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1.
Thirty-five zeranol-implanted (I) and nonimplanted (NI) ram and wether lambs representing four treatments (implanted rams [IR], nonimplanted rams [NIR], implanted wethers [IW], and nonimplanted wethers [NIW]) were evaluated for meat palatability and muscle collagen characteristics. Rib (longissimus muscle, LM) chops from I lambs were juicier (P less than .05) than rib chops from NI lambs. Chops from IR lambs had more (P less than .05) detectable connective tissue and lower myofibrillar and overall tenderness scores than chops from NIR, IW, or NIW lambs. Warner-Bratzler shear (WBS) values tended to be higher (P = .06) for LM chops from rams than for those from wethers, but WBS values for Biceps femoris (BF) chops were similar (P greater than .05) for rams and wethers. Implanting did not affect (P greater than .05) WBS values. Rams had more (P less than .05) LM heat-labile (soluble, SC), nonheat-labile (insoluble, IC), and total collagen (TC) and a higher (P less than .05) percentage of SC (SC/TC) than did wethers. Soluble collagen, TC, and percentage of SC for the BF were higher (P less than .05) and IC tended (P = .09) to be higher in chops from rams than in those from wethers. Implanting did not affect (P greater than .05) collagen amount or solubility. Serum nonprotein hydroxyproline (NPHP) was higher (P less than .05) in rams than in wethers throughout the feeding period and tended (P = .05) to be higher at slaughter. Implanting did not affect (P greater than .05) serum NPHP.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

2.
Calpains are crucial for the degradation of myofibrillar proteins in muscle. Calpastatin is a specific inhibitor of calpains. The objective of this study was to elucidate the effect of nutrient restriction on the activity of calpains and calpastatin in the skeletal muscle of both cows and fetuses. Beginning 30 d after conception, 20 cows were fed either a control diet consisting of native grass hay fortified with vitamins and minerals at recommendations for a mature cow to gain 0.72 kg/d or half the vitamins and minerals and millet straw at 68.1% of NEm requirements. Cows were slaughtered on d 125 of gestation, and the LM was sampled at the 12th rib for calpain and calpastatin measurement. When comparing the muscle samples from nutrient-restricted and control cows, no difference in the activity of calpain I and II was observed; however, there was a significant difference (P < 0.05) in calpastatin activity. Muscle samples from control cows had greater calpastatin content than those of nutrient-restricted cows (P < 0.05); in contrast, the calpastatin content of fetal muscle was greater in fetuses gestated by nutrient-restricted cows than those of control cows (P < 0.05). Further, there were three calpastatin isoforms of 125, 110, and 70 kD detected in fetal muscle, whereas only the110-kD isoform was detected for cow muscle. These results indicate that the activity of the calpain system in skeletal muscle is mainly controlled through the expression of calpastatin. Alternating the calpastatin content in muscle and thereby modulating calpain activity may provide a mechanism for the maintenance of fetal muscle growth during nutrient restriction, whereas skeletal muscle loss in cows is upregulated.  相似文献   

3.
We developed a ribonuclease protection assay (RPA) to quantify the mRNA mass of calpastatin and the catalytic subunits of calpains I and II in ovine and bovine tissues. The method is based on constructing standard curves using predetermined amounts of in vitro synthesized sense cRNA of the calpain system, hybridized with excess radiolabeled antisense counterprobes. This is possible because the vectors used for riboprobe preparation can be used to transcribe the sense cRNA required to generate the standard curves to quantify absolute calpain I, calpain II, and calpastatin mRNA levels. We used the RPA to study calpain I, calpain II, and calpastatin gene expression in ovine liver, heart, and skeletal muscle. The results revealed that calpain II gene expression was similar in the three tissues. However, the expression of calpain I and calpastatin genes indicates that each tissue has its unique pattern. We also analyzed the activity of calpain I, calpain II, and calpastatin by the conventional DEAE chromatographic method for comparison. The results indicated that the RPA is more repeatable than the DEAE method. Special features of the RPA as compared with DEAE chromatography are as follows; 1) the RPA is a reliable method for quantifying the expression of calpains in all tissues because it is not affected by the presence of inhibitors or activators, 2) the RPA method can be expanded to analyze the expression of the tissue-specific calpains simply by designing specific probes for them, and 3) the RPA requires a small amount of tissue. The described method will facilitate future studies on the gene expression of calpains and will contribute to determining their physiological functions.  相似文献   

