Establishing an appropriate mode of comparison for measuring the performance of marbling score output from video image analysis beef carcass grading systems.

A beef carcass instrument grading system that improves accuracy and consistency of marbling score (MS) evaluation would have the potential to advance value-based marketing efforts and reduce disparity in quality grading among USDA graders, shifts, and plants. The objectives of this study were to use output data from the Video Image Analysis-Computer Vision System (VIA-CVS, Research Management Systems Inc., Fort Collins, CO) to develop an appropriate method by which performance of video image analysis MS output could be evaluated for accuracy, precision, and repeatability for purposes of seeking official USDA approval for using an instrument in commerce to augment assessment of quality grade, and to use the developed standards to gain approval for VIA-CVS to assist USDA personnel in assigning official beef carcass MS. An initial MS output algorithm was developed (phase I) for the VIA-CVS before 2 separate preliminary instrument evaluation trials (phases II and III) were conducted. During phases II and III, a 3-member panel of USDA expert graders independently assigned MS to 1,068 and 1,242 stationary carcasses, respectively. Mean expert MS was calculated for each carcass. Additionally, a separate 3-member USDA expert panel developed a consensus MS for each carcass in phase III. In phase II, VIA-CVS stationary triple-placement and triple-trigger instrument repeatability values (n = 262 and 260, respectively), measured as the percentage of total variance explained by carcasses, were 99.9 and 99.8%, respectively. In phases II and III, 95% of carcasses were assigned expert MS for which differences between individual expert MS, and for which the consensus MS in phase III only, was < or = 96 MS units. Two differing approaches to simple regression analysis, as well as a separate method-comparability analysis that accommodates error in both dependent and independent variables, were used to assess accuracy and precision of instrument MS predictions vs. mean expert MS. Method-comparability analysis was more appropriate in assessing the bias and precision of instrument MS predictions. Ether-extractable fat percentages (n = 257; phase II) differed among MS (P < 0.05) but were not suitable to predict or validate assigned MS. The performance and reproducibility of expert MS assignment in future evaluations was considered, and an official USDA performance standard was established, to which an instrument must conform to be approved for official on-line MS assessment. The VIA-CVS subsequently was approved to assign MS to carcasses on-line after completion of a 2006 USDA instrument approval trial conducted according to methods developed during completion of this study.

Filament overlap affects TnC extraction from skinned muscle fibres.

Recent studies on calcium regulation of muscle contraction selectively extract troponin C (TnC) from skinned skeletal muscle fibres with a low ionic strength rigor solution containing a Ca2+/Mg2+ chelator. As previous results from this laboratory and others demonstrate a crossbridge effect, especially rigor, on many of the properties of TnC, the effects of filament overlap on TnC extraction from skinned rabbit psoas muscle fibres were investigated. Tension-pCa relationships at a sarcomere length of 2.7 microns were determined before and after a 5 min TnC extraction at sarcomere lengths of 2.3, 2.5, 2.7, 3.1, 3.3 or 3.5 microns with 20 mM Tris, pH 7.8, 5 mM EDTA. The decrease in the post-extraction maximum Ca2+ activated tension, an indicator of the amount of TnC extracted, was linearly related to the overlap of the thick and thin filaments with decreases in tension being associated with a decrease in filament overlap. The smaller fibre diameter at the longer sarcomere length could facilitate diffusion of TnC from fibre segments. However, the wide range of measured diameters, 40-120 microns, accounted for only 14% of the observed tension decrement and shrinking the fibre with polyvinylpyrrolidone did not increase the tension decrement. Increasing the sarcomere length before extraction was also found to decrease the TnC content of fibre segments along with the post-extraction maximum tension. Thus, TnC appears to be preferentially extracted from non-overlap than overlap regions of the sarcomere. These results further indicate that rigor crossbridges affect TnC other than through increased Ca2+ binding and that under the conditions used here, they retard its extraction.

Effects of time on feed on beef nutrient composition.

