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The calmodulin/[Ca.sup.2+]-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. Key words: calcineurin, fiber type, gene sequence, pig, protein phosphatase 2B doi: 10.2527/jas.2009-1832

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-l[beta]-D-ribofuranoside (AICAR) as a model. Four-weekold 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 con comitant 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-l[alpha], 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. Key words: 5'-AMP-activated protein kinase, 5-aminoimidazole-4-carboxamide-l-[beta]-D-ribofuranoside, myosin heavy chain, skeletal muscle

The objective of this study was to evaluate the influence of dietary protein and fat from distillers dried grains with solubles (DDGS) on feedlot performance, carcass characteristics, and meat quality in finishing steers. Angus-cross steers (n = 105; 443 [+ or -] 20 kg of BW) were blocked by BW and randomly assigned to 1 of 5 dietary treatments: 1) corn-based diet with DDGS included at 25% of DM (CON), 2) CON with DDGS included at twice the amount of CON (50% of DM; 50DDGS), 3) CON with added corn protein to equal the CP in the 50DDGS diet (CON+CP), 4) CON with added vegetable oil to equal the fat in the 50DDGS diet (CON+VO), and 5) CON with protein and fat added to equal the CP and fat in the 50DDGS diet (CON+CPVO). Steers were fed to a common 12th-rib fat depth endpoint (1.3 [+ or -] 0.2 cm; 68 to 125 d on trial). Loins and rounds were collected from 44 carcasses for Warner-Bratzler shear force (WBSF), ether extract, and case-life analyses. Data were analyzed using the MIXED procedure of SAS. Contrasts between 1) CON vs. elevated CP diets (50DDGS, CON+CP, and CON+CPVO; EP), 2) CON vs. elevated fat diets (50DDGS, CON+VO, and CON+CPVO; EF) and 3) CON vs. diets with elevated CP and fat (50DDGS and CON+CPVO; EPF) were analyzed. There were no dif ferences in days on feed or DMI among treatments. Steers fed CON had greater ADG (P [less than or equal to] 0.03) than EP, EF, and EPF diets. Steers fed CON also had greater G:F (P [less than or equal to] 0.04) than EP and EPF steers. Final BW was greater for CON than EP and EPF diets (P [less than or equal to] 0.03). Likewise, CON steers had heavier HCW than EPF steers (P = 0.04). Dressing percent, 12th-rib fat depth, LM area, KPH, and yield grade were not affected by treatment (P [greater than or equal to] 0.06). Steers fed the CON diet had greater marbling scores (P [less than or equal to] 0.03) and quality grades (P [less than or equal to] 0.02) compared with those fed EP, EF, and EPF diets. There were no differences in WBSF, ether extract, or lipid oxidation due to treatment (P [greater than or equal to] 0.44). However, CON steers had greater (P = 0.02) [L.sup.*] values than EF-fed steers and greater [b.sup.*] values than EP, EF, and EPF steers (P [less than or equal to] 0.02) during retail display of ground product. Data from this study illustrate that live animal performance, marbling and quality grade, and color stability of ground product during retail display are negatively affected when DDGS are increased from 25 to 50% of the diet DM. This response appears to be due to elevated dietary fat, elevated CP, and a combination of elevated fat and protein within in the diet. Key words: distillers grain, feedlot, marbling, meat quality, steer

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 rain 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 [micro]m [+ or -] 0.03 vs. 2.12 [+ or -] 0.03 [micro]m, respectively, means [+ or -] 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 [micro]-calpain. Therefore, it is unlikely that the mechanism by which proteolysis is limited in short sarcomeres involves an actomyosin-mediated interference of TnT. Key words: beef. [micro]-calpain, sarcomere length, tenderness, troponin-T

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. Key words: halothane, myosin heavy chain, porcine, Rendement Napole, skeletal muscle

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-l-[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. adenosine 5'-monophosphate-activated protein kinase; glucose transporter; C2C12 cells; caffeine; ryanodine receptor

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 pm, 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. Key words: tenderness, sarcomere length, proteolysis, troponin-T, 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], [[beta].sub.1]-adrenergic receptor (AR), and [[beta].sub.2]-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 [[beta].sub.1]-AR was not affected by feeding RAC, whereas [[beta].sub.2]-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 [[beta].sub.2AR. Key words: fiber type, myosin heavy chain, ractopamine