4.
Sixty-four white-faced rams and wethers were dressed with the aid of a commercial pelt puller. The effects of age, castration, and season on difficulty of pelt removal and pelt damage were evaluated. Lambs were divided into two age groups (5 and 12 mo) within gender (ram and whether) and season (spring and fall). A greater force (P less than .05) was required to remove pelts from rams than from wethers in both 5- and 12-mo-old groups. Older lambs slaughtered in the fall required more force (P less than .05) to remove their pelts than did those slaughtered in the spring, but differences by season did not exist for 5-mo-old lambs. The difference between rams and wethers in percentage of live weight that was closely shorn pelt weight was not significant (P greater than .05). The area of grain crack in the flank expressed as a percentage of total area of the skin was lower (P less than .05) for skins from 5-mo-old lambs and ram lambs than it was for skins from 12-mo-old lambs and wether lambs, respectively. Factors involved in difficulty of pelt removal in ram lambs included crosscut shoulder weight, fat firmness, and carcass weight. Difficulty of pelt removal in wether lambs was best predicted by including crosscut shoulder weight and bodywall thickness in multiple regression equations.  相似文献   

5.
The calpain system in three muscles of normal and callipyge sheep   总被引：19，自引：0，他引：19  
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.  相似文献   

6.
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.  相似文献   

7.
In two separate 2 x 2 factorial arrangements, 69 male, crossbred lambs were allotted randomly to the following treatments: 1) nonimplanted (NI) rams, 2) implanted (I) rams, 3) NI wethers, and 4) I wethers. In Trial 1, 36 lambs were allotted to treatment groups at birth (n = 9) and I lambs were implanted with 12 mg of zeranol between 1 and 3 d of age and again at weaning (average age of 62 d). Lambs were slaughtered at three time-constant end points of 78, 93, and 107 d on feed postweaning (average age of 155 d). Rams grew faster postweaning, were more efficient in their feed conversion, were heavier at slaughter, and had lower numerical yield grades than did wethers (P less than .05). Implanted lambs tended (P = .08) to grow faster and were (P less than .05) more efficient in their feed conversion than NI lambs. Rams produced heavier (P less than .05) trimmed subprimal shoulders, loins, and legs and had (P less than .05) a higher percentage of their carcass weight in the subprimal shoulder than did wethers. During Trial 2, NI rams (n = 8), I rams (n = 8), NI wethers (n = 8), and I wethers (n = 9) were allotted to treatment groups, and I lambs were implanted at average ages of 14, 55, and 98 d. After weaning, lambs were weighed every 14 d and were slaughtered 7 d after reaching a minimum weight of 50 kg (average age of 148 d).(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

8.
The biochemistry of intermuscular variation in tenderness is not fully understood. To investigate the role of the calpains in this process we performed two experiments using bovine and ovine species. In the bovine experiment, two distinct muscles, longissimus thoracis et lumborum (LT) and psoas major (PM), were used. In the ovine experiment, four muscles, LT, PM, semimembranosus (SM), and semitendinosus (ST), were used. Muscles were sampled at death for the determination of the steady-state mRNA level of calpains and calpastatin and the activities of calpain 1, 2, and calpastatin. Muscles were also sampled to determine the temporal changes in pH, tenderness, and the activity of the ubiquitous calpain system during postmortem aging. The results of the relative rate of tenderization in both species was found to be related to muscle type; LT had the highest value in both species. Within species, the mRNA steady-state levels of calpain 1 and calpastatin were similar in various bovine and ovine muscles. Bovine calpain 2 mRNA level was significantly lower in the LT than in the PM. Ovine calpain 2 mRNA level was lower, but not significantly different, in the LT compared to the other muscles. The mRNA level of bovine calpain 3 was significantly higher in the LT muscle than in the PM. In the ovine, the mRNA level of calpain 3 was highest in the LT, followed by SM, PM, and ST. Results on the activity of the ubiquitous calpain system in various muscles at death were dependent on muscle type and species. Temporal changes in the activity of calpains and calpastatin during the first 24 h of postmortem aging were similar in the muscles studied: calpain 1 and calpastatin declined significantly and calpain 2 remained relatively unchanged. The temporal changes in muscle pH in both experiments indicated that the extent and rate of pH decline during aging was related to muscle type. Correlation analysis between the relative rate of tenderization and mRNA expression of calpains revealed a strong relationship with calpain 3 in both species.  相似文献   

