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Stearoyl CoA desaturase (SCD) is responsible for converting SFA into MUFA and plays an important role in regulating the fatty acid composition of tissues. Although the number of SCD isoforms differs among species, SCD-1 is the predominant isoform expressed in the major lipogenic tissues of all species studied. The SCD-1 gene promoter region has been cloned for several species, including the human, mouse, pig, and recently, the cow. In this study, we cloned and partially characterized the ovine SCD promoter region. Sequence alignment showed a high degree of similarity with published bovine (94%) and porcine (92%) sequences. This included a highly conserved PUFA response region, which was also similar to that found in the human SCD and mouse SCD-1 promoters. Previous studies have indicated that there may be species differences in the regulation of SCD promoter activity by fatty acids. Using promoter-reporter gene (luciferase) constructs transfected into both HEK 293 and McA-RH7777 cells (kidney- and liver-derived cell lines, respectively), we showed the activity of the SCD promoter from 4 different species (mouse, human, pig, and sheep) to be reduced in a dose-dependent manner by addition of unsaturated fatty acids to the media, with linoleic acid being more potent than oleic acid after a 24-h treatment at 60 [micro]M. This effect was dependent on the presence of the PUFA response region. In each of the species studied, the PUFA response region of the SCD promoter was shown to have an active sterol response element, which responded to treatment of cells with sterol or overexpression of the truncated active form of sterol regulatory element binding protein-1c. Thus, any species differences in previously reported regulation of SCD expression by fatty acids are not due to differences in promoter structure between species, but are more likely to depend on the cell type being studied or the relative concentrations and distribution of sterol regulatory element binding proteins or other transcription factors. Key words: fatty acid, pig, promoter activity, sheep, stearoyl coenzyme A desaturase, sterol doi: 10.2527/jas.2009-2603

The contractile and associated metabolic characteristics of muscles are determined by their myosin heavy chain (MHC) isoform expression. In large mammals, the level of MHCIIB expression, which is associated with fast glycolytic-type muscle fibers, has not been fully characterized. In this study, quantitative reverse transcription-PCR and SDS-PAGE methodologies were developed for the analyses of adult ovine MHC isoform expression and used to characterize MHC expression in 3 skeletal muscles [LM, semitendinosus, and supraspinatus) from 66-d-old lambs. Three MHC isoforms (MHCI, MHCIIA, and MHCIIX) were detected at both the protein and messenger RNA levels in all 3 muscles, with greater proportions of type II than type I MHC. The expression of MHCIIB could not be detected at the protein level in any of the muscles and was detectable (in semitendinosus muscle) only at the messenger RNA level by using semiquantitative reverse transcription-PCR, indicating that MHCIIX is the predominant fast glycolytic fiber type in the sheep muscles studied. The methodologies developed are suitable for studying fiber type transformations at the molecular level, as well as allowing analyses of very small samples, including biopsies, when histochemical analysis may not be possible. Key words: fiber type, myosin heavy chain, real-time polymerase chain reaction, sheep, skeletal muscle, sodium dodecyl sulfate-polyacrylamide gel electrophoresis doi: 10.2527/jas.2009-2067