The molecular mechanisms controlling [beta]-adrenergic receptor agonist (BA)-induced skeletal muscle hypertrophy are not well known. We presently report that BA exerts a distinct muscle- and muscle fiber type-specific hypertrophy. Moreover, we have shown that pharmacologically or genetically attenuating extracellular signal-regulated kinase (ERK) signaling in muscle fibers resulted in decreases (P < 0.05) in fast but not slow fiber type-specific reporter gene expressions in response to BA exposure in vitro and in vivo. Consistent with these data, forced expression of MAPK phosphatase 1, a nuclear protein that dephosphorylates ERK1/2, in fast-twitch skeletal muscle ablated (P < 0.05) the hypertrophic effects of BA feeding (clenbuterol, 20 parts per million in water) in vivo. Further analysis has shown that BA-induced phosphorylation and activation of ERK occurred to a greater (P < 0.05) extent in fast myofibers than in slow myofibers. Analysis of the basal level of ERK activity in slow and fast muscles revealed that ERK1/2 is activated to a greater extent in fast- than in slow-twitch muscles. These data indicate that ERK signaling is differentially involved in BA-induced hypertrophy in slow and fast skeletal muscles, suggesting that the increased abundance of phospho-ERK1/2 and ERK activity found in fast-twitch myofibers, compared with their slow-twitch counterparts, may account, at least in part, for the fiber type-specific hypertrophy induced by BA stimulation. These data suggest that fast myofibers are pivotal in the adaptation of muscle to environmental cues and that the mechanism underlying this change is partially mediated by the MAPK signaling cascade. muscle fiber type; mitogen-activated protein kinase signaling pathways; mechanism doi: 10.1152/ajpcell.00466.2006.

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 ([R.sup.2] = 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 energyspecific genes differs greatly across porcine muscles with different functions. Key words: myosin heavy chain, porcine, skeletal muscle

Feeding increased levels of dietary vitamin E can inhibit lipid oxidation. The aim of this study was to investigate the effect of levels of dietary [alpha]-tocopherol acetate (VE) and feeding duration on meat quality and lipid oxidation. Eighty-one pigs were allocated to 1 of 3 diets containing 40, 200, or 400 IU of VE/kg of feed, and each diet group was divided into 3 feeding periods (3, 6, or 9 wk). Carcass characteristics and meat quality were evaluated. Oxidative stability of fresh and cooked pork patties and pork chops was determined after chilled or frozen storage. Increasing dietary concentrations of VE did not affect any growth performance parameter. Drip loss, however, decreased (P < 0.05) with increased dietary VE levels. Moreover, an increased duration of VE feeding improved (P < 0.05) pH and drip loss. Less lipid oxidation (P < 0.05) was detected in fresh ground pork from pigs fed greater concentrations of VE after 4 d of storage. A greater (P < 0.05) resistance to oxidation in cooked ground pork was observed in pigs fed 200 or 400 IU of VE/kg at 2 and 6 d of storage. Fresh and cooked pork patty oxidation decreased (P < 0.05) linearly as feeding duration increased from 3 to 9 wk. After 6 mo of freezer storage, lipid oxidation of pork chops from pigs fed 200 or 400 IU of VE/kg was lower (P < 0.05) than for pigs fed 40 IU of VE/kg. Likewise, lipid oxidation of pork chops of pigs fed VE for an extended period of time (6 wk) was lower (P < 0.05) after 9 mo of storage. Fatty acid profiles of neutral lipid fraction of the LM became more unsaturated (P < 0.05) with added VE to the feed. These results indicate an increased intake of dietary VE concentration, and prolonged feeding of VE can improve drip loss and reduce lipid oxidation in ground pork and pork chops. This study suggests that supplementation with 200 IU of VE/kg of feed for 6 wk before market is beneficial in improving lipid stability and pork quality. Key words: lipid oxidation, meat quality, pork, vitamin E