Forty-eight Angus x Hereford yearling steers were used to assess the effect of time on feed (TOF) on the nutrient composition of beef longissimus muscle (LM). Steers were fed a high-concentrate diet with the exception of the d-0 group, which served as a grass-fed control, and then were serially slaughtered at 28-d intervals during the 196-d feeding period. Steaks were removed from the 10th rib and trimmed of exterior fat and epimysial connective tissue before nutrient analysis. Intramuscular fat content doubled (P < .05) between d 84 and 112 but did not differ (P > .05) from d 0 to 84 or from d 112 to 196. This increase in fat content resulted in decreased (P < .05) concentrations of moisture, protein, and ash in the LM. Concentrations of Mg, K, and Fe in the LM increased (P < .10) with advanced TOF. The increase in the total lipid (TL) content of the LM stemmed from a proportional increase (P < .05) in neutral lipid (NL). Polar lipid (PL) remained constant (P = .33) throughout TOF. The NL and TL became more unsaturated as TOF increased, primarily due to a linear (P < .01) increase in oleic (C18:1) acid concentration. In contrast, the polyunsaturated fatty acid (PUFA) concentration in the PL exhibited a linear (P < .01) decrease across TOF. As a result, advanced TOF increased the monounsaturated fatty acid (MUFA) content by 22% and decreased the PUFA content by 72% in the LM. The ratio of hypercholesterolemic (C14 + C16):hypocholesterolemic (MUFA+PUFA) fatty acids was unaffected by increasing TOF from d 28 to 196; however, this ratio was lower (P < .05) for grass-fed controls (d 0) than for d 28 to 84 and d 196. Cholesterol content (milligrams/100 grams) changed cubically (P = .06) across TOF. Ultimately, by limiting TOF to 112 d, the beef industry could provide consumers a palatable beef product that easily fits into a healthy diet and at the same time diminishes the costs associated with external fat trim.

Ca(2+)-dependence of structural changes in troponin-C in demembranated fibers of rabbit psoas muscle.

The Ca(2+)-dependence of structural changes in troponin-C (TnC) has been detected by monitoring the fluorescence from TnC labeled at Methionine-25, in the NH2-terminal domain, with danzylaziridine (TnC-DANZ) and then exchanged for endogenous TnC in glycerinated single fibers. The fluorescence-pCa relation obtained from fibers stretched to a sarcomere length greater than 4.0 microns evidenced two transitions: a small one, attributable to the binding of Ca2+ to the high affinity, Ca(2+)-Mg(2+)-binding sites of TnC; and a large one, attributable to the binding of Ca2+ to the low affinity, Ca(2+)-specific binding sites of TnC. In the fluorescence-pCa relation determined with fibers set to a sarcomere length of 2.4 microns, hence obtained in the presence of cycling cross-bridges, the large transition had the same Ca(2+)-dependence as did the development of tension. These results indicate that the NH2-terminal globular domain of TnC is modified by the binding of Ca2+ to sites located in both globular domains and that the structural changes in TnC resulting from the binding of Ca2+ to the low-affinity sites, but not to the high-affinity sites, are directly associated with the triggering of contraction.

Muscle force and stiffness during activation and relaxation. Implications for the actomyosin ATPase.

Isolated skinned frog skeletal muscle fibers were activated (increasing [Ca2+]) and then relaxed (decreasing [Ca2+]) with solution changes, and muscle force and stiffness were recorded during the steady state. To investigate the actomyosin cycle, the biochemical species were changed (lowering [MgATP] and elevating [H2PO4-]) to populate different states in the actomyosin ATPase cycle. In solutions with 200 microM [MgATP], compared with physiological [MgATP], the slope of the plot of relative steady state muscle force vs. stiffness was decreased. At low [MgATP], cross-bridge dissociation from actin should be reduced, increasing the population of the last cross-bridge state before dissociation. These data imply that the last cross-bridge state before dissociation could be an attached low-force-producing or non-force-producing state. In solutions with 10 mM total Pi, compared to normal levels of MgATP, the maximally activated muscle force was reduced more than muscle stiffness, and the slope of the plot of relative steady state muscle force vs. stiffness was reduced. Assuming that in elevated Pi, Pi release from the cross-bridge is reversed, the state(s) before Pi release would be populated. These data are consistent with the conclusion that the cross-bridges are strongly bound to actin before Pi release. In addition, if Ca2+ activates the ATPase by allowing for the strong attachment of the myosin to actin in an A.M.ADP.Pi state, it could do so before Pi release. The calcium sensitivity of muscle force and stiffness in solutions with 4 mM [MgATP] was bracketed by that measured in solutions with 200 microM [MgATP], where muscle force and stiffness were more sensitive to calcium, and 10 mM total Pi, where muscle force and stiffness were less sensitive to calcium. The changes in calcium sensitivity were explained using a model in which force-producing and rigor cross-bridges can affect Ca2+ binding or promote the attachment of other cross-bridges to alter calcium sensitivity.