The calpain proteinases and their specific inhibitor calpastatin have been proposed to influence both the rates of myofibrillar protein turnover in vivo and meat tenderization postmortem. Elevated calpastatin concentrations in particular are associated with certain forms of hypertrophic growth and meat toughness. In the 5' region of the porcine calpastatin gene, there are 3 calpastatin promoters upstream of exons 1xa, 1xb, and 1u, respectively, each of which contain transcription factor-binding motifs, suggesting sensitivity to a variety of growth-promoting stimuli. This study examined the effect of the [beta]-adrenergic agonist clenbuterol and porcine ST (pST) treatment on calpastatin promoter usage in porcine LM in vivo using real-time PCR and also the responsiveness of transfected calpastatin promoter sequences to cyclic adenosine monophosphate (cAMP) and calcium ([Ca.sup.2+]) related stimuli in reporter gene systems in cell studies. The effect of clenbuterol and pST on potential signaling pathways in vivo was also assessed by monitoring protein phosphatase 2B (calcineurin), NFATc3, calpain 3, I[kappa]B[alpha], and NF[kappa]B by quantitative immunoblotting. Total calpastatin mRNA was increased by 52% (P < 0.05) after treatment with clenbuterol for 1 d and reduced by 35% (P < 0.01) after pST treatment for 7 d. Whereas clenbuterol had no significant differential effects on individual mRNA transcripts (types 1 to 3) derived from the 3 upstream promoters, pST significantly reduced all of these by 51, 39, and 40% (P < 0.001, 0.05, and 0.05), respectively. Promoter activity was increased in rat L6G8 cells transfected with a construct derived from exon 1u after treatment with dibutyryl cAMP (68%, P < 0.05) or forskolin (43%, P < 0.05), whereas 1xa activity was reduced by both of these agents (47 and 33%, respectively, P < 0.05). Treatment of cells with the calcium ionophore calcimycin reduced the activity of the 1u promoter by 40% (P < 0.01), with no effect on the other promoter constructs. Cyclosporin A had no effect on any promoter construct. The only signaling pathway component to be significantly altered by the in vivo treatments was calcineurin, which was decreased by 24% (P < 0.05) in clenbuterol-treated animals. In conclusion, 2 types of growth promoter in pigs had contrasting effects on calpastatin expression in LM. Transfected calpastatin promoters were differentially sensitive to cAMP- and [Ca.sup.2+]-related stimuli, in agreement with the proposed mode of action of the 2 growth promoters. Key words: calpastatin, calpastatin expression, growth promoter, pig, skeletal muscle

The objective of this study was to investigate the protease family caspases in skeletal muscle and their potential contribution to postmortem proteolysis and meat tenderization. Ten Large White gilts were slaughtered, and samples of LM were taken at 0, 2, 4, 8, 16, 32, and 192 h after slaughter and immediately snap frozen in liquid nitrogen. Samples were subsequently analyzed for caspase 3/7 and caspase 9 activity, protein levels of known caspase substrates, alpha II spectrin and poly (ADP-ribose) polymerase (PARP), as well as, at 192 h, shear force. Specific degradation products of alpha II spectrin and PARP, which are known indicators of caspase activity, and apoptosis were detected on immunoblots of muscle samples taken over the postmortem period. The relationships between the changes observed in caspase activities and protein levels of PARP and spectrin across the entire postmortem conditioning period were investigated (n = 70). Protein levels of alpha II spectrin cleavage products across the conditioning period were found to correlate positively to caspase 3/7 activity (r = 0.38, P = 0.003) and caspase 9 activity (r = 0.32, P = 0.012), indicating that caspase-mediated cleavage was occurring in situ. There was a negative relationship between shear force and the 0 to 32 h ratio of caspase 3/7 (r = -0.62, P = 0.053) and caspase 9 activities (r = -0.68, P = 0.044). In addition, there was also a negative relationship between shear force and the level of the caspase-generated alpha II spectrin 120 kDa degradation product (r = -0.75, P = 0.012). The findings of this study indicate that changes in caspase activity and caspase-mediated cleavage take place in muscle during the conditioning period, and this could be associated with the development of tender meat. Key words: caspase, meat, pig, skeletal muscle, tenderization

