Effect of the beta-adrenergic agonist L644,969 on muscle growth, endogenous proteinase activities, and postmortem proteolysis in wether lambs.

To examine the effect of a beta-adrenergic agonist (BAA) on muscle growth, proteinase activities, and postmortem proteolysis, 16 wether lambs were randomly assigned to receive 0 or 4 ppm of L644,969 in a completely mixed high-concentrate diet for 6 wk. Weight of the biceps femoris was 18.6% heavier in treated lambs. At 0 h after slaughter, treated lambs had higher cathepsin B (35.6%), cathepsins B + L (19.1%), calpastatin (62.8%), and m-calpain (24.6%) than control lambs, but both groups had similar mu-calpain activities. In both longissimus and biceps femoris muscles, treated lambs had higher protein and RNA and lower DNA concentrations. However, total DNA was not affected, indicating that the increase in muscle mass was probably due to muscle hypertrophy rather than to hyperplasia. The pattern of postmortem proteolysis was significantly altered by BAA feeding. In treated lambs, postmortem storage had no effect on the myofibril fragmentation index and degradation of desmin and troponin-T. These results indicate that the ability of the muscle to undergo postmortem proteolysis has been dramatically reduced with BAA feeding. Similar proteolytic systems are thought to be involved in antemortem and postmortem degradation of myofibrillar proteins, so BAA-mediated protein accretion is probably due, at least in part, to reduced protein degradation. To examine whether protein synthesis was altered with BAA feeding, the level of skeletal muscle alpha-actin mRNA was quantified. Longissimus muscle alpha-actin mRNA abundance was 30% greater in BAA-fed lambs. Collectively, these results indicate that dietary administration of BAA increases muscle mass through hypertrophy and that the increase in muscle protein accretion is due to reduced degradation and possibly to increased synthesis of muscle proteins.     

Effect of the callipyge gene on muscle growth, calpastatin activity, and tenderness of three muscles across the growth curve

Changes in muscle growth, calpastatin activity, and tenderness of three muscles were assessed in 20 callipyge and 20 normal wether lambs slaughtered at live weights (LW) of 7, 20, 36, 52, and 69 kg. At 24 h postmortem, the longissimus (LM), semimembranosus (SM), and supraspinatus (SS) muscles were removed and weighed and samples were obtained for calpastatin activity (CA; 24 h) and Warner-Bratzler shear force (WBS; aged 6 d). For muscle weights and calpastatin activity, the weight group x muscle x phenotype interaction was significant (P < 0.05). Muscle weights were similar (P > 0.05) between phenotypes for all three muscles at 7 kg LW. At 20 kg LW, the LM and SM muscles from the callipyge lambs were heavier (P < 0.05) than those from normal lambs; however, the SS did not differ (P > 0.05) between phenotypes at 7, 20, or 52 kg. From 20 to 69 kg LW, the LM and SM weights were 42 and 49% heavier (P < 0.05) for callipyge than for normal lambs. Calpastatin activity of the callipyge LM was greater (P < 0.05) than that of normal LM at 36, 52, and 69 kg. In the callipyge LM, CA was similar (P > 0.05) at 20, 36, and 52 kg LW and did not differ (P > 0.05) from 7-kg or 69-kg values. Calpastatin activity declined (P < 0.05) across the growth curve for the SM and SS, but values were higher (P < 0.05) in the SM in callipyge than in normal lambs. Shear force values of the LM were lower (P < 0.05) for normal lambs at 36, 52, and 69 kg LW than for callipyge lambs. In the SM and SS, WBS values decreased (P < 0.05) across the growth curve, but values were higher (P < 0.05) for callipyge lambs in the SM only. These data indicate that the selective muscular hypertrophy of the callipyge phenotype develops during the postnatal growth period between 7 and 20 kg LW (19 and 100 d of age). Longissimus and semimembranosus muscles in the callipyge lambs were over 40% heavier from 20 to 69 kg LW; however, they also had higher levels of calpastatin activity and Warner-Bratzler shear force during this time period, indicating the need for postmortem tenderization treatments to improve palatability.     

Effects of age and castration on activities of calpains and calpastatin in sheep skeletal muscle.

