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A perfused ruminant muscle preparation
- B. Jane Coward, P. J. Buttery
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- Journal:
- The Journal of Agricultural Science / Volume 95 / Issue 1 / August 1980
- Published online by Cambridge University Press:
- 27 March 2009, pp. 7-16
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- Article
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A technique was developed for the continuous retrograde perfusion of the muscle of the right crus. The hemidiaphragm was perfused for 3 h with a semi-synthetic medium, containing fresh sheep erythrocytes. The metabolic integrity of the perfused muscle was examined. Visual appearance was satisfactory. Perfusion pressure remained constant throughout the perfusion period. An adequate perfusate flow rate (approximately 8 ml/min) was maintained over the perfusion period. Perfusion pressure and flow rate were proportional. Perfusion was complete as indicated by staining with dye and by latex casts of perfused vessels, but there were indications of heterogeneity of perfusion (the dorsal and ventral ends of the organ). Histological changes following perfusion were minor, and the perfused muscle appeared normal under an electron microscope, with the exception of a loss of cytoplasmic granules. Increases in muscle water content (2·3%) and extracellular space were small. Muscle adenosine triphosphate (ATP) and adenosine diphosphate (ADP) concentrations declined significantly following perfusion but ATP/ADP ratio increased significantly. Muscle glycogen content was maintained only when exogenous insulin was added to the medium; significant losses occurred following perfusion in the absence of insulin. Only 2–3% of tissue glutamate-pyruvate transaminase and glutamate-oxaloacetate transaminase were released into the medium over the perfusion period, but approximately 20% of muscle potassium was lost. Lactate was produced by the perfused sheep hemidiaphragm at a rate higher than those that have been reported for the perfused rat hind-limb, but lower than those reported for perfused rat diaphragm. Ratio of lactate/pyruvate produced by the perfused muscle was also higher than values reported for the rat hind-limb. The perfused muscle actively incorporated labelled amino acids into protein, but estimated protein synthetic rate in the absence of exogenous insulin was only approximately 50% of rates reported for diaphragm muscle in vivo. While the isolated, perfused sheep hemidiaphragm shows several deficiencies, it does provide a useful method for the study of ruminant muscle metabolism under carefully controlled conditions in vitro, and overcomes many of the disadvantages of other techniques that are currently available.
Metabbolism of perfused ruminant muscle
- B. Jane Coward, P. J. Buttery
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- Journal:
- The Journal of Agricultural Science / Volume 98 / Issue 2 / April 1982
- Published online by Cambridge University Press:
- 27 March 2009, pp. 307-316
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- Article
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Substrate and nitrogen metabolism of ruminant muscle was studied in vitro using a perfused sheep hemidiaphragm preparation. Glucose, acetate, propionate and butyrate were taken up by the perfused muscle at a constant rate over 2 h. Rate of acetoacetate uptake declined over the perfusion period. Net production of 3-hydroxybutyrate over the perfusion period was observed.
Release of alanine and glycine by the perfused muscle could not be accounted for by protein breakdown, implicating muscle as a site of synthesis of these amino acids. The perfused muscle metabolized aspartate and arginine to a considerable extent, but the branched-chain amino acids only to a small extent. Only a small proportion, approximately 5%, of the alanine produced by the perfused muscle was derived from glucose.
In the presence of amino acids, at twice the concentration found in plasma, and insulin, some amino acids showed a net uptake by the diaphragm but when cysteine was omitted from the perfusate, all amino acids with the exception of arginine showed a net output. Evidence was obtained that protein synthesis was reduced in the absence of cysteine. The output of Nτ methylhistidine by the preparation was much higher than could be accounted for by estimates of protein breakdown using isotope dilution techniques. These data are, however, consistent with the view that Nτ methylhistidine is not a good index of muscle protein breakdown in sheep.