Effects of polymorphisms in the calpastatin and µ-calpain genes on meat tenderness in 3 French beef breeds.

The objectives of the study were to evaluate allelic frequencies and to test the association of polymorphisms in the calpastatin (CAST) and µ-calpain (CAPN1) genes with meat tenderness in 3 French beef breeds. A total of 1,114 Charolais, 1,254 Limousin, and 981 Blonde d'Aquitaine purebred young bulls were genotyped for 3 SNP in the CAST gene and 4 SNP in the CAPN1 gene. Two of these markers, 1 in each gene, can be found in Australian or American commercial genetic tests. Others have previously been reported in American studies or are newly evidenced SNP. The quantitative traits studied were Warner-Bratzler shear force and a tenderness score evaluated by trained sensory panels. All the SNP were informative in the 3 breeds. Associations of individual markers or haplotypes with traits were analyzed. The results differed in the 3 breeds. The G allele of a CAST marker (position 97574679 on Btau4.0) was found to exert a significant effect on the shear force (+0.18 phenotypic SD; RSD) and tenderness score (-0.22 RSD) in the Blonde d'Aquitaine breed. In the same breed, this marker was associated with another CAST SNP (position 97576054 on Btau4.0) such that the GA haplotype appeared to be associated with tougher meat. Two CAPN1 markers (positions 45221250 and 45241089 on Btau4.0) had a significant effect on both traits in the Charolais breed (from |0.11| to |0.25| RSD). In the same breed, these markers were associated with another CAPN1 SNP (position 45219395 on Btau4.0) such that the ACA and AGG haplotypes appeared to be associated with a tender meat and a tougher meat, respectively. Consequently, the present results indicate that the effects of the markers studied are breed-specific and cannot be extended to all Bos taurus breeds. Further studies are also required to identify other more appropriate markers for French beef breeds.

The two mutations, Q204X and nt821, of the myostatin gene affect carcass and meat quality in young heterozygous bulls of French beef breeds.

The availability of genetic tests to detect different mutations in the myostatin gene allows the identification of heterozygous animals and would warrant the superiority of these animals for slaughter performance if this superiority is confirmed. Thus, 2 mutations of this gene, Q204X and nt821, were studied in 3 French beef breeds in the program Qualvigène. This work was done with 1,114 Charolais, 1,254 Limousin, and 981 Blonde d'Aquitaine young bulls from, respectively, 48, 36, and 30 sires and slaughtered from 2004 to 2006. In addition to the usual carcass traits recorded at slaughter (e.g., carcass yield, muscle score), carcass composition was estimated by weighing internal fat and dissecting the 6th rib. The muscle characteristic traits analyzed were lipid and collagen contents, muscle fiber section area, and pH. Regarding meat quality, sensory qualities of meat samples were evaluated by a taste panel, and Warner-Bratzler shear force was measured. Deoxyribonucleic acid was extracted from the blood samples of all calves, the blood samples of 78% of the dams, and the blood or semen samples of all the sires. Genotypes were determined for 2 disruptive mutations, Q204X and nt821. Analyses were conducted by breed. The superiority of carcass traits of calves carrying one copy of the mutated allele (Q204X or nt821) over noncarrier animals was approximately +1 SD in the Charolais and Limousin breeds but was not significant in the Blonde d'Aquitaine. In the Charolais breed, for which the frequency was the greatest (7%), young bulls carrying the Q204X mutation presented a carcass with less fat, less intramuscular fat and collagen contents, and a clearer and more tender meat than those of homozygous-normal cattle. The meat of these animals also had slightly less flavor. Also in the Charolais breed, 13 of 48 sires were heterozygous. For each sire, the substitution effect of the wild allele by the mutant allele was approximately +1 SD for carcass conformation and yield, showing that the estimate of the substitution effect was independent of family structure, as it ought to be for a causal mutation. These results illustrate the challenge of using genetic tests to detect animals with the genetic potential for greater grades of carcasses and meat quality.

Front-face fluorescence spectroscopy as a tool to classify seven bovine muscles according to their chemical and rheological characteristics.

Potential of front-face fluorescence spectroscopy was evaluated to classify muscles according to their chemical and rheological characteristics. Seven bovine muscles (Semitendinosus, Semimembranosus, Tensor fasciae latae, Rectus abdominis, Longissimus thoracis et lumborum, Triceps branchii and Infraspinatus) were taken from 14 animals of the Charolais breed. Chemical characteristics and rheological properties of the meat were determined including dry matter, fat, collagen, protein, peak load, energy required to rupture and cooking loss. Emission spectra in the 305-400nm, 340-540nm and 410-700nm ranges were recorded using front-face fluorescence spectroscopy by fixing the excitation wavelengths at 290, 322 and 382nm, respectively. Analysis of variance (ANOVA) applied on chemical and rheological parameters showed that these muscles were significantly different (P<0.01) from each other. Chemical and rheological data were divided into low, medium and high range groups for each variable. The results of PLSDA showed that 305-400nm spectra were responsible for 67% (calibration), 53% (validation), 96% (calibration) and 55% (validation) of good classification for protein and cooking loss, respectively, while 340-540nm spectra allowed 75% of good classification (validation samples) for fat content.

Sarcomere length determination using front-face fluorescence polarization.