The objectives of this experiment were to assess effects of animal age and castration on activities of calpain I, calpain II, and calpastatin in sheep skeletal muscle. Ten newborn male lambs (2.9 kg), six weaned wethers (23.2 kg), six weaned rams (22.2 kg), six market wethers (55.4 kg), and six market rams (60.2 kg) were slaughtered and samples of biceps femoris were taken for assay of calpain I (micromolar calcium-dependent proteinase), calpain II (millimolar calcium-dependent proteinase), and calpastatin. Preweaning weight gain was similar for rams and wethers; however, postweaning ram growth exceeded (P less than .05) that of wethers. Ram biceps femoris weights at market were greater than those of wethers (P less than .05). Irrespective of age or gender, activity of calpain II was two- to threefold greater than that of calpain I. Muscle calpastatin activity was severa fold higher than calpain I and II activities. Activities of calpains and calpastatin declined (P less than .05) between birth and weaning. A portion of these losses were due to a dilution effect caused by accumulation of muscle proteins. Neonatal attenuation of calpain activities may underlie age-related attenuation of fractional rates of muscle protein degradation. Although ram muscle growth exceeded that of wethers, no differences (P greater than .05) in activities of muscle calpains or calpastatin were detected between these two groups at weaning or at market weight. Hence, castration did not influence lamb muscle growth by altering muscle calpain or calpastatin activities.     

Postmortem proteolysis and calpain/calpastatin activity in callipyge and normal lamb biceps femoris during extended postmortem storage.

The present experiment was conducted to determine whether calpastatin inhibits only the rate, or both the rate and extent, of calpain-induced postmortem proteolysis. Biceps femoris from normal (n = 6) and callipyge (n = 6) lamb was stored for 56 d at 4 degrees C. Calpastatin activity was higher (P < .05) in the callipyge muscle at 0 and 14 d postmortem, but not at 56 d postmortem. The activity of mu-calpain did not differ between normal and callipyge biceps femoris at 0 and 56 d postmortem (P > .05), but was higher at 14 d postmortem in the callipyge muscle (P < 0.05). The activity of m-calpain was higher in the callipyge muscle (P < 0.05). Western blot analyses of titin, nebulin, dystrophin, myosin heavy chain, vinculin, alpha-actinin, desmin, and troponin-T indicated that postmortem proteolysis was less extensive in callipyge than in normal biceps femoris at all postmortem times. The results of this experiment indicate that calpastatin inhibits both the rate and extent of postmortem proteolysis.     

Electrical stimulation effects on tenderness of five muscles from Hampshire x Rambouillet crossbred lambs with the callipyge phenotype

The objective of this study was to determine effects of electrical stimulation (ES) on muscle quality and sensory traits of 12 Hampshire x Rambouillet callipyge lambs. One side of each carcass was randomly assigned to an ES treatment of 550 V and 60 Hz of electricity for 2 s on and 2 s off 15 times. The other side was a nonstimulated control (NES). Heated calpastatin, sarcomere length, myofibrillar fragmentation index (MFI), Warner-Bratzler shear (WBS), and trained sensory panel values were measured on the semitendinosus (ST), semimembranosus (SM), longissimus (ML), supraspinatus (SP), and triceps brachii (TB) muscles. Electrically stimulating the carcass sides induced a more rapid (P = .001) pH decline in the longissimus muscle, and ES sides had a brighter (P = .001) red color of loineye than nonstimulated sides. At d 14 of storage (2 degrees C), the TB had the highest (P < .05) MFI value, indicating more protein degradation, and the ST and ML muscles had the lowest MFI (P = .008). Regardless of ES treatment, SM and ML had the highest (P < .05) WBS values. The ST muscle had higher (P < .05) WBS values than the SP but did not differ (P > .05) from the TB muscle. Electrical stimulation had no effect on WBS or any trained sensory panel values (P > .05). The percentage of loin chops rated slightly tender or better was improved 30 to 34% by electrical stimulation (P < .05). The ML muscle was scored lower (P < .05) in sustained juiciness compared with the SM, SP, and TB but did not differ (P > .05) from the ST muscle. The SM and ML muscles were rated lower (P < .05) in initial and sustained tenderness scores than other muscles. Tenderness scores were higher (P < .05) for the TB than for the SP but did not differ (P > .05) from the ST muscle. Electrically stimulating callipyge carcasses improves the tenderness of loin chops by increasing the percentage of chops rated from slightly tough to slightly tender.     