9.
Relationships of implanted testosterone, dihydrotestosterone and estradiol-17 beta to collagen degradation and intramuscular collagen concentration and stability were determined. Intramuscular collagen content, solubility and shrinkage temperature and serum hydroxyproline were analyzed in groups of six rams, wethers, and wethers implanted with various levels of testosterone or dihydrotestosterone and groups of 10 rams, wethers and wethers implanted with estradiol-17 beta, dihydrotestosterone or a combination of these two steroids. Intramuscular collagen content in both experiments was higher (P less than .05) in muscles of rams than in muscles of wethers. Administration of the highest level of testosterone to wethers raised (P less than .05) total and insoluble intramuscular collagen to concentrations noted in rams. Administration of the testosterone metabolite, dihydrotestosterone, to wethers had no effect on intramuscular collagen. Administration of estradiol-17 beta to wethers tended to raise concentrations of intramuscular collagen so that they were no longer lower (P less than .05) than those in rams. Collagen stability as measured by solubility and thermal shrinkage temperature did not differ among rams, wethers or implanted wethers (P greater than .05). Increases in collagen accretion due to hormone administration were observed to be the result of increases in the insoluble portion of the intramuscular collagen (P less than .05).  相似文献   

10.
Two trials examined genetic and environmental influences on muscle fiber type proportions. Trial 1 compared Polypay and Coopworth x Polypay male lambs either left intact or castrated early, mid or late in growth. Trial 2 compared Hampshire-sired lambs (females, early castrate wethers and late castrate wethers) from Suffolk x Coopworth dams and whiteface x Coopworth dams. Half the lambs in each trial were raised from weaning to 52 kg slaughter weight in drylot; the other half were reared to 41 kg on pasture before being finished in drylot. Analysis of longissimus tissue samples revealed no significant differences among rams, wethers and ewes in proportions of muscle fiber types. Early castration increased the proportion of alpha red fibers in Trial 1 (P less than .01) but not in Trial 2. Polypay lambs had a higher proportion of beta red fibers (P less than .05) than the Coopworth x Polypay lambs, but no differences were seen between the genotypes of Trial 2. Single-born lambs in Trial 1 had a 6% higher proportion of alpha white fibers (P less than .05) than twin-born lambs; however, this difference was not detected in Trial 2. Drylot lambs had a higher proportion of beta red fibers than pasture-reared lambs, the difference being 5% (P less than .01) in Trial 2. Muscle fiber type proportions were not found to be related to growth rate and carcass fatness, and no evidence of differential fiber transformation was found in this trial.  相似文献   

11.
Steers of known percentage Brahman (B) and Angus (A) breeding (100% A, n = 6; F1 B x A, n = 6; and 100% B, n = 6) were used to determine the effect of calcium chloride injection on the calpain proteinase system and meat tenderness. The steers were slaughtered in six replications (at either 9 or 14 mm of backfat, determined ultrasonically), with each breed type represented. Calpains and calpastatin activities were measured on fresh, prerigor longissimus muscle samples. Carcass data were collected after a 24-h chill, and the short loin (IMPS #180), top sirloin (IMPS #184), and top round (IMPS #168) were removed from both sides of each carcass. The cuts from the right side were then injected at 5% (wt/wt) with CaCl2 solution (2.2%). Longissimus muscle calpain and calpastatin activities were also measured at 48 h postmortem from the injected and control sides of each carcass. Warner-Bratzler shear force was measured on steaks from the three subprimals aged 1, 2, 5, 15, or 31 d. Marbling scores and USDA quality grades were higher (P<.05) in A than in F1 B x A and B carcasses. Calpastatin activity was higher (P<.05) in muscle from B than in muscle from A and F1 B x A steers, and postmortem storage (O vs 48 h) and CaCl2 injection reduced (P<.05) the activity of the calpains and calpastatin. Strip loin and top sirloin steaks from A and F1 B x A steers were more tender (P<.05) than steaks from B steers; however, top round steak tenderness did not differ (P>.05) across breed type. Calcium injection improved strip loin and top sirloin steak tenderness, but it did not affect top round steak tenderness. Collectively, these data show that CaC12 injection can be used to improve meat tenderness, with similar responses shown in cattle containing 0, 50, and 100% B inheritance. However, even with CaCl2 injection, B steaks are less tender than their A and F1 B x A counterparts.  相似文献   