The effects of dietary vitamin E (VE, [alpha]-tocopherol acetate) and fat supplementation on growth and carcass quality characteristics, oxidative stability of fresh and cooked pork patty in storage, fatty acid profiles of muscle and adipose tissue, and VE concentrations of plasma, muscle, and adipose tissue were studied. Six hundred pigs were allocated to i of 6 diets and fed for 63 d in a 3 x 2 factorial design. The dietary treatments included 3 fat levels (normal corn, high oil corn, high oil corn plus added beef tallow) and 2 levels of VE supplementation (40 IU/kg, normal VE supplementation; and 200 IU/kg, high VE supplementation). At 113 kg of BW, 54 pigs were slaughtered as a subsample to evaluate dietary effects on pork quality. Growth performance and meat quality characteristics did not differ (P > 0.05) among treatment groups. The high level of VE supplementation had a beneficial effect on the oxidative stability of pork as indicated by thiobarbituric acid reactive substance (TBARS) values. Lean tissue had lower (P < 0.05) TBARS in the group fed the high VE than in those fed the normal VE level. The TBARS values differed among storage periods (0 to 6 d) and also between fresh and cooked ground ham. Fat type did not significantly affect total saturated and unsaturated fatty acids proportions in the neutral and polar fraction of muscle. Adding VE acetate led to greater (P < 0.05) monounsaturated and total unsaturated fatty acid proportions in neutral lipids of muscle and adipose tissues. Increasing dietary levels of VE acetate increased the concentration of VE in plasma and muscle. These results indicate that dietary VE acetate supplementation increased (P < 0.05) lipid stability and the VE concentration of muscle. Key words: [alpha]-tocopherol, fatty acid, high oil corn, lipid oxidation, pork, supplemental fat

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. Key Words: [beta]-Adrenergic Agonists, Muscle Fibers, Myosins

Objectives were to determine 1) effects on traits measured from birth to slaughter in [F.sub.2] 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 [F.sub.1] calves that were inter se-mated within sire breed to produce [F.sub.2] 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 ([P.sub.0]), one ([P.sub.1]), or two ([P.sub.2]) copies of msP (H, n = 227; L, n = 207; [P.sub.0], n = 40; [P.sub.1], n = 107; and [P.sub.2], n = 37). Limousin-cross [F.sub.2] 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 [F.sub.2] calves. Normal-muscled Piedmontese-cross [F.sub.2] calves ([P.sub.0]) were similar to Hereford-cross [F.sub.2] 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 ([P.sub.1] and [P.sub.2]) 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) [P.sub.2] 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. Key Words: Animal Production, Cattle, Muscular Hypertrophy, Pleiotropy

Research is presented concerning the effectiveness of sodium bicarbonate and sodium phosphate in reducing the pH decline in early postmortem pork. The prevention of pale, soft, exudative pork by this technique is discussed.

Cell-mediated gene transfer is a potential tool for studying muscle growth, but efficient genetic manipulation and implantation strategies have not been developed for pigs. The objectives of the present study were to determine methods for transient and stable incorporation of reporter genes into porcine muscle cells and to investigate their use for cell-mediated gene transfer in pigs. Porcine myoblasts and fibroblasts were isolated from muscle of 2-wk-old male pigs. Myogenic cell lines were identified using muscle-specific monoclonal antibodies, myotube fusion assays, and the presence of muscle-specific markers (MyoD and desmin). Four commercial cationic liposomes (lipofectAMINE, lipofectin, cellFECTIN, and DMRIE-C) were tested at different DNA:lipid ratios for their ability to transfect myoblasts and fibroblasts transiently with a luciferase reporter plasmid. LipofectAMINE resulted in the greatest (P < .01) transient luciferase activity for both cell types. Electroporation of cells for transient transfection resulted in less luciferase activity than cationic transfection. Stable transfections were conducted using a green fluorescence protein (GFP) reporter plasmid containing the neomycin resistance gene. LipofectAMINE transfection resulted in stable GFP expression in 1:16,000 myoblasts and 1:33,000 fibroblasts. Stable electroporation resulted in efficiencies that were significantly lower than established with cationic liposomes. Porcine cells were transduced with GFP using vesicular stomatitis virus glycoprotein G pseudotyped retrovirus and resulted in efficiencies of 1:1.2 for myoblasts and 1:1.1 for fibroblasts. These results show that cationic liposomes are superior to electroporation for transfection, but retroviral transduction produced stable reporter gene expression in > 80% of porcine muscle cells. Transduced GFP-positive cells were separated from GFP-negative cells by fluorescence-activated cell sorting and implanted into 2-wk-old male pigs. On d 4, implanted muscles were removed and subjected to immunodetection of GFP protein. Fibroblast implantation resulted in limited GFP expression within muscle, whereas myoblast implantation resulted in GFP within muscle fibers. This suggests that cell-mediated gene transfer is possible in porcine muscle and may be useful as an approach for studying muscle growth in pigs. Key Words: Gene Transfer, Muscles, Pigs, Retroviridae, Transfection

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 Montbeliard 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. Key Words: Cattle, Skeletal Muscle, Growth, Castration, Androgens, Messenger RNA, Polymerase Chain Reaction

The developmental pattern and tissue specificity of porcine myostatin expression was explored and the expression in skeletal muscle during circumstances in which muscle growth was altered was examined. Results show that myostatin is expressed significantly in developing muscle fasciculi, in skeletal muscles and in lactating mammary gland. However, it was not expressed in other organs and tissues such as adipose, brain, tongue and heart. It was also observed that food intake, growth hormone and safflower have no significant effect on myostatin expression.