Tryptophan is the major intrinsic fluorophore in muscle and is a constituent of proteins that have two preferential alignments both parallel and perpendicular to muscle fibre direction. A simple theoretical model and an experimental method based on front-face fluorescence polarization technique for tryptophan fluorescence anisotropy measurements were used for the estimation of post-rigor sarcomere length in beef in the range 1.6-3.4µm. Fluorescence anisotropy and structure-related model variables displayed changes in cold-shortened samples compared with normal and stretched ones. The anisotropy of contracted samples was lowered by misalignment of fibres in the sample. This method can therefore be used for in-line detection of cold shortening which has meat toughness as a consequence.

A theoretical approach of the relationships between collagen content, collagen cross-links and meat tenderness.

This work concerns the relationship between meat tenderness and the rubber-like properties, i.e. pressure and elastic modulus, that endomysium and perimysium connective tissues develop when meat has been heated to a temperature above which collagen contracts. For rest length meats with similar intramuscular connective tissue morphology, and which are at the same ageing state and pH, the elastic modulus of the collagenous fraction of connective tissues is approximately proportional to the total number of collagen cross-links present per volume of meat. Calculations from various published experiments concerned with the effect on tenderness of muscle type, animal age, type, and sex from different species show that this modulus follows most of the variations of meat toughness. Moreover, the proportionality between the increase in this elastic modulus and the increase in meat toughness approaches unity in situations where toughness mainly depends on connective tissues. This work demonstrates the decisive role of rubber-like properties of connective tissues in meat tenderness variations.

Serine peptidase inhibitors, the best predictor of beef ageing amongst a large set of quantitative variables.

For consumers, tenderness is the most important sensory attribute of beef meat and, though to a lesser extent, of pork. Tenderness is therefore by far the most common cause of its unacceptability. The major challenge for the beef industry is to evaluate the toughness of the meat as soon as possible after death. In this context, the aim of the present work was to develop an equation to predict the myofibrillar ultimate resistance of raw meat. The study was done on the Longissimus muscle from twenty three 19 months-old Charolais bulls grown in the same INRA farm. Muscles excised within 1h post-mortem were vacuum packed and stored at 15°C during 24h and then transferred to 4°C until used. The activities of lactate dehydrogenase, citrate synthase and myofibrillar Mg-Ca dependent ATPase, the levels of lactate dehydrogenase enzyme, myoglobin, myosin types I, IIa and IIb, cysteine and serine peptidase inhibitors, the pH, the osmolarity, the expressible juice, µ-calpain, m-calpain and calpastatin and meat toughness were measured. According to the physical method used here, the force measured on raw meat represents the resistance of the myofibillar structure. Stepwise linear regression was used to determine the best equation (p<0.05) for predicting toughness at 6 days post-mortem. A 6-variables predictive equation including serine peptidase inhibitors (partial R(2)=0.4), the rate (partial R(2)=0.25), and the extent of pH decline (partial R(2)=0.03), the at death LDH activity (partial R(2)=0.24), the extent of increase in osmotic pressure (partial R(2)=0.13), and the rate of µ-calpain activity loss (partial R(2)=0.09), explained 70% of the variability in meat toughness at 6 days post-mortem. This equation was developed from 20 animals and the other 3 animals, chosen randomly, were used to validate it. The absolute need for a predictive model of meat toughness and the nature of the serine peptidase inhibitors together with their potential target enzymes are discussed.

A simple cryogenic holder for tensile testing of soft biological tissues.

To overcome the difficulty of gripping soft biological materials for tensile test, a simple inexpensive cryogenic holder was developed which allows rapid (3 min) preparation of samples. It is made of 6 parts, built in a bakelite cloth, which is an excellent thermal isolant, and is used with rectangular (8x10(-2)x10(-2)x10(-2)m) samples. The holder with the sample inside is completely immersed in liquid nitrogen for 50 s. This duration allows the freezing of the sample ends on a 10(-2)m length and gives a very flat freezing surface throughout the sample cross section. The 6x10(-2)m central part of the sample remained at ambient temperature. Two parts of the holder help maintain the sample until its ends are vertically gripped in the tensile machine thus avoiding any sample deformation during this step. No pressure was applied on the frozen part of the sample by grips of the tensile machine and this avoids breaks in this region. The sample is fixed by adhesion forces (>1 kN) between its frozen parts and 2 pieces of the holder. The procedure has been successfully tested with bovine and salmon muscle samples and results show tensile breaks randomly distributed in the unfrozen region of the samples. Particular attention has been paid to obtain a very flat freezing surface so that the axial strain is equal throughout the sample and therefore any strain-related mechanical parameters can be accurately determined. The dimensions of the holder can be easily modified to fit other sample geometries and can be used with other biological materials.

Effect of cooking temperature and cooking time on Warner-Bratzler tenderness measurement and collagen content in rabbit meat.

The effects of cooking temperature (50-90 °C) and time (10-120 min) on Warner-Bratzler (WB) tenderness measurement of longissimus lumborum (LL) muscle in 70-day-old rabbits were investigated. Cooking losses, total collagen content and collagen solubility of LL muscle were measured in parallel. Increasing cooking temperature caused a four-phase effect on WB measurement. Stress and total energy were significantly increased between raw meat and cooked meat at 50 °C, then they dramatically decreased to a minimum observed at 60-65 °C, and increased again to reach a maximum at 80-90 °C. Cooking losses exhibