagged insulin-like growth factor-I (TIGF-I). Significant quantities of TIGF-I were detected by ELISA and confirmed by western blotting. Both IGFBP-2 and IGFBP-2 mRNA were increased in treated muscle compared with controls. We conclude that increased expression of IGF-I in muscle results in increased IGFBP-2. Furthermore, these data indicate that this in vivo approach of gene transfer results in biologically active recombinant protein production in porcine skeletal muscle, and provides an excellent in vivo model for studying the autocrine and (or) paracrine effects of locally produced growth factors in skeletal muscle.

Development of a recombinant insulin like growth factor I (IGF-I) that is distinguishable from its endogenous counterpart would provide a powerful tool for delineating the role of IGF in myogenesis. Therefore, the objective of this study was to create an epitope-tagged IGF-I that retains biological activity and determine whether expression of this construct is possible in muscle tissue following direct DNA injection. Expression vectors were created that encoded porcine IGF-I containing a T7 (11-amino acid) epitope-tag (TIGF). Immunoreactivity of the purified recombinant TIGF was confirmed using monoclonal antibodies. Biological activity was evaluated by examining differentiation of myoblasts cultured with TIGF or transfected with TIGF plasmid DNA. Addition of purified TIGF to myoblast cultures stimulated (P < 0.05) muscle creatine kinase levels similar to insulin (10(-5) M). Likewise, transfection of L6A1 with TIGF DNA hastened (P < 0.01) differentiation compared to control pcDNA-transfected myoblasts. The integrity of the recombinant protein was confirmed using a sandwich-configured enzyme linked immunosorbent assay. Finally, recombinant TIGF DNA was injected in porcine muscle and the ability to detect TIGF protein was evaluated. TIGF expression was detected in muscle fibers of injected porcine muscle. These data show that a T7 amino acid tag placed on the amino terminus of the IGF-I protein remains intact during processing and does not interfere with the biological activity of the molecule. Use of this DNA construct is an excellent tool for investigating the role of IGFs in control muscle development and provides a model to investigate other regulators of animal growth.

The effect of testosterone on sexual dimorphism is evident by differential growth of forelimb and neck muscles in bulls and steers. Divergent hormone sensitivites may account for the differential growth rates of individual muscles. Therefore, the objective of this study was to compare androgen receptor (AR) expression in three different muscles of bulls and steers at various ages and growth rates. Thirty Montbéliard bulls and 30 steers were assigned to four slaughter age groups. Four or five animals of each sex were slaughtered at 4 and 8 mo of age. Animals in the remaining two slaughter groups (12 and 16 mo) were divided into groups of either restricted (R) or ad libitum (AL) access to feed. Five animals of each sex and diet were slaughtered at the end of the restricted intake period at 12 mo of age. To simulate compensatory growth, the remaining animals (R and AL) were allowed ad libitum access to feed until slaughter at 16 mo of age. Total RNA was extracted from samples of semitendinosus (ST), triceps brachii (TB), and splenius (SP) muscles. Androgen receptor mRNA was quantified in 200-ng total RNA preparations using an internally standardized reverse transcription (RT) PCR assay. Data were analyzed using 18S ribosomal RNA concentrations as a covariable. Steers had higher AR mRNA levels per RNA unit than bulls (P < .01). Androgen receptor mRNA levels differed between muscles (P < .05), with lowest expression in the SP. The pattern of AR expression differed (P < .05) for each muscle with increasing age. Between 4 and 12 mo of age, AR mRNA levels increased (P < .05) in SP but remained unchanged in the ST and TB. Feeding regimen had no effect on muscle AR expression, but steers exhibiting compensatory growth had higher AR mRNA levels than AL steers (P < .01) or bulls (P < .01). Our results show that AR expression is muscle-specific and may be modulated by circulating testicular hormones. These data suggest that the regulation of AR expression may be linked to allometric muscle growth patterns in cattle and compensatory gain in steers.