Muscle cross-bridge attachment: effects on calcium binding and calcium activation.

Data from intact and skinned muscle fibers support the hypothesis that cross-bridge interaction modifies TnC structure and calcium activation. Barnacle single muscle fibers microinjected with the calcium bioluminescent photoprotein, aequorin, show extra light (calcium) when shortened during the declining phase of the calcium transient. The extra calcium is increased by increases in muscle force, and its decline is delayed at higher forces. This extra calcium occurs probably because calcium binding to the activating sites is increased by cross-bridge interaction. In rabbit muscle, TnC structure is modified by cross-bridge interaction, since in skinned rabbit psoas muscle fibers TnC extraction is slower at shorter sarcomere lengths, where cross-bridge attachment is increased. Thus the rigor bridges formed in the extraction solution strengthen the attachment of TnC to the thin filament. Reintroduction of TnC, labeled with fluorescent probes near the Ca specific binding sites (Danzylaziridine-DANZ) and Ca-Mg sites (Rhodamine), into the partially TnC extracted fibers allows us to assess the structural changes (total fluorescence for the DANZ probes, linear dichroism for the RHOD probe) in response to calcium binding and cross-bridge attachment. At sarcomere lengths beyond overlap, calcium binding increases the DANZ-TnC fluorescence and disorders the RHOD-TnC label. At full overlap of filaments, rigor cross-bridges also increase the DANZ-TnC fluorescence and RHOD-TnC disorder. The addition of calcium in rigor increases the DANZ-TnC fluorescence little but causes additional RHOD-TnC disorder, although both fluorescence and disorder are increased further in the presence of calcium plus MgATP. In fibers containing DANZ-TnC, decreasing MgATP in the absence of calcium increases both the force and the fluorescence as rigor cross-bridges activate the muscle. In the presence of calcium, an increase in MgATP to 0.75 microM produces a small fluorescent enhancement, but an increase in MgATP to 10 microM and to 3 mM produces a substantial enhancement. The data imply that calcium activates the thin filament, but that the filament is activated further by rigor cross-bridges. Active cross-bridges activate the thin filament still further. Thus, cross-bridges modify TnC structure and calcium activation, with active cross-bridges being more effective than rigor cross-bridges.

Quantitative determination of myosin and actin in rabbit skeletal muscle.

The myosin and actin content of muscle tissue and purified myofibrils from rabbit psoas muscle has been determined. Myofibrils were purified using Percoll gradients, which allowed rapid separation from nuclei and connective tissue proteins. Myosin and actin were quantitated by amino acid analysis of the appropriate bands from sodium dodecyl sulfate/polyacrylamide gels. Muscle tissue contained 94 and 619 nmol/g wet weight of myosin and actin, respectively, while myofibrils had 0.82 and 5.37 mumol/g protein. Thus myosin contributed 43% and actin 22% of the myofibril protein mass. The value of 2.5 myosins per 14.3 nm repeat as calculated from these results suggests that thick filament models with mixtures of two and three crossbridges per repeat should be considered.

Troponin subunit stoichiometry and content in rabbit skeletal muscle and myofibrils.

The quantity and molar ratio of the three troponin subunits to actin were determined in rabbit psoas muscle, muscle homogenates (800 X g pellet), and purified myofibrils. Proteins were separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The quantities of the separated proteins were determined directly from the gel slices by amino acid analysis after correction for losses and background. The molar ratio of actin, troponin T, troponin I, and troponin C was found to be 6.99:1:05:1:04:0.92 in purified myofibrils and was not significantly different (p greater than 0.05) from those obtained from 800 X g pellets of muscle homogenates or intact muscle tissue. Isolated troponin purified by several different procedures also had a 1:1:1 subunit ratio although the variability was much greater than that found in myofibrils. The troponin content of rabbit psoas muscle and myofibrils was 91 +/- 16 and 770 +/- 110 pmol/mg, respectively.