12.
Evidence has indicated that mu-calpain, m-calpain, and calpastatin have important roles in the proteolytic degradation that results in postmortem tenderization. Simple assays of these 3 proteins at different times postmortem, however, has shown that calpastatin and mu-calpain both rapidly lose their activity during postmortem storage, so that proteolytic activity of mu-calpain is nearly zero after 3 d postmortem, even when assayed at pH 7.5 and 25 degrees C, and ability of calpastatin to inhibit the calpains is 30% or less of its ability when assayed at death. m-Calpain, however, retains much of its proteolytic activity during postmortem storage, but the Ca(2+) requirement of m-calpain is much higher than that reported to exist in postmortem muscle. Consequently, it is unclear how the calpain system functions in postmortem muscle. To clarify this issue, we have initiated attempts to purify the 2 calpains and calpastatin from bovine semitendinosus muscle after 11-13 d postmortem. The known properties of the calpains and calpastatin in postmortem muscle have important effects on approaches that can be used to purify them. A hexyl-TSK hydrophobic interaction column is a critical first step in separating calpastatin from the 2 calpains in postmortem muscle. Dot-blot assays were used to detect proteolytically inactive mu-calpain. After 2 column chromatographic steps, 5 fractions can be identified: 1) calpastatin I that does not bind to an anion-exchange matrix, that does not completely inhibit the calpains, and that consists of small polypeptides <60 kDa; 2) calpastatin II that binds weakly to an anion-exchange matrix and that contains polypeptides <60 kDa; all these polypeptides are smaller than the native 115- to 125-kDa skeletal muscle calpastatin; 3) proteolytically active mu-calpain even though very little mu-calpain activity can be detected in zymogram assays of muscle extracts from 11- to 13-d postmortem muscle; this mu-calpain has an autolyzed 76-kDa large subunit but the small subunit consists of 24-, 26- and a small amount of unautolyzed 28-kDa polypeptides; 4) proteolytically active m-calpain that is not autolyzed; and 5) proteolytically inactive mu-calpain whose large subunit is autolyzed to a 76-kDa polypeptide and whose small subunit contains polypeptides similar to the proteolytically active mu-calpain. Hence, loss of calpastatin activity in postmortem muscle is due to its degradation, but the cause of the loss of mu-calpain activity remains unknown.  相似文献   

13.
Fifty wethers and 51 spring-born rams were divided into five groups and slaughtered at different seasons of the year at average ages of 271, 361, 459, 557 or 652 d to determine the age and season at which differences in secondary sex characteristics could be detected. Serum testosterone concentrations and testes weights were low in January when the rams were 271 d of age and again in April at 361 d of age. By July, at 459 d of age, testosterone concentrations and testes weights had peaked and then decreased the following November at 557 d and February at 652 d. In contrast with plasma testosterone concentrations and testes weights, buckiness scores, splenius to semimembranosus or semitendinosus muscle ratios, splenius muscle weights and neck and shoulder percentages were not seasonal. All of these measures increased significantly up to July and continued to increase slowly, but not significantly, thereafter. Muscle color and texture scores and rib eye color scores tended to increase in a linear manner for both rams and wethers as age increased. Subcutaneous fat from rams was yellower and softer than that from wethers over all age groups. Ram fat firmness did not change (P greater than .05) with age, and the only significant change in ram fat color was between the groups at 271 and 361 d of age. Overall, season of year coupled with higher levels of serum testosterone was related to initial development of secondary sex characteristics in ram lambs.  相似文献   