A detailed methodology is described for fluorescence-activated cell sorting (FACS) of porcine muscle cells that have been transfected to express green fluorescent protein (GFP). Cells are liberated from porcine skeletal muscle and primary cultures are transfected with DNA encoding GFP. Primary cultures are subjected to immunocytochemistry using a primary muscle-specific monoclonal antibody followed by incubation with a phycoerythrin-conjugated second antibody. Transfected myoblasts are sorted from fibroblasts using forward angle light scatter and ninety degree light scatter, phycoerythrin fluorescence, and GFP fluorescence. These procedures allow for isolation of genetically- engineered porcine muscle cells more rapidly than traditional clonal selection procedures. Consequently, FACS provides porcine myoblast populations that retain the majority of their replicative capacity and are not contaminated with non-myogenic cells.

1. Early post-mortem pH was used to identify PSE-like turkey meat, characterised by a low water-holding capacity and pale colour. In a commercial turkey processing facility, the pH value of turkey breast muscle was measured at 0, 9, 10, 14, 154, 231, and 246 min post mortem. 2. At 14 min post mortem, the carcases were separated into 3 pH classes: low (<5.70), medium (5.70 to 6.18), and high (>6.18). Low pH carcasses (<5.70) had higher drip loss(1.75%) than medium (0.81%) and high pH carcases (0.75%). Furthermore, high pH carcases (>6.18) had a lower L* value (darker) than low and medium pH carcases. 3. The effects of using CO2 'snow' on turkey breast muscle quality were also investigated. Light CO2 'snow' treatment (57 g snow/kg breast) and heavy CO2 treatment (113 g snow/kg breast) resulted in higher drip losses than the control no CO2 snow) group.

The objective of this study was to establish the developmental pattern and tissue specificity of porcine myostatin expression and to evaluate expression in skeletal muscle during circumstances in which muscle growth was altered. Northern blot analysis revealed two transcripts (1.5 and 0.8 kb). Myostatin mRNA was detected in whole fetuses at 21 and 35 days and was markedly increased (P < 0.05) by 49 days. At birth, mRNA abundance in longissimus muscle had declined significantly (P < 0.05) from that at day 105 of gestation and continued to decrease (P < 0.05) to its lowest level 2 wk postnatally (4 kg body wt). Myostatin expression was higher (P < 0. 05) at 55, 107, and 162 kg body wt than at 4 kg body wt. Postnatally, myostatin mRNA was detected in skeletal muscle and mammary gland. Expression at birth was 65% higher (P < 0.04) in longissimus muscle of low-birth-weight piglets (0.57 +/- 0.052 kg body wt) vs. normal (1.37 +/- 0.077 kg body wt) littermates, irrespective of gender. However, suppression of longissimus muscle growth by food deprivation (3 days) did not alter (P > 0.15) myostatin expression in either 4- or 7-wk-old piglets. Additionally, myostatin mRNA abundance was not changed by porcine growth hormone administration in growing animals. These data indicate that myostatin expression in skeletal muscle peaks prenatally and that greater expression is associated with low birth weight. Expression in mammary gland indicates a possible role for myostatin in mammary gland development and/or lactation.

Skeletal muscle IGF-I and alpha-actin mRNA responses to increased amino acid availability were investigated in young, rapidly growing steers. Four Holstein steers (208 kg BW) were surgically implanted with an abomasal cannula and jugular catheters and allowed 2 wk to recover. Steers were offered hourly a 43:57 forage:concentrate diet at 95% of ad libitum intake supplemented with continuous abomasal infusion of glucose (to replace 12.5% of metabolizable ad libitum energy intake) for 13 d before the start of abomasal infusion of 67 g of casein N/d. Biopsies of the liver and both semimembranosus muscles were removed and frozen in liquid N, and casein infusion was begun. Muscle biopsies were collected at 8, 16, 24, and 48 h, and on d 7 and 14. Nitrogen balance increased from 23.6 to 71.5 g/d (P < .001) within 24 h and remained elevated (mean = 58.4 g/d) during the 14 d of casein infusion. Plasma urea N increased from 4 to 9.5 mg/dL at 24 h and remained unchanged to d 14. Muscle IGF-I mRNA abundance increased to 215% of basal values at 16 h (P < .01), 244% of basal values at 24 h, and 222% of basal values at 48 h after initiation of casein infusion. Values reached a maximum of 274% of basal values on d 7 and then declined to near preinfusion levels on d 14. The IGF-I mRNA abundance was approximately 100 times higher in liver than in skeletal muscle and was not different on d 0 and 14. Although plasma IGF-I concentrations were numerically higher during the first 24 h of abomasal casein infusion, they were not significantly higher during the chronic phase of treatment. Plasma IGF binding protein (BP)-2 concentrations were higher at 16, 24, and 48 h after casein infusion was begun, but IGFBP-3 concentrations were not altered at these sampling times. Neither acute (first 24 h) nor chronic (daily) plasma insulin concentrations were altered by abomasal casein infusion. Plasma somatotropin concentrations were lower (P = .008) at 24 h of casein infusion and beyond. Results suggest that enhanced amino acid availability may modulate skeletal muscle protein synthesis and accretion through an autocrine or paracrine IGF-I influence.