The calpain system in three muscles of normal and callipyge sheep

Activities of mu- and m-calpain and of calpastatin were measured at four different times during postmortem storage (0, 1, 3, and 10 d) in three muscles from either callipyge or noncallipyge (normal) sheep. The weights of two muscles, the biceps femoris and the longissimus, are greater in the callipyge phenotype, whereas the weight of the infraspinatus is not affected. The activity of m-calpain was greater (P < 0.05) in the biceps femoris and longissimus from callipyge than in those from normal sheep, but it was the same in the infraspinatus in the two phenotypes. The extractable activity of m-calpain did not change (biceps femoris and infraspinatus) or decreased slightly (longissimus) during postmortem storage. Extractable activity of mu-calpain decreased to zero or nearly zero after 10 d postmortem in all muscles from both groups of sheep. The rate of decrease in mu-calpain activity was the same in muscles from the callipyge and normal sheep. At all time points during postmortem storage, calpastatin activity was greater (P < 0.05) in the biceps femoris and longissimus from the callipyge than from the normal sheep, but it was the same in the infraspinatus from callipyge and normal sheep. Calpastatin activity decreased (P < 0.05) in all three muscles from both phenotypes during postmortem storage; the rate of this decrease in the callipyge biceps femoris and longissimus and in the infraspinatus from both the callipyge and normal sheep was slow, especially after the first 24 h postmortem, whereas calpastatin activity in the biceps femoris and longissimus from the normal sheep decreased rapidly. During postmortem storage, the 125-kDa calpastatin polypeptide was degraded, but the 80-kDa subunit of mu-calpain was cleaved only to 76- and 78-kDa polypeptides even though extractable mu-calpain activity declined nearly to zero. Approximately 50 to 60% of total mu-calpain became associated with the nonextractable pellet after 1 d postmortem. The myofibril fragmentation index for the biceps femoris and longissimus from normal sheep increased significantly during postmortem storage. The fragmentation index for the infraspinatus from the callipyge and normal sheep increased to an intermediate extent, whereas the index for the biceps femoris and longissimus from the callipyge did not change during 10-d postmortem storage. The results suggest that postmortem tenderization is related to the rate of calpastatin degradation in postmortem muscle and that calpastatin inhibition of the calpains in postmortem muscle is modulated in some as yet unknown manner.     

Feeding high levels of vitamin D3 does not improve tenderness of callipyge lamb loin chops

The objective of this study was to determine whether feeding high doses of vitamin D3 7 d before slaughter would increase muscle Ca++ levels and result in more tender loin chops. Market lambs (n = 4 callipyge and 4 normal in Exp. 1, and n = 16 calipyge and 16 normal in Exp. 2) were randomly and equally assigned to feeding groups based on callipyge genotype and experimental diet, (vitamin D3 or control). Serum Ca++, muscle Ca++, Warner-Bratzler shear force, and troponin-T degradation data were analyzed. In Exp. 1, vitamin D3 was supplemented at 1 or 2 x 10(6) IU/d. The 2 x 10(6) IU dose resulted in the greatest serum Ca++ reponse and was chosen for Exp. 2. In Exp. 2, serum Ca++ concentration was higher (P < 0.05) for normal and callipyge lambs fed the vitamin D3 diet than for the control diet fed lambs. Muscle Ca++ concentrations, however, were not higher (P = 0.28) for the vitamin D3-fed lambs. Warner-Bratzler shear values were higher (P < 0.05) for callipyge than for normal lambs, but no differences were observed with vitamin D3 supplementation. These data were supported by results from Western blot analysis of troponin-T degradation, in which no differences were observed for vitamin D3 vs control diet lambs at 14 d postmortem. This experiment showed that feeding 2 x 10(6) IU/d of vitamin D3 to market lambs, callipyge or normal, raised serum Ca++ concentration, but did not increase muscle Ca++ concentration. This lack of response in muscle Ca++ was likely the reason that no differences were observed for Warner-Bratzler shear force values or troponin-T degradation data between the vitamin D3 and control loin chops. A higher dose of vitamin D3 may be required to improve tenderness.     