Three main fiber types (one slow [type I] and two fast [type IIA and IIB] can be distinguished using conventional actomyosin ATPase (AM-ATPase) histochemistry after acidic pretreatment in mature pig skeletal muscle. We report the isolation, characterization, and identification of four adult 3'-untranslated regions corresponding to types I, IIA, IIB, and IIX myosin heavy chains (MyHC) from a cDNA library. Identification of different type II clones was based on sequence homology, in situ hybridizations (ISH), AM-ATPase histochemistry, and immunocytochemistry. Enzyme histochemistry, immunocytochemistry, and ISH were performed on serial transverse sections of longissimus and red portion of semitendinosus muscle. Results showed that all three fast MyHC transcripts were expressed in the longissimus, whereas only type IIA and IIX transcripts were present in deep red semitendinosus muscle. Type I and IIA fibers contained mostly type I and IIA transcripts, respectively, whereas type IIB fibers contained a heterogeneous population of transcripts. In longissimus muscle, 18, 31, and 51% of conventional IIB fibers were pure IIX, hybrid IIX/IIB, and pure lib fibers, respectively. Conversely, conventional lib fibers were actually IIX in deep red semitendinosus muscle. Expression of the three fast adult MyHC isoforms in longissimus was spatially regulated around the typical islets of type I fibers encountered in pig skeletal muscle. Thus, IIA fibers were contiguous to type I fibers, pure IIX fibers were in the direct vicinity of type I and IIA fibers, and hybrid IIX/IIB fibers were located mostly within primary fascicles between the islets of type I fibers; however, pure IIB fibers were located mainly at the periphery of the rosettes near the edges of primary fascicles. In light of the present study, conventional IIB fibers, as defined with AM-ATPase staining, are a heterogeneous population that should be split into pure IIX, hybrid IIX/IIB, and pure IIB fibers for a more accurate fiber typing. Key Words: Myosins, Muscle Fibers, Skeletal Muscle, Pigs, Hybridization

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. Inverse]y, muscle IGF-I expression increased (P < .05) to maximal levels around birth. For in situ hybridization, frozen muscle tissue sections (10 [[micro]meter]) 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. Key Words: Insulin-like Growth Factor, Muscles, Pigs

The synergistic activity of mechanically recovered neck bone lean (MRNL) and ascorbic acid (Asc) preserves the color of ground beef patties for four days during retail display. The amount of surface metmyoglobin (metMb) is less in patties containing Asc as compared to those prepared without Asc. The level of surface metMb decreases linearly with an increase in MRNL level in patties containing Asc but is unaffected in patties without Asc. The color stabilizing effect of Asc and MRNL is lost after five days of display probably due to microbial activity.

Color image processing techniques are used to determine the muscle color and marbling scores of beef ribeye steaks. The method correctly determines the sensory scores corresponding to color and marbling as 0.86 and 0.84, respectively. Steak images are processed for the evaluation of color and marbling. A beef color guide and USDA marbling score cards are used to initially allot marbling and color scores to each steak.