There is a need to improve the lean tissue content of ruminant animals destined for meat production. Muscle fiber number is set during fetal development. The effect of undernutrition of pregnant ewes on subsequent muscle fiber characteristics of their offspring was investigated. The trial involved 32 pregnant ewes carrying twins. The ewes were allocated randomly to one of four groups: three different treatment groups (n = 8) and a control group (n = 8). The diet of the treatment groups was dropped to 50% of their daily requirement to support the ewe and allow for conceptus growth for varying periods before being returned to 100% of their daily requirement until term. Group d 30-70 ewes were fed 100% of their daily requirement until d 30, the diet was then decreased to 50% until d 70; it was then returned to 100% of their daily requirement until term. Group d 55-95 ewes were similarly restricted from d 55 through 95, and Group d 85-115 ewes were restricted from d 85 through 115. The control group was fed 100% of their daily requirement to support the ewe and allow for conceptus growth throughout gestation. After parturition, the lactating ewes were fed a normal commercial diet. On d 14 (after parturition), the lambs were slaughtered and the LM, semitendinosus (ST), and vastus lateralis (VL) were dissected and snap frozen. The immunochemical determination of myosin heavy-chain slow (MHC-slow) and myosin heavy-chain fast (MHC-fast) proteins was measured by immunoprobing of Western blots. The number of fast and slow fibers and the diameter of these fibers also were measured in each muscle sample by histochemical techniques. Decreased maternal nutrition before fiber formation (d 30 through 70) was observed to change the muscle characteristics of the newborn lambs. These lambs had significantly fewer fast fibers (P < 0.001) and significantly more slow fibers (P < 0.001) in both the LM and VL compared with the other groups. Maternal nutrient restriction at the other periods had no effect on the number of muscle fibers in the newborn lambs; however, a decrease (LM, P < 0.05; VL, P < 0.01; ST, P = 0.08) in muscle weight was observed in the lambs born to the ewes restricted between d 85 and 115 of gestation compared with the other groups. This study has shown that decreased maternal diet before muscle fiber formation will alter the muscle fiber development in the fetus. Key Words: Maternal Nutrition, Muscle Fiber Formation, Sheep

The number of muscle fibers within a muscle has been found to be of high importance for the growth potential of an animal, and this number is set during fetal development. The objective of this study was to identify the ontogeny of muscle cell differentiation and fiber formation by observing the changes in expression of factors known to influence myoblast proliferation and differentiation. Twenty-one Swaledale x Leicester Blue Face ewes carrying twins were allotted to this trial. From d 40 of gestation, three ewes were killed every 15 d until term. At each time point, the fetuses were located, removed, and total muscle from both hind limbs was dissected from each fetus and snap frozen in liquid [N.sub.2]. Ribonuclease protection assays were used to quantify transcripts for IGF-I, IGF-II, GH receptor (GHR), and myostatin genes in the muscle samples, whereas quantitative real-time PCR was used to quantify myogenin transcripts. Histological sections also were taken from the fetal muscle samples and observed for evidence of muscle differentiation resulting in fiber formation. The abundance of mRNA for ovine IGF-II and ovine myogenin peaked at d 85 of gestation (P < 0.001). The abundance of ovine IGF-I transcripts peaked at d 100 of gestation, whereas the abundance of ovine GHR mRNA increased throughout gestation (P < 0.05). No change (P = 0.87) in the abundance of myostatin mRNA was observed. The histological sections from the muscle samples demonstrated a clear change in the appearance of the muscle tissue at each time period. Major fiber formation was observed around d 85. The results obtained from the analysis of gene expression and the histological sections suggest that the majority of muscle differentiation and fiber formation takes place around d 85, with myoblast proliferation mainly occurring before this time. It may be possible to manipulate the number of muscle fibers formed by targeting treatments during this proliferation stage immediately before the period of major fiber formation. Key Words: Muscle Differentiation, Myogenin, Sheep

The calpain proteolytic system was examined in the longissimus muscle (LD) of heterozygote pigs carrying a single copy of a mutation in the skeletal muscle ryanodine receptor gene (RyR1) that is associated with porcine stress syndrome and reduced meat quality. Conventional British White-type pigs (n = 30) were selected from a commercial line on the basis of slaughter weight, backfat depth, and pH at 45 min postmortem > 6.0; based on DNA analysis, 11 were heterozygous RyR1 mutants (Nn), and 19 were normal genotype (NN). The LD samples were taken from carcasses at 2, 4, and 24 h postmortem for calpain analysis with enzyme assay and immunoblotting, using specific antisera raised against recombinant polypeptides derived from calpain large subunits and calpastatin. Shear force (SF) was measured after conditioning for 8 d at 2 degrees C and did not differ between Nn andNN groups. The extractable activity of (mu)-calpain decreased over 24 h postmortem (P < .001), with no significant difference in activity between NN and Nn animals at any time. The activity of m-calpain also decreased with time (F < .001), but it was lower at all times in Nn than in normal genotypes (P < .001). After Western blotting, the immunoreactivity of (mu)- and m-calpain large subunit bands declined over 24 h postmortem (P < .001); values for (mu)-calpain were higher (P < .05) and for m-calpain were lower (P < .001) in heterozygotes than in normal animals at each sampling time. The calpastatin antibody detected a major band of 135 kDa that declined with time postmortem but did not differ between Nn and NN genotypes at any sampling time. These data indicate that the levels of extractable (mu)- and m-calpain, but not calpastatin, may be different in pigs that carry the RyR1 mutation. Key Words: Pigs, Muscles, Calpain, Calpastatin