Effect of dietary protein on calpastatin in canine skeletal muscle

The cysteine proteinases, mu- and m-calpain, along with their inhibitor, calpastatin, have been hypothesized to play a role in skeletal muscle protein degradation. Because nutrition has previously been shown to influence the expression of calpastatin, the working hypothesis of this study was that the quantity and source of dietary protein could influence regulation of the calpain system in muscle. The objectives to support this hypothesis were to determine the effects of dietary protein (amount and source) on the expression of calpastatin in canine skeletal muscle. This study comprised eight diets with seven dogs per diet. A biopsy was taken from the biceps femoris of all 56 dogs before and after 10 wk on their respective diets. This experimental design allowed examination of change within individual dogs. Diets 1 to 4 contained 12% total protein derived from chicken and/or corn gluten meal in ratios of 100:0, 67:33, 33:67, and 0:100%, respectively. Diets 5 to 8 contained 28% total protein with protein sources and ratios identical to Diets 1 to 4. Differences in calpastatin were examined qualitatively using SDS-PAGE and immunoblotting, and semiquantitatively with densitometric analyses. The majority of the calpastatin blots showed three distinct calpastatin bands, the uppermost appearing at approximately 110 kDa. Diet 5 (28% CP, 100% chicken) resulted in an increase in the expression of the 110-kDa calpastatin band compared with the other two lower molecular weight bands in the same samples. Muscle from dogs fed Diet 5 showed greater increase in (P < 0.05) calpastatin intensity of the topmost band than those fed Diet 8 (0:100; chicken:corn gluten meal). Diet 5 (100:0; chicken:corn gluten meal) showed greater total calpastatin intensity than Diet 8 (0:100; chicken:corn gluten meal). These data suggest that dogs fed a diet containing a higher total percentage of chicken protein may have a greater potential to regulate calpain-mediated degradation of muscle protein than dogs fed diets containing corn gluten meal.     

Muscle protein metabolism in finishing pigs fed ractopamine

Forty crossbred barrows (average initial weight, 66.4 kg) were utilized to determine the effects of ractopamine (a phenethanolamine/beta adrenergic agonist) on protein accretion and synthesis, activities of cathepsins B, H, L and calcium-dependent proteinase and nucleic acid content of semitendinosus muscle (ST). All pigs were offered a 16% protein, mineral and vitamin fortified corn-soybean meal diet supplemented with either 0 or 20 ppm ractopamine for 14, 21, 28, 35 or 42 d. Protein synthesis (fractional rates) was studied in pigs at d 21 and 35; ST protease activities, protein and nucleic acid content were measured on d 14, 28 and 42. Ractopamine increased (P less than .01) ST total protein content and maintained RNA muscle concentration and total ST muscle RNA content. DNA content (mg/g ST) declined (P less than .05) upon ractopamine feeding, but total DNA per muscle remained unchanged except for d 42, when the ST muscles were largest. Fractional accretion rates (FAR) were 1.0 and 1.2% for control and ractopamine-fed pigs, respectively. Fractional protein synthesis rate (FSR) was higher (P less than .06) in ractopamine-fed pigs (6.1%/d) than in control pigs (4.4%/d). Fractional protein synthesis rate could account for the observed muscle hypertrophy and increased FAR. Estimated fractional breakdown rates (FBR = FSR - FAR) were 3.4%/d and 4.9%/d for control and ractopamine-fed pigs, respectively. The activities of the catheptic proteases and calcium-dependent proteinase were not affected by the treatments.     

Level of nutrition and visceral organ protein synthetic capacity and nucleic acid content in sheep.

Thirty-two crossbred wether lambs were assigned to a feed intake level of either ad libitum (ADLIB) for maximum rate of growth or restricted to maintain body weight (MAINT) throughout a 21-d period. At 7-d intervals (d 0.7, 14, and 21), four lambs per treatment were slaughtered to obtain measurements of visceral organ protein synthetic capacity and tissue composition. Protein synthetic capacity was assessed by in vitro [14C]valine incorporation and tissue RNA, DNA, and protein contents. Concentrations of protein and RNA were not significantly affected in most tissues measured. However, for liver, duodenal, and jejunal tissue, DNA concentrations in ADLIB lambs were lower (P less than .05) than in MAINT lambs. Ratios of protein:DNA in most organs were higher (P less than .05) in ADLIB than in MAINT lambs. During the 21-d period, liver and small intestinal protein and RNA mass were higher (P less than .10) in ADLIB than in MAINT lambs, and DNA mass was unaffected. Also during the 21-d period, the average total mass of ruminal protein, RNA, and DNA in ADLIB lambs was higher (P less than .05) than in MAINT lambs. Estimates of valine incorporation and ratios of RNA:protein seemed to reflect protein synthetic capacity of the visceral tissues measured; however, the effect of level of feed intake on these measurements was equivocal. These data suggest that the level of feed intake affected visceral organ mass through changes in cellular hypertrophy.     

