Effect of postmortem storage on mu-calpain and m-calpain in ovine skeletal muscle.

Casein zymography was used to determine the effect of postmortem storage on the proteolytic activity of mu-calpain and m-calpain in lamb longissimus. Casein zymography assays were conducted on crude muscle extracts (only one centrifugation). Six market weight crossbred lambs were slaughtered and a portion of the longissimus lumborum was removed at death (within 15 min of exsanguination) and after 3, 6, 9, 12, 24, 72, and 360 h postmortem. Muscle samples were snap-frozen in liquid nitrogen and stored at -70 degrees C. Soluble muscle proteins were extracted from muscle samples and analyzed by in-gel casein assay to measure calpain proteolytic activity. There was a gradual decline in mu-calpain activity (P < 0.05) such that after 24 and 72 h postmortem, mu-calpain had lost 42 and 95% of its activity, respectively. After 360 h postmortem, no mu-calpain activity could be detected (under the conditions used in this study). Autolysis of mu-calpain could be detected as early as 3 h postmortem. It was demonstrated that the detectable level of mu-calpain activity is a function of the amount of muscle protein electrophoresed. Hence, the activity data reported are in relative terms, rather than absolute values. Furthermore, it was demonstrated that the activity data also are a function of the assay methods used. Different methods have different lower detection limits. Of the three assays examined, 14C-labeled casein was the most sensitive, then the in-gel casein assay, and the least-sensitive method was the standard casein assay. Unlike mu-calpain, postmortem storage had no effect on m-calpain (P > 0.05). When the calcium concentration of a muscle extract was increased to the level that induces m-calpain autolysis, m-calpain was autolyzed and its autolysis was readily detected by the in-gel casein assay. Collectively, these results demonstrate that calcium concentration in postmortem muscle is only high enough to activate mu-calpain. These results support the widely believed conclusion that mu-calpain-mediated proteolysis of key myofibrillar and cytoskeletal proteins is responsible for postmortem tenderization. Hence, understanding the regulation of mu-calpain in postmortem muscle should be the focus of future studies.     

Effect of pH and ionic strength on mu- and m-calpain inhibition by calpastatin

The objectives of this study were to determine the extent to which pH and ionic strength influence mu- and m-calpain activity and the inhibition of calpains by calpastatin. Calpastatin, mu-calpain, and m-calpain were purified from at-death porcine semimembranosus. Mu-calpain or m-calpain (0.45 U) were incubated with the calpain substrate Suc-Leu-Leu-Val-Tyr-7-amino-4-methyl coumarin in the presence of calpastatin (0, 0.15, or 0.30 U of calpain inhibitory activity) under the following pH and ionic strength conditions: pH 7.5 and 165 mM NaCl or 295 mM NaCl; pH 6.5 and 165 mM NaCl or 295 mM NaCl; and pH 6.0 and 165 mM NaCl or 295 mM NaCl. The reactions were initiated with addition of 100 microM (mu-calpain) or 1 mM CaCl2 (m-calpain), and calpain activity was recorded at 30 and 60 min. Mu-calpain had the greatest (P < 0.01) activity at pH 6.5 at each ionic strength. Higher ionic strength decreased mu-calpain activity (P < 0.01) at all pH conditions. Inhibition percent of mu-calpain by calpastatin was not affected by pH; however, it was influenced by ionic strength. Inhibition of mu-calpain by calpastatin was higher (P < 0.01) at 295 mM NaCl than at 165 mM NaCl when 0.3 units of calpastatin were included in the assay. Activity of m-calpain was greater (P < 0.01) at pH 7.5 than at pH 6.5. m-Calpain activity was not detected at pH 6.0. Inhibition of m-calpain was greater (P < 0.01) when 0.15 and 0.3 U calpastatin were added at pH 6.5 than 7.5 at 165 mM NaCl, whereas percentage inhibition of m-calpain was greater (P < 0.01) at 295 mM than 165 mM NaCl at pH 7.5 and 6.5. These observations provide new evidence that defines further the influence of pH decline and increased ionic strength on mu-calpain, m-calpain, and calpastatin activity, thereby helping to more accurately define a role for these enzymes in the process of postmortem tenderization.     