14.
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15.
Concentration and maturation of collagen and serum concentrations of hydroxyproline and testosterone were determined in growing rams and wethers to characterize developmental changes in collagen associated with a representative testicular steroid. Groups of eight rams and eight wethers were slaughtered at 12, 18, 24 and 30 wk of age. Concentrations of collagen in longissimus, supraspinatus and infraspinatus muscles and serum hydroxyproline were greater (P less than .05) in rams than in wethers at all ages. Collagen stability, as measured by collagen solubility and thermal shrinkage temperature, was greater (P less than .05) in wethers than in rams. Differences in collagen stability and serum hydroxyproline concentration indicated that collagen synthesis and turnover were more rapid in rams than in wethers. Serum hydroxyproline decreased (P less than .05) and collagen solubility decreased (P less than .05) with age, indicating that collagen turnover was occurring most rapidly in 12-wk-old lambs and that collagen maturation was predominant in 24- to 30-wk old lambs. Testosterone parameters measured in rams were unrelated within groups to collagen characteristics, possibly reflecting the high variability in testosterone secretion and the slow development of collagen. However, rams as young as 12 wk of age were under the influence of testosterone, and differences in collagen between rams and wethers were apparent at that time.  相似文献   

16.
Effects of Dorset, Finnsheep, Romanov, Texel, and Montadale breeds for performance as sires were estimated in the initial phase of a comprehensive evaluation of these breeds as contributors to sheep crossbreeding systems. Objectives were to evaluate the effects of ram breed, ewe breed, season of mating, and two-way interactions. Rams from the five breeds were single-sire-mated with ewes from two breed types to produce lambs over a 3-yr period. Ewes were assigned to one of three distinct 35-d mating seasons initiated each year in August, October, and December. A different sample of six rams per breed was used each year across all three seasons, and each ram was penned with ewes of both breeds. Traits evaluated and number of ewe records were conception rate and litter weaning weight per ewe exposed (n = 3,261) and number born, litter birth weight, average birth weight, number weaned, and litter weaning weight per ewe lambing (n = 2,751). Ram breed and ewe breed interacted (P < .01) for conception rate and litter weaning weight per ewe exposed, implicating mating preferences, particularly of Romanov rams. In mixed groups of ewes exposed to Romanov rams, conception rate was 12.7% lower and litter weight weaned was 8.4 kg lower in the ewe breed presumably less preferred for mating by the rams. On a per ewe exposed basis, Romanov-sired litters produced either the largest or the smallest values for litter weaning weight, depending on the breed of ewe. Effects of ram breed on number born and litter birth weight interacted (P < .05) with season of mating. The largest litters within each ram breed were associated with the October mating season. Montadale and Romanov rams sired larger and heavier litters from August matings than from December matings, whereas the opposite was true for Dorset-sired litters. Texel- and Finnsheep-sired litters were similar in size and weight from August and December matings. Breed of ram differences affected per ewe lambing productivity measurements (P < .01). Differences between ram breeds for ewe productivity were noted, with increased number born and improved survival of crossbred progeny to weaning for Romanov-sired litters. These results may have implications for using these ram breeds as sires in different crossbreeding systems. Structured mating systems or the creation of new composite populations involving these breeds could be used to match the resources, environment, and market of specific production situations.  相似文献   

17.
Forty-eight Suffolk x white-faced ram and wether lambs approximately 5, 7, or 9 mo of age were slaughtered to evaluate the effects of age and gender on difficulty of pelt removal, pelt damage, and leg damage. A commercial belt-type pelt puller and a scale that recorded force required to remove the pelt from the thickest part of the legs was used as lambs hung suspended from their front legs. Rams required more force (P less than .05) to remove the pelt than wethers, and the difference between genders became larger as age increased. Neither pelt damage due to grain crack nor leg damage judged by amount of fell and fat removed by the pelt puller changed with age. Rams possessed thicker pelts (P less than .05) than wethers; this plus a greater amount of collagen crosslinking in ram skins could be responsible for the slightly smaller amount of grain crack observed in ram pelts. Factors involved in difficulty of pelt removal in ram lambs included age, splenius weight, and overall maturity. Difficulty of pelt removal in wether lambs was best predicted by including age and splenius weight in the model. These data tend to support packers' common practice of discounting rams over 5 mo of age because rams develop masculine characteristics and become harder to dress with increasing age.  相似文献   