Characterization and consumer acceptance of three muscles from Hampshire x Rambouillet cross sheep expressing the callipyge phenotype

Eight Hampshire x Rambouillet crossbred wethers expressing the callipyge phenotype and eight Hampshire x Rambouillet half-sibling wethers with a normal phenotype were slaughtered when they reached 59 kg. The supraspinatus (SPM), longissimus (LM), and semitendinosus (STM) muscles were analyzed to determine callipyge effects on calpain and calpastatin activities, sarcomere length, percentage of muscle fiber types, and muscle fiber areas. After 14 d of aging, chops were frozen until analyses for trained sensory panel evaluations, Warner-Bratzler shear force values, and consumer perceptions of tenderness, flavor, juiciness, and overall satisfaction of chops were conducted. Calpastatin activity was 57% greater (P < 0.05) and m-calpain activity was 33% greater (P < 0.05) in muscles from carcasses of callipyge than normal sheep. Sarcomeres were shorter (P < 0.001) in the LM than the SPM or STM, regardless of phenotype. Muscle fiber area was 76% larger (P < 0.05) in the LM of callipyge than normal sheep, but muscle fiber area was not affected (P > 0.05) by phenotype in the SPM or STM. Phenotype had no effect (P = 0.12) on the percentage of slow-twitch, oxidative fiber types in any of the three muscles. In STM and LM from callipyge lambs, the percentage of fast-twitch, oxidative/glycolytic fibers was lower (P < 0.05) and that of fast-twitch-glycolytic fibers was higher (P < 0.05) than in their normal counterparts. Phenotype did not affect (P = 0.90) the fiber type percentage in the SPM. Callipyge LM were less tender and normal LM were more tender than other chops (P < 0.05). Callipyge loin chops had higher Warner-Bratzler shear force values than other chops (P < 0.001). Consumers rated fewer (P < 0.05) callipyge loin and shoulder chops acceptable in juiciness, tenderness, and overall acceptability than normal chops, but phenotype did not affect (P > 0.05) consumer acceptability of leg chops. These results indicate that LM from Hampshire x Rambouillet sheep displaying the callipyge phenotype had higher calpastatin activity and were less tender than the LM from normal sheep. In addition, consumer perceptions indicated that only one in 10 leg chops, one in five shoulder chops, and one in four loin chops from callipyge sheep were unacceptable.     

Cimaterol-induced muscle hypertrophy and altered endocrine status in lambs

The objectives of this study were 1) to determine how cellular growth of skeletal muscle is altered by the repartitioning agent cimaterol and 2) to determine if cimaterol alters endocrine status in association with its repartitioning effects. Thirty Dorset wether lambs were randomly assigned to a pre-treatment baseline group or received 0 or 10 ppm cimaterol in a complete, mixed, high-concentrate diet for 7 or 12 wk. Weights of biceps femoris (BF), semimembranosus (SM) and semitendinosus (ST) muscles were 32.8, 27.1 and 31.5% greater, respectively, in treated lambs at 7 wk, and were 22 to 24% greater at 12 wk. Longissimus (LD) cross-sectional area was 26 and 32% greater at these treatment intervals. Percent type I fibers declined significantly over the course of the experiment in ST, SM and LD, and cimaterol caused a small but significant reduction in percent type I fibers in the ST at 7 and 12 wk. Muscles from lambs fed cimaterol contained 50 and 75% more fibers that exhibited negative staining for phosphorylase activity. Mean cross-sectional area of type I and type II fibers in the combined portions of the ST were 30.4 and 29.3% greater, respectively, in cimaterol-fed lambs after 12 wk, while type I and type II fiber areas in the longissimus were only 13 and 15% greater, respectively. Cimaterol-induced hypertrophy of the ST resulted in both protein and RNA content being 30 to 35% greater (P less than .01) at 7 and 12 wk, while DNA concentration was 22% less (P less than .01) at 7 wk. DNA concentration returned to normal by 12 wk. These results indicate that cimaterol elicits a rapid increase in muscle RNA and protein accretion without concurrent incorporation of satellite cell nuclei. Plasma insulin and insulin-like growth factor-1 (IGF-1) concentrations were 55 and 34% lower, respectively, in cimaterol-fed lambs. Plasma somatotropin concentration and area under the curve were 2.3 times greater (P less than .01) in lambs fed cimaterol for 6 wk, while plasma cortisol, prolactin and glucose concentration were unaffected at 6 or 12 wk. The significant changes in endocrine status may be important in the mechanism(s) of cimaterol in altering muscle accretion.     