Effect of oxidation, pH, and ionic strength on calpastatin inhibition of mu- and m-calpain

The objective of this study was to evaluate the effect of oxidation on mu- and m-calpain activity at varying pH and ionic strength conditions in the presence of calpastatin. In 2 separate experiments, purified porcine skeletal muscle mu- or m-calpain (0.45 units of caseinolytic activity) was incubated in the presence of calpastatin (0, 0.15, or 0.30 units) at pH 7.5, 6.5, or 6.0 with either 165 or 295 mM NaCl. The reactions were initiated with the addition of CaCl2 (100 microM for mu-calpain; 1 mM for m-calpain). In Experiment 1, mu- or m-calpain was incubated with the calpain substrate Suc-Leu-Leu-Val-Tyr-AMC (170 microM). Either 0 or 16 mu microM H2O2 was added to each assay. Activity was measured at 60 min. In Experiment 2, calpain was incubated with highly purified porcine myofibrils (4 mg/mL) under conditions described. Either 0 or 100 microM H2O2 was added immediately prior to the addition of calpain. Degradation of desmin was determined on samples collected at 2, 15, 60, and 120 min. Results from Experiment 1 indicated that oxidation decreased (P < 0.01) activity of mu-calpain. Mu-calpain had the greatest (P < 0.01) activity at pH 6.5, and m-calpain had the greatest (P < 0.01) activity at pH 7.5 at 60 min. m-Calpain activity was not detected at pH 6.0. Mu- and m-calpain activity were lower (P < 0.01) at 295 mM NaCl than at 165 mM NaCl at all pH conditions. Oxidation lowered (P < 0.01) calpastatin inhibition of mu-and m-calpain at all pH and ionic strength combinations. In Experiment 2, oxidation decreased proteolytic activity of mu-calpain against desmin at pH 6.0 (P < 0.05 at 15, 60, and 120 min) and decreased m-calpain at all pH conditions. However, desmin degradation by mu-calpain was not as efficiently inhibited by calpastatin at pH 7.5 and as at pH 6.5 (P = 0.03 at 60 min) when oxidizing conditions were created. This is consistent with the results from Experiment 1, which indicated that oxidation decreased the ability of calpastatin to inhibit mu-calpain. These studies provide evidence that oxidation influences calpain activity and inhibition of calpains by calpastatin differently under varying environmental conditions. The results suggest that, at the higher pH conditions used, calpastatin may limit the possibility of oxidation-induced inactivation of mu-calpain.     

Effect of sire on mu- and m-calpain activity and rate of tenderization as indicated by myofibril fragmentation indices of steaks from Brahman cattle

The objectives of this study were to assess the influence of sire on mu- and m-calpain activities, to evaluate the relationships of activities of these enzymes to other traits related to beef palatability, and to assess the influence of sire on the rate of tenderization (as measured by myofibril fragmentation index [MFI]) in Brahman longissimus muscle. Brahman calves (n = 87), sired by nine bulls, were born, weaned, fed, and slaughtered in central Florida. Traits evaluated were mu- and m-calpain activities and MFI after 1, 7, 14, and 21 d of aging. Other traits were analyzed to determine their associations with mu- and m-calpain activity and MFI, including calpastatin activity, percentage of raw and cooked lipids, Warner-Bratzler shear force (WBSF) values after 7, 14, and 21 d of aging, and sensory panel rating of tenderness, juiciness, and connective tissue amount after 14 d of aging. Data were analyzed using a model with sire, sex, year, and slaughter group (calves of the same sex slaughtered on the same date) as fixed effects, and adjusted to a constant adjusted 12th-rib fat thickness. Sire affected mu-calpain activity (P < 0.04), calpastatin activity (P < 0.01), d-14 MFI (P < 0.02), d-7 WBSF (P < 0.05), d-14 WBSF (P < 0.04), and sensory panel juiciness score (P < 0.01), but not (P < 0.75) m-calpain activity. Measures of tenderness and palatability were generally moderately to strongly correlated (both simple and residual correlations) with calpastatin and m-calpain activity. Myofibril fragmentation index residuals (adjusted for all model components except sire) after all aging periods were fitted using nonlinear regression to the exponential curve (MFI(i) = kappa0 + kappa1 exp[kappa2 t(i)] + epsilon(i), where t(i) represents aging in days, k0 is ultimate MFI after aging, kappa1 is the difference between initial and ultimate MFI, kappa2 is the rate of increase in MFI, and epsilon(i) is the error term associated with the ith observation, assumed to be independent and identically distributed normally). Sires had different estimates and combinations of estimates, which were used to plot MFI change with time. These curves visually differed for sires and suggested that postmortem tenderization extent and rate differ as well. Use of a combination of these estimated parameters in a selection/carcass sorting program represents an alternative consideration for tenderization improvement programs.     

Effect of storage on bovine erythrocyte cholinesterase activity.