18.
Lactation and growth of three contemporary lines of grade Targhee sheep developed from the same genetic base were characterized by three experiments performed over a period of 2 yr. Two lines (HW and DH) had been selected for 120-d weaning weights for 24 yr prior to beginning these experiments. A third line (C) was a randomly selected control. Year I experiment contrasted 10 DH with 7 C ram lambs fed to 58 kg. Year II experiments utilized 9 C, 14 DH and 10 HW ram lambs and 11 ewes suckling twins from each line. All Year II ram lambs were born and weaned as twins, then fed to 50 kg. Mature DH and HW ewes were heavier (P less than .05) than C ewes (65.2 and 68.8 vs 54.9 kg), and the DH and HW lambs grew faster than C lambs both before (P less than .05) and after weaning (P less than .05). While both DH and HW lambs drank more milk (2,419 and 2,368 vs 2,059 g X d-1 X pair-1; P less than .10) only HW ewes showed a trend towards greater potential milk production than controls (HW = 2,774 vs C = 2,155 g X d-1 X ewe-1 P less than .12). The HW lambs tended to be leaner than C lambs (P less than .05), but DH lambs did not differ from either line. Lambs from DH and HW lines required less post-weaning feed (121.9 and 129.3 vs 152.0 kg P less than .05) and exhibited 17 and 16% greater weight per day of age at 50 kg than controls (P less than .05). The DH line displayed lower feed: gain ratios than controls in both post-weaning trials (6.68 vs 7.30 to 58 kg; 5.83 vs 6.24 at 50 kg; P = .06).  相似文献   

19.
Stepwise and continuous gradient ion-exchange chromatography were compared for yield of calpains and calpastatin from ovine muscle in a study designed to quantify their activities for comparative purposes. In Exp. 1, a continuous (25 to 400 mM NaCl) gradient and a two-step gradient method (200 mM NaCl to coelute mu-calpain and calpastatin together and then 400 mM NaCl to elute mu-calpain) were compared. For the two-step method, mu-calpain activities were determined by subtracting calpastatin activities before and after heat inactivation of mu-calpain. Both the two-step and the continuous gradient method yielded similar results over a broad range of activities. The stepwise gradient method does not require the use of fraction collectors and pumps, and it can be completed in a fraction of the time required for the continuous gradient method. In Exp. 2, the two-step method was compared with a three-step method (100 mM NaCl to elute calpastatin, then 200 mM NaCl to elute mu-calpain, and then 400 mM NaCl to elute m-calpain). Unlike the continuous gradient method, calpastatin and mu-calpain could not be completely separated using the three-step chromatography method. Thus, the three-step gradient method should not be used to quantify the components of the calpain proteolytic system. The present results indicate that the two-step gradient method is a fast and inexpensive method to determine calpain and calpastatin activities in studies designed to quantify the components of the calpain proteolytic system in skeletal muscle.  相似文献   

20.
Our objective was to evaluate whether small (biopsy-sized) samples could be used to measure calpain and calpastatin activities in skeletal muscle. The accuracy of different separation and assay methods for the quantification of calpains and calpastatin from small (1.0 and 0.2 g) skeletal muscle samples was tested. In Exp. 1, the LM was removed from six lambs, and a 50-g subsample was processed using the reference method (DEAE-Sephacel chromatography and casein assay). Subsamples (1.0 and 0.2 g) also were processed using the two-step separation (1 mL DEAE-Sephacel and bulk elution using 200 and 400 mM NaCl) and heated calpastatin methods; in both cases, fractions were assayed with Bodipy-labeled and [14C]-labeled casein microassays. Finally, casein zymography was used to separate and quantify the calpain proteases from 1.0-and 0.2-g samples. The values obtained after processing the 50-g sample using the reference method were judged most accurate, and the alternative approaches were compared with these. For each extraction and assay approach, we considered: 1) the effect of the sample size on the mean activity; 2) increased or decreased variation of data; and 3) the correlation relative to the reference method. Where possible, we compared the ratio of calpain to calpastatin activities determined using the alternative approaches with the ratios found using the reference method. These methodologies were further investigated in Exp. 2, where single homogenates from different tissues (heart, spleen, lung, and muscle) were assayed using the alternative approaches. Experiment 1 established that most of the approaches suffered from poor correlations and/or unacceptable variation. By using a large, homogenous sample in Exp. 2, however, we determined that this error was not due to the methodologies themselves. Therefore, the unacceptable variation found in Exp. 1 resulted from the small sample size, and we recommend that large tissue samples (e.g., 50 g) should be used for calpain and calpastatin activity measurements in skeletal muscle instead of small tissue biopsies (e.g., 0.2 and 1.0 g).  相似文献   

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