Effects of birth weight and postnatal nutrition on neonatal sheep: II. Skeletal muscle growth and development

This study investigated effects of birth weight and postnatal nutrition on growth and development of skeletal muscles in neonatal lambs. Low (L; mean +/- SD 2.289 +/- .341 kg, n = 28) and high (H; 4.840 +/- .446 kg, n = 20) birth weight male Suffolk x (Finnsheep x Dorset) lambs were individually reared on a liquid diet to grow rapidly (ad libitum fed, ADG 337 g, n = 20) or slowly (ADG 150 g, n = 20) from birth to live weights (LW) up to approximately 20 kg. At birth, weight of semitendinosus (ST) muscle in L lambs was 43% that in H lambs; aggregate weights of ST and seven other dissected muscles were similarly reduced. In ST muscle of L lambs, mass of DNA, RNA, and protein were also significantly reduced to levels 67, 60, and 34%, respectively, of those in H lambs. However, myofiber numbers of ST, tibialis caudalis, or soleus muscles did not differ between the L and H birth weight lambs and did not change during postnatal growth. During postnatal rearing, daily accretion rate of dissected muscle was lower in L than in H lambs. Accretion of muscle per kilogram of gain in empty body weight (EBW) was reduced in the slowly grown L lambs compared with their H counterparts, although the difference was less pronounced between the rapidly grown L and H lambs. Throughout the postnatal growth period, ST muscle of L lambs contained less DNA with a higher protein:DNA ratio at any given muscle weight than that of H lambs. Slowly grown lambs had heavier muscles at any given EBW than rapidly grown lambs. Content of DNA and protein:DNA ratio in ST muscle were unaffected by postnatal nutrition, but RNA content and RNA:DNA were greater and protein:RNA was lower at any given muscle weight in rapidly grown lambs. Results suggest that myofiber number in fetal sheep muscles is established before the presumed, negative effects of inadequate fetal nutrient supply on skeletal muscle growth and development become apparent. However, proliferation of myonuclei may be influenced by fetal nutrition in late pregnancy. Reduced myonuclei number in severely growth-retarded newborn lambs may limit the capacity for postnatal growth of skeletal muscles.     

Ubiquitin gene expression and ubiquitin conjugation in chicken muscle do not reflect differences in growth rate between broiler and layer birds.

Previous work has shown that chicken strains selected for growth (broilers) degrade muscle proteins less rapidly than those selected for egg laying. They also have decreased calpain and increased calpastatin content in breast muscle. This study aimed to test the hypothesis that these differences correlate with changes in the ATP- and ubiquitin-dependent proteolytic system. Chickens of a broiler strain (Ross 1) and a layer strain (ISABrown) were reared to the age of 4 wk under identical conditions with ad libitum access to feed and water. Mean fractional growth rates were 10.4%/d for broilers and 7.4%/d for layers. Feed intake measured in the last week of the trial was slightly greater in layer birds (.11 and .12 g x g body weight(-1) x d(-1) for broilers and layers respectively; P < .006). Polyubiquitin (UbI) messenger RNA was abundant in the muscles of these well-fed birds, but it showed little difference between strains. Muscle did not significantly express the UbII polyubiquitin gene. The ATP-dependent system conjugating ubiquitin to endogenous proteins had greatest activity in the gastrocnemius muscle of broiler birds but was not significantly different between breeds. Proteins cross-reactive with antisera to recombinant human proteasome regulatory subunits MSS1 (multicopy suppressor of SUG 1; S7) and TBP1 (tat binding protein 1; S6') were present in muscle homogenates from both strains of bird. The chick equivalent of TBP1 was more abundant in breast muscle of broiler birds than in leg muscle, or in either muscle of layers. Antiserum to recombinant yeast subunit mts2 (mitosis temperature sensitive gene 2; S4) did not react with any protein of the expected size but detected a 30-kDa peptide that was not associated with the 26S proteasome; this was found only in muscle from the layer strain. Hence, during normal growth of chickens, rates of protein degradation are not controlled by the expression of ubiquitin mRNA or the conjugation of ubiquitin. However, the composition of the 26S proteasome may be a regulatory factor.     