Tenderization of skeletal muscle in meat animals has been closely linked to the postmortem activity of the calpain proteolytic enzyme system, which includes the specific inhibitor calpastatin. Increased understanding of the skeletal muscle-specific calpain isoform p94 has prompted suggestions as to whether it too could have a role in the tenderization process. In this study, two groups of pigs were identified in which shear force measurements after 8 d of conditioning indicated a large variation in the tenderness of longissimus muscle. The quantity of p94 in the muscle was monitored by immunoblotting, using a porcine-specific polyclonal antibody raised against a recombinant peptide fragment generated as a fusion protein. The antiserum recognized a 94-kDa protein associated with myofibrils in skeletal but not cardiac muscle, as expected for this calpain isoform, although it could not be tested with the native protein because of the extreme instability of p94. In the first experiment, the mean shear force for the tough group was 6.71 [+ or -] .28 kg (n = 12, SEM) and that of the tender group was 3.87 [+ or -] .12 kg (n = 12), but there was no difference in the normalized absorbance of the immunopositive 94 kDa band on Western blots from samples collected at approximately 40 min postmortem. In the second experiment, the stability of p94 in chilled carcasses was investigated over 24 h, using a further two groups of 10 tough and 10 tender pigs of mean shear force values 5.36 [+ or -] .14 kg and 2.81 [+ or -] .15 kg, respectively. In tough and tender animals, there was a decline (P < .05) in the 94-kDa immunostaining material of mean half-lives of 13.8 and 12.9 h, respectively, although there was considerable variability. Despite this variability in half lives and shear force values, no correlation was seen between these factors. Thus, in porcine longissimus muscle, the variability in tenderness after 8 d of conditioning cannot be attributed to an underlying variability in p94. Key Words: Pigs, Skeletal Muscle, Calpain, Meat Quality

The relationship between plasma epinephrine and postmortem activity of the calpain system in porcine longissimus muscle was investigated. Two groups of Large White pigs were continuously infused intravenously with either placebo (control) or epinephrine (treated) at a rate of .15 [[micro]gram][center dot][kg.sup.-1][center dot][min.sup.-1] for a period of 1 wk before slaughter. Samples of longissimus muscle were taken at 0, 1, 2, 4, 8, 24, 48, and 192 h (to to [t.sub.192]) after slaughter and immediately snap-frozen in liquid nitrogen for subsequent analysis of [micro]-calpain, m-calpain, and calpastatin activity. Epinephrine infusion had no effect on the activities of [micro]- and m-calpain at [t.sub.0]. Calpastatin activity at [t.sub.0] was increased (P < .01) in treated pigs by 97%. The ratio of total calpain:calpastatin activity at to was reduced (P < .01) in treated pigs. The activity of [micro]-calpain decreased rapidly after slaughter, irrespective of treatment, dropping to less than 10% of the initial activity within 8 h. The activity of m-calpain also decreased over the first 8 h, although the rate of decrease was less (P < .05) in treated pigs. Consequently, m-calpain activity remained greater in treated pigs compared with controls throughout the period normally associated with tenderization. Postmortem values for calpastatin activity tended to be highly variable, with activities being similar between control and treated pigs within 1 h after slaughter. Over the entire 192 h sampling period, calpastatin activity decreased (P < .001), although the effect was independent of treatment. In general the results imply that variations in plasma epinephrine concentrations, which may naturally occur as part of the stress response, perturb the calpain enzyme system. Key Words: Pigs, Calpain, Calpastatin, Skeletal Muscle, Beta-Adrenergic Agonists, Epinephrine