Interaction of monomeric and oligomeric soybean agglutinins with pig lymphocytes and plasma membranes.

The pig lymphocyte surface contains two distinct receptor classes for monomeric (nonmitogen) and oligomeric (mitogen) soybean agglutinins. The major class (70-80%) for which both forms compete effectively binds them with a similar weak avidity in a non-cooperative monovalent reaction. This class appears to be one or more plasma membrane glycolipids and not involved in the mitogenic process. The minor class (20-30%) binds the monomer weakly and non-cooperatively. It binds the oligomers strongly via positively cooperative interactions involving mobile surface molecules in a metabolically energy-independent clustering process. The minor class appears to be one or more of seven plasma-membrane glycopolypeptides. The monomer does not compete effectively for this class nor does it specifically inhibit the mitogenicity of the oligomers, suggesting that receptors involved in the mitogenic process fall into the minor class and are likely to be glycopolypeptides.

Effect of temperature and pH on the post-mortem degradation of myofibrillar proteins.

Incubation of bovine muscle at 37°C promoted a more drastic proteolytic change in myofibrillar proteins, as determined from sodium dodecyl sulphate polyacrylamide gels of isolated myofibrils, than incubation at 4°C. Degradation of myosin and troponin-T were the most noticeable changes at 37°C. Loss of α-actinin was observed in the 4°C incubated muscle. Ground bovine muscle incubated at pH 5·4 and 7 revealed that alterations in myosin and troponin-T were the most noticeable changes at ph 5·4 while troponin-T and α-actinin were altered at pH7. More troponin-T degradation occurred at pH 5·4 and 37°C than at pH7 and 4°C (similar to the degradation of myosin), indicating that troponin-T degradation in post-mortem muscle may be an indicator of overall myofibrillar proteolysis and not responsible for post-mortem tenderisation per se. Myosin degradation in the ground samples incubated at pH 5·4 and in whole samples incubated at 37°C was compared with the digestion of myofibrillar myosin by papain. Both pyrophosphate and Guba-Straub extracts of the 37°C and pH 5·4 treated samples confirmed that myosin degradation did occur to a much greater extent at this temperature and pH than at 4°C and pH7, and, in addition, at pH 5·4 frequent cleavage occurred near the papain sensitive site of myosin.

The binding of lectins to components of plasma membranes from porcine submaxillary lymph node lymphocytes.

By sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis the plasma membranes from porcine lymphocytes contain at least 30--35 glycopolypeptides and one or more glycolipids to which one or more of 12 purified lectins bind. The specificities of binding generally followed the same pattern as those of the reaction of the lectin with intact pig lymphocytes. Some lectins (e.g., the isolectin pair, Agaricus bisporus lectins A and B and a group consisting of the Lens culinaris A and B isolectins and the closely related Pisum sativum lectins) bind to almost identical populations of plasma membrane components and compete with each other for all their binding sites. Others (e.g., Concanavalin A and the Lens culinaris-Pisum sativum group and a group consisting of phytohemagglutinin-L, Ricinus communis lectin-60 and Ricinus communis lectin-120 bind in a cross reactive manner to some common binding moieties but, in addition, to certain nonshared ones. Still others (e.g., soybean agglutinin, peanut agglutinin and wheat germ agglutinin) do not share any common binding moieties with the other lectins. The amount of lectin binding and the number of membrane components to which a lectin binds is directly related to the Ka of binding of the lectin to the intact lymphocyte. Those with high Ka (Cocanavalin A Lens culinaris lectins, Pisum sativum lectins, phytohemagglutinin-L), bind to 20-30 different components giving very complex binding patterns while those with lower Ka (Agaricus bisporus lectins, wheat germ agglutinin, peanut agglutinin, and soybean agglutinin) bind to 8--13 components with easily distinguishable patterns. Soybean agglutinin binds almost exclusively to a glycolipid fraction while for the others one or more glycopolypeptides served as the major lectin-binding molecule. The Ricinus lectins, two lymphocyte toxins, bind to essentially every plasma membrane component to which the mitogen phytohemagglutinin-L binds, in fact competing for most of those plasma membrane moieties which bind phytohemagglutinin-L.