Bovine whole blood and washed erythrocyte samples with either sodium heparin or EDTA as the anticoagulant were stored at -24, 2 to 3, 21, or 37 C for up to 8 weeks to determine the stability of erythrocyte cholinesterase (ChE) activity. An automated colorimetric method was used to measure ChE activity. Regression analysis was used to estimate the rate of decline of ChE activity of samples to 95% of normal. Samples stored at -24 and 2 to 3 C were very stable, and ChE activity was maintained for several weeks or longer. Samples stored at 21 C maintained an activity level not less than 95% of normal for at least 9 days and those stored at 37 C for at least 2 days. The type of anticoagulant used did not appear to affect significantly the stability of activity.     

Calpain activation and proteolysis in postmortem goose muscles

Meat tenderness is considered as the most important criterion for meat quality by consumers and can be improved by the actions of endogenous proteases, mainly calpains, during postmortem storage at 0–5°C. The purpose of this study, therefore, was to examine the postmortem calpain activation and proteolysis in breast (BM) and leg and thigh (LM) muscles of White Roman goose. BM and LM were taken from goose carcasses (n = 15) at 0 (10–15 min postmortem), 1, 3, and 7 days of storage at 5°C. The decrease in postmortem pH, calpain‐1 and ‐11 activities, and contents of the calpain‐1 80 kDa subunit and desmin was more rapid (p < .05) in BM than in LM. Our results show that postmortem proteolysis was more extensive in BM than in LM of White Roman goose, not only because the difference in fiber type composition between two muscles, but because the rate and extent of calpain activation were greater in BM as well. These results may provide useful information to optimize meat processing for different muscles in goose industry.     

Isolation and characterization of mu-calpain, m-calpain, and calpastatin from postmortem muscle. I. Initial steps

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.     

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.     

Assessment of organoleptic postmortem inspection techniques for bovine offal

OBJECTIVE: To evaluate organoleptic postmortem inspection techniques for bovine livers and kidneys. STUDY DESIGN: At Australian export abattoirs, bovine liver and kidneys are assessed and graded by qualified meat inspectors during normal operations. Over a 12-month period at a large abattoir in eastern Australia during 1997 and 1998, a sample of these organs was reassessed independently using a range of pathological and microbiological methods. Agreement between routine inspection and independent assessment was evaluated using methods of inter-rater agreement. RESULTS: A total of 944 livers and 1374 kidneys were included in the study. All of these organs had been classified during routine inspection. The authors examined 363 livers and 329 kidneys both grossly and histologically, including 36 livers and 14 kidneys that were also subjected to microbiological examination. All other organs were only examined grossly. There was only a moderate level of agreement between the routine and independent assessment methods. For livers, the percentage agreement was 80.2%, McNemar's test of symmetry 55.2 (3 degrees of freedom, P < 0.001) and kappa 0.63. For kidneys, the percentage agreement was 67.8%, McNemar's test of symmetry 9.9 (1 df, P = 0.002) and kappa 0.35. CONCLUSIONS: The results reinforce concerns from a number of authors about organoleptic postmortem inspection. Risk assessment methodologies offer the opportunity to modify inspection techniques in a manner that is most relevant to current public health concerns.     

Assessment of organoleptic postmortem inspection techniques for bovine offal

Objective To evaluate organoleptic postmortem inspection techniques for bovine livers and kidneys.
Study design At Australian export abattoirs, bovine liver and kidneys are assessed and graded by qualified meat inspectors during normal operations. Over a 12-month period at a large abattoir in eastern Australia during 1997 and 1998, a sample of these organs was reassessed independently using a range of pathological and microbiological methods. Agreement between routine inspection and independent assessment was evaluated using methods of inter-rater agreement.
Results A total of 944 livers and 1374 kidneys were included in the study. All of these organs had been classified during routine inspection. The authors examined 363 livers and 329 kidneys both grossly and histologically, including 36 livers and 14 kidneys that were also subjected to microbiological examination. All other organs were only examined grossly. There was only a moderate level of agreement between the routine and independent assessment methods. For livers, the percentage agreement was 80.2%, McNemar's test of symmetry 55.2 (3 degrees of freedom, P < 0.001) and kappa 0.63. For kidneys, the percentage agreement was 67.8%, McNemar's test of symmetry 9.9 (1df, P = 0.002) and kappa 0.35.
Conclusions The results reinforce concerns from a number of authors about organoleptic postmortem inspection. Risk assessment methodologies offer the opportunity to modify inspection techniques in a manner that is most relevant to current public health concerns.     

5种青藏高原中药材对磷酸二脂酶4活性的影响

目的：从青藏高原地产中药材中筛选磷酸二脂酶4（PDE4）特异性抑制剂。方法：选取甘草、羌活、柴胡、大黄、红景天5种药材为试验对象,以PDE4特异性抑制剂Rolipram为阳性对照组,从猪中性粒细胞中提取PDE4进行PDE4活性实验。结果：5 mg/mL甘草、羌活、柴胡、大黄、红景天水提物、10μmol/L Rolipram组PDE4活性分别为6.8%、11.02%、11.07%、12.7%、18.7%、7.61%;抑制率分别为38.18%、0.02%、-0.47%、-15.21%、-69.66%、30.90%。与空白对照组PDE4活性相比较,甘草组P〈0.05;红景天组P〈0.001。与Rolipram组抑制率相比较,柴胡组P〈0.05;大黄组P〈0.01;红景天组P〈0.001。甘草组对PDE4抑制率高于PDE4特异性抑制剂Rolipram。结论：5mg/mL甘草水提物能显著抑制PDE4活性,且高于PDE4特异性抑制剂Rolipram,5 mg/mL柴胡、羌活水提物对PDE4活性的影响不明显,5 mg/mL大黄水提物对PDE4的活性具有促进作用,5 mg/mL红景天对PDE4的活性具有极显著的促进作用。     

Early postmortem biochemical factors influence tenderness and water-holding capacity of three porcine muscles

The objective of this study was to determine whether differences in pork tenderness and water-holding capacity could be explained by factors influencing calpain activity and proteolysis. Halothane-negative (HAL-1843 normal) Duroc pigs (n = 16) were slaughtered, and temperature and pH of the longissimus dorsi (LD), semimembranosus (SM), and psoas major (PM) were measured at 30 and 45 min and 1, 6, 12, and 24 h postmortem. Calpastatin activity; mu-calpain activity; and autolysis and proteolysis of titin, nebulin, desmin, and troponin-T were determined on muscle samples from the LD, SM, and PM at early times postmortem. Myofibrils from each muscle were purified to assess myofibril-bound (mu-calpain. Percentage drip loss was determined, and Warner-Bratzler shear (WBS) force was analyzed. Myosin heavy-chain (MHC) isoforms were examined using SDS-PAGE. The pH of PM was lower (P < 0.01) than the pH of LD and SM at 30 and 45 min and 1 h postmortem. The PM had a higher (P < 0.01) percentage of the MHC type IIa/IIx isoforms than the LD. The-LD had the greatest proportion of (P < 0.01) MHC IIb isoforms of any of the muscles. The PM had the lowest (P < 0.01) percentage of MHC IIb isoforms and a greater (P < 0.05) percentage of type I MHC isoforms than the LD and SM. The PM had less (P < 0.01) drip loss after 96 h of storage than the SM and LD. The PM had more desmin degradation (P < 0.01) than the LD and SM at 45 min and 6 h postmortem. Degradation of titin occurred earlier in the PM than the LD and SM. At 45 min postmortem, the PM consistently had some autolysis of mu-calpain, whereas the LD and SM did not. At 6 h postmortem, some autolysis of mu-calpain (80-kDa subunit) was observed in all three muscles. The rapid pH decline and increased rate of autolysis in the PM paralleled an earlier appearance of myofibril-bound mu-calpain. The SM had higher calpastatin activity (P < 0.05) at 45 min, 6 h, and 24 h and had higher WBS values at 48 h (P < 0.01) and 120 h (P < 0.05) postmortem than the LD. At 48 and 120 h postmortem, more degradation of desmin, titin, and nebulin were observed in the LD than in the SM. These results show that mu-calpain activity, mu-calpain autolysis, and protein degradation are associated with differences in pork tenderness and water-holding capacity observed in different muscles.     

Effect of medium additives during liquid storage on developmental competence of in vitro matured bovine oocytes

Our aim was to improve the developmental competence of bovine oocytes during their liquid storage by using additives. In vitro matured oocytes were stored for 20 h at 25°C in HEPES buffered TCM 199 medium (base medium). After storage, in vitro embryo development after in vitro fertilization was compared to those of non‐stored (control) ones. Addition of 10% (v/v) newborn calf serum or 10.27 mmol/L pyruvate alone to the base medium did not improve blastocyst formation rates in stored oocytes; however, their simultaneous addition significantly improved the rate compared with those stored in base medium (P < 0.05). Supplementation of the holding medium with dithiothreitol (DTT) at any concentrations did not improve embryo development from stored oocytes. Although supplementation with cyclosporine A (CsA) significantly reduced apoptosis and membrane damage rates during storage, it did not improve the developmental competence of oocytes. 1,2‐bis(2‐aminophenoxy) ethane N,N,N’,N’‐tetraacetic acid tetrakis‐acetoxymethyl ester and ruthenium red had no effect on oocyte apoptotic rates. Blastocyst formation rates in all stored groups remained significantly lower than that of the control. In conclusion, pyruvate and serum had a synergic effect to moderate the reduction of oocyte quality during storage, whereas mitochondrial membrane pore inhibitor CsA and the antioxidant DTT did not affect their developmental competence.     

Effect of time delay and storage temperature on blood gas and acid-base values of bovine venous blood

The aim of this study was to investigate possible changes in the gas composition and acid-base values of bovine venous blood samples stored at different temperatures (+4, 22 and 37 degrees C) for up to 48 h. Five healthy cattle were used in the study. A total of 15 blood samples collected from the animals were allocated into three groups, which were, respectively, then stored in a refrigerator adjusted to +4 degrees C (Group I, n=5), at a room temperature of about 22 degrees C (Group II, n=5) and in an incubator adjusted to 37 degrees C (Group III; n=5) for up to 48 h. Blood gas and acid-base values were analysed at 0 (baseline), 1, 2, 3, 4, 5, 6, 12, 24, 36 and 48 h of storage. A significant decrease (p<0.001) was found, in the pH of the refrigerated blood after 5 h and its maximum decrease was recorded at 48 h as 0.04 unit. There were also significant alterations (p<0.001) in the blood pH of the samples stored at room temperature and in the incubator after 2 and 3 h, respectively. The maximum mean alteration in pCO(2) value for Group I was -0.72 kPa during the assessment, while for groups II and III, maximum alterations in pCO(2) were detected as +2.68 and +4.16 kPa, respectively. Mean pO(2) values increased significantly (p<0.001) for Group I after 24 h and for Group II after 6 h, while a significant decrease was recorded for Group III after 24 h (p<0.001). Base excess (BE) and bicarbonate (HCO(3)) fractions decreased significantly for all the groups during the study, compared to their baseline values. In conclusion, acid-base values of the samples stored at 22 and +4 degrees C were found to be within normal range and could be used for clinical purposes for up to 12 and 48 h, respectively, although there were small statistically significant alterations.     

Sarcomere length influences postmortem proteolysis of excised bovine semitendinosus muscle

The interaction between sarcomere length and postmortem proteolysis as related to meat tenderness is not clear. The extent of thick and thin filament overlap alters actomyosin binding and may alter substrate availability during aging-induced tenderization. The objective of this study was to determine the influence of sarcomere length on proteolytic degradation in beef. Strips from bovine semitendinosus were either stretched 40% and restrained or allowed to shorten unrestrained in an ice bath. After rigor completion, 0.6-cm cross sections were fabricated and were randomly assigned to 2, 4, 7, or 10 d of aging treatments. Myofibrils were isolated for sarcomere length determination. Samples were collected and frozen for shear force analysis, and muscle proteins were extracted for SDS-PAGE and Western blotting analyses to determine troponin T (TnT) proteolysis. Sarcomere length was greater (P < 0.01) in stretched muscle samples compared with shortened samples (2.57 vs. 1.43 microm, respectively). Correspondingly, shear force values were greater (P < 0.05) in shortened samples than stretched samples. Western blots revealed the presence of 3 major intact TnT bands that diminished with time postmortem and 4 bands (TnT degradation products) that accumulated during postmortem storage. Quantification of intact TnT showed increased (P < 0.05) proteolysis at 4 and 7 d postmortem in samples with long sarcomeres. By 10 d, only traces of the greatest molecular weight intact TnT band were evident in both shortened and stretched samples, suggesting this TnT band may be more susceptible to proteolysis than other intact TnT bands. Degradation products of TnT appeared earlier postmortem in samples with long sarcomeres. The 30-kDa TnT fragment appeared after 7 d of postmortem storage in samples with long sarcomeres but not until 10 d in muscle containing short sarcomeres. Collectively, these data show that postmortem TnT proteolysis is sarcomere length-dependent and suggest that thick and thin filament overlap may influence the postmortem aging process in beef.     

Effects of storage conditions on bovine carrier erythrocytes

Bovine carrier RBC containing [14C]sucrose as a marker were stored for 23 days in protein-free media. Cell recovery was highest with a phosphate buffer (pH 7.0) containing 2 mM MgCl2, 5 mM inosine, 5 mM adenosine, and 10 mM glucose. A cell recovery of 60% was observed after 23 days of storage at 4 C. Stored carrier erythrocytes reinjected into cattle had an 18-day half-life, which is less than that for fresh carrier RBC.