Developmental fall in whole body protein turnover of chick embryos during incubation

Whole body protein turnover was measured in chick embryos during incubation to investigate whether or not there is a fall in fractional rates of protein synthesis and degradation during development. Stable isotopically labelled [15N]phenylalanine was injected intraperitoneally into embryos on days 12 and 19. From 60 to 90 min after injection the isotope enrichment in free and protein-bound phenylalanine was measured with a selected-ion gas-chromatograph mass-spectrometer. The results showed that from days 12 to 19 of incubation, there was a remarkable reduction in fractional rates of protein synthesis and degradation in the whole body of chick embryos. During embryonic growth, protein synthesis per unit of RNA that is, the minimum amino acid translation rate of RNA, did not change significantly, whereas the RNA:protein ratio was reduced to one-third from days 12 to 19 of incubation. It was concluded, therefore, that the dramatic fall in fractional synthesis rate in chick embryos would be entirely attributable to the rapid increase in protein content, thereby changing the RNA:protein ratio in parallel with the fractional synthesis rate.     

Skeletal muscle protein synthesis and growth hormone secretion in young lambs treated with clenbuterol

To determine effects of clenbuterol (CB) on muscle protein turnover and growth hormone (GH) secretion, 16 crossbred wether lambs (14.4 kg) were randomized into two groups designated to receive daily oral boluses of gelatin capsules containing corn starch with either 0 (control, CTL) or 1.87 mg/kg body weight CB for either 14 (n = 8) or 28 d (n = 8). This calculates to be approximately 40 mg CB/kg diet. Lambs had ad libitum access to a 16% crude protein corn-soy diet and feed consumption (FC) was measured. After 14 and 28 d, lambs were slaughtered and semitendinosus (ST), longissimus (LD) and brachialis (BR) muscles were exercised, weighed and analyzed for protein (TP) content. For 6 h prior to slaughter of 28-d lambs, 2.5 microCi L-[U-14C]tyrosine/kg was infused intravenously, blood was sampled and plasma was analyzed for specific radioactivity of tyrosine. Plasma GH concentrations were assessed by radioimmunoassay. No differences due to treatment were found in FC, rate of gain or GH concentrations. Semitendinosus and BR weights of control lambs at 14 d did not differ between treatments. At 28 d, ST and BR weights of control lambs (58.8 and 18.5 g, respectively) were less (P less than .10) than those of lambs treated with CB (74.3 and 23.1 g, respectively). The TP per ST and BR at 28 d for control lambs was 71.5 and 85.1% (P less than .10) that of muscles of lambs treated with CB. Fractional protein synthesis rates (FSR) of the BR (9.4 vs 6.1%/d) and total protein synthesized in ST muscle per day (1.4 vs .8 g) were elevated (P less than .10) in lambs treated with CB compared to controls. These data suggest that the increased fractional accretion rate observed in lambs treated with CB for 28 d was caused by increased FSR.     