The objective of this study was to determine the effects of recombinant porcine somatotropin (rpST) on placental size, fetal growth, and maternal and fetal IGF-I and IGF-II concentrations. Twenty-four pregnant gilts received daily injections of either 1 mL of saline (control) (n = 12) or 5 mg of rpST (n = 12) from d 30 to 43 of gestation. Gilts were slaughtered on d 44 of gestation, reproductive tracts were removed, and fetal weight and length, placental weight, and implantation length were recorded. There was no effect of rpST on fetal or implantation length. Placental weight increased with rpST administration (71.20 [+ or -] 3.52 vs 58.35 [+ or -] 3.41 g; P < .02), as did fetal weight (18.06 [+ or -] .55 vs 16.44 [+ or -] .53 g; rpST vs control, respective]y; P < .05). Implantation lengths were partitioned into quartiles to determine the effect of rpST on fetuses with different implantation lengths. The effect of rpST on fetal weight was greater in the first quartile (< 137.5 mm) than in the fourth quartile (> 240 mm) (16.04 vs 13.86 g compared with 19.47 vs 18.21 g, respectively). Analy-sis using a modified Brody curve suggests that the effect of rpST treatment on fetal weight is equivalent to the effect of increasing implantation length by 58.8 mm. Administration of rpST numerically raised IGF-I (P = .07) and IGF-II (P = .12) concentrations in fetal serum. Although maternal serum IGF-I concentrations were similar at d 30, treatment with rpST increased these concentrations over time (77.76, 247.75, 267.85 vs 82.59, 79.59, 77.97 ng/mL on d 30, 37, 43, respectively; P < .001, SE = 14.09). Maternal IGF-II concentrations were also similar at d 30 but decreased over time with rpST treatment (265.78, 219.61, 191.05 vs 285.44, 284.72, 283.05 ng/mL; P < .03, SE = 14.03). Increased maternal IGF-I concentrations may exhibit negative feedback on maternal IGF-II concentrations. The more pronounced effect of rpST on growth in fetuses with shorter implantation lengths suggests that rpST may increase uptake or utilization of nutrients by fetuses. In addition, nutrient transfer across placental membranes may be enhanced by rpST. Key Words: Somatotropin, Fetal Growth, Insulin-Like Growth Factor, Pigs

Double-muscled fetuses develop more muscle fibers than normal-muscled fetuses. To examine whether serum growth factors modulate muscle development in cattle, twin pregnancies were induced in eight Holstein heifers using embryos from Belgian Blue and Holstein genetics representing heavy (HM) and light (LM) muscled cattle, respectively. Twin combinations were 1) two pairs of Belgian Blue fetuses that were designated as HM (HM), 2) two pairs of Holstein fetuses that were designated as LM (LM), and 3) four pairs of mixed fetuses; the four Holstein fetuses were designated as LM (HM) and the four Belgian Blue fetuses were designated as HM (LM). Pregnancies were terminated at 175 [+ or -] 5 d after conception and fetuses, with evidence of vascular anastomosis, were dissected. Carcass weights weregreatest (P < .05) for HM fetuses. Total bone and individual femur weights were greatest (P < .05) for LM (LM) fetuses. Total skeletal muscle mass and mass of semitendinosus, quadriceps femoris, infraspinatus, and longissimus muscles were in the order of HM (HM) > HM (LM) > LM (HM) = LM (LM) (P < .05). Estimated apparent muscle fiber number determined from a cross-section of semitendinosus muscle was in the order of HM (LM) > HM (HM) > LM (HM) = LM (LM) (P < .05). These data show that the presence of a co-twinned fetus with a lower genetic propensity for muscle development reduces the capacity of heavily muscled fetuses to develop muscle mass by 175 d after conception and strongly support the idea that blood-borne factors regulate muscle hypertrophy in fetal cattle. Key Words: Myogenesis, Twinning, Growth Factors, Bovidae

: Double-muscled (DM) cattle possess more muscle fibers than do normal-muscled (NM) beef or dairy cattle. Serum-borne growth factors have been shown to modulate myogenesis. Media containing serum from 12 DM and 60 NM fetuses grouped by crown-rump lengths (CRL) of [is less than or equal to] 25, 26 to 50, 51 to 75, or > 75 cm were used to test the effect of fetal serum on L6 myoblast proliferation. Because DM and NM fetuses were similar in CRL at ages corresponding to 20- to 60-cm CRL, CRL was used to determine fetal age. Normal-muscled fetal serum-induced thymidine incorporation in L6 myoblasts was greater (P < .05) at CRL > 50- than at [is less than or equal to] 50-cm CRL. Mean incorporation tended to increase with CRL. Thymidine incorporation was 56, 41, and 41% greater (P < .05) with serum from DM fetuses than with that from NM fetuses at CRL of < 25, 26 to 50, and 51 to 75 cm, respectively. Morphological examination of cross-sections of the semitendinosus muscle showed that apparent muscle fiber number was greater (P < .05) for DM fetuses than for NM fetuses. These results confirm greater apparent muscle fiber number in DM cattle and show the existence of greater growth factor activity in serum of DM fetuses during early fetal development. This greater growth factor activity may play a role in bovine muscle fiber hyperplasia. Key Words: Bovine, Fetus, Double Muscling, Myoblast Proliferation, Muscle Fibers, Serum