To investigate the role of IGF in muscle development in vivo, developmental expression and location of IGF-I and -II protein and mRNA were examined in fetal, postnatal, and adult skeletal muscle. Muscle tissue was collected from 30-, 44-, 59-, 68-, 75-, 89-, and 109-d porcine fetuses, 21-d neonatal pigs, and 6-mo-old (adult) pigs. Relative amounts of IGF-II mRNA peaked (P < .05) in 59-d fetal muscle and decreased thereafter. Inversely, muscle IGF-I expression increased (P < .05) to maximal levels around birth. For in situ hybridization, frozen muscle tissue sections (10 microm) were hybridized with a hydrolyzed form of the same riboprobes or incubated with polyclonal or monoclonal antibodies to IGF-I or -II, respectively. The majority of IGF-I and IGF-II mRNA was localized to developing muscle fibers, whereas little signal was found in the surrounding connective tissues. Immunofluorescent localization of IGF-I and -II confirmed that muscle IGF are present in developing muscle fibers. Collectively, these data show that IGF-I and -II are expressed and produced primarily in muscle cells within developing muscle tissue and support the hypothesis that IGF-I and -II modulate fetal muscle development.

Double muscled (DM) cattle possess nearly 40% more muscle fibers than normal muscled (NM) beef or dairy cattle. Previous work showed that serum from DM fetuses stimulated proliferation of L6 myoblasts to a greater extent than serum from NM fetuses. Although the exact role of insulin-like growth factor-II (IGF-II) in the regulation of fetal myogenesis is unknown, it has been shown to serve as a autocrine-acting growth factor during terminal differentiation of myoblasts in mitogen-depleted culture media. Delay of IGF-II expression may alter the ultimate number of muscle fibers formed during fetal development. To investigate this, forty-seven skeletal muscle and twenty-nine liver samples were collected from NM fetuses representing fetuses grouped by crown-rump lengths (CRL) of < 20, 21-30, 31-40, 41-50, 51-60, 61-70 or > 70 cm. Twelve DM fetuses representing 20, 40, 50, and 85 cm were compared to NM groups with the same CRL. Total RNA preparations from these samples were subjected to northern and dot blot analysis using rat IGF-II and beta actin cDNAs and a human 28S rRNA oligomer. IGF-II transcripts of 4.5, 3.6, 2.75, 2.5, and 1.15 kilobases (kb) were detected in liver and muscle RNA from both DM and NM fetuses. Liver IGF-II expression increased (P < 0.05) in both DM and NM fetuses with CRL. Mean concentrations of muscle IGF-II mRNA initially increased (P < 0.05), then decreased (P < 0.05) with CRL in DM and NM fetuses. Muscle IGF-II mRNA was greater (P < 0.05) for NM fetuses compared to DM fetuses at 20 cm CRL, whereas at 53 cm CRL, DM muscle IGF-II was greater (P < 0.05) than that of NM fetuses. These results show that the maximum expression of muscle IGF-II is delayed in DM fetuses compared to NM fetuses. This delayed expression may play an explicit role in controlling myogenesis in the development of double muscle cattle.