Effects of manipulation of the caspase system on myofibrillar protein degradation in vitro

Apoptosis via the intrinsic caspase 9 pathway can be induced by oxidative stressors hydrogen peroxide (H?O?) and N-(4 hydroxyphenol) rentinamide (fenretinide), a synthetic retinoid. Accelerated muscle atrophy and proteolysis in muscle-wasting conditions have been linked to oxidative stress and activated protease systems. Therefore, the hypothesis of this study was that proteolysis of myofibrillar proteins could be manipulated through the induction or inhibition of the caspase system. After slaughter, LM and supraspinatus muscles from callipyge (n = 5) and normal (n = 3) lambs were excised, finely diced, and incubated with treatment buffers containing oxidative stressors fenretinide or H?O?, recombinant caspase 3, caspase-specific inhibitor N-acetyl-Asp-Glu-Val-Asp-CHO (DEVD), or control solution. Muscle samples were incubated for 1, 2, 7, and 21 d at 4°C. Activation of the initiator caspase, caspase 9, and myofibrillar protein degradation was determined by SDS-PAGE and Western blotting. Results showed that fenretinide, H?O?, and recombinant caspase 3 increased (P < 0.05) proteolysis of myofibril proteins, whereas DEVD inhibited degradation (P < 0.05). Proteolysis of myofibrillar proteins increased with incubation time (P < 0.0001), and incubation time × treatment interactions (P < 0.05) indicated that the treatment effects did not all occur at the same rate. This study has shown that manipulation of the caspase system through induction or inhibition of activity can affect degradation of myofibrillar proteins, providing further evidence that the caspase system could be involved in postmortem proteolysis and tenderization. However, these stimulated changes were not sufficient to overcome the lack of proteolysis that is characteristic of muscle from callipyge lambs.     

Effects of a parenteral supplement of folic acid and its interaction with level of feed intake on hepatic tissues and growth performance of young dairy heifers.

Forty-seven dairy heifers of approximately 10 d of age were assigned to a factorial experiment in which a supplement of folic acid (0 or 40 mg) administered weekly by i.m. injection and level of feed intake were the two factors studied. The heifers were weaned after 5 wk of experimentation. Following weaning, and until the end of the experiment, 11 wk later, they had ad libitum access to grass hay and concentrates at two different levels, ad libitum or restricted, to allow a body weight gain of 700 g/d. A supplement of folic acid (P less than .05) and ad libitum access to feed (P less than .05) increased the mean concentration of serum folates. Blood hemoglobin and packed cell volume were not affected by the level of feed intake. However, they were both increased (P less than .05) by the supplement of folic acid. Average daily gain was analyzed over three different periods: 0 to 5 wk (before weaning), 5 to 10 wk, and 10 to 16 wk. Average daily gain was increased by the supplement of folic acid during the second period (P less than .05) and by ad libitum access to feed during the last two periods (P less than .05). Ad libitum access to feed increased (P less than .05) weight of the liver, decreased the (P less than .05) concentrations of RNA and DNA, and increased (P less than .05) the ratios of protein/DNA and RNA/DNA. The supplement of folic acid decreased (P less than .05) weight of the liver and increased the ratio RNA/DNA (P less than .05). These effects of supplement of folic acid on growth performance and on hematological cells may reflect a lack of folic acid during the weeks after weaning.     

Muscle protein degradation in bull calves with compensatory growth

Twelve 5-month-old bull calves were allocated to two feeding strategies: AA, 6 calves were fed ad libitum 34 weeks; and RA, 6 calves were fed restrictively for 14 weeks with an intake of 50% of the metabolic energy and protein eaten by the AA calves, followed by ad libitum feeding for 20 weeks. At the end of the 14-week restriction period, and after 2, 5, 8, 11, 14 and 17 weeks of re-alimentation, urine, blood and muscle biopsy samples from M. longissimus dorsi (LD) were collected. The urine was analysed for 3-methylhistidine for determination of the fractional breakdown rate of muscle protein (FBR). The FBR was depressed during restricted feeding (1.4 versus 2.0%/day; P = 0.05), however, during re-alimentation where the calves exerted compensatory growth, the FBR increased and reached a maximum after 5 weeks into this period (3.1 versus 1.9%/day; P < 0.001). The maximal FBR in the RA calves coincided with a maximum concentration of RNA and DNA in LD, and a maximal fractional rate of growth. The activity of μ-calpain in LD and the concentration of IGF-I in serum were decreased at the end of the restriction period but increased as soon as energy was offered ad libitum. The results support the hypothesis that muscle protein turnover is affected by a restriction/re-alimentation feeding strategy, and that muscle protein degradation reaches a maximum during the re-alimentation period, which exceeds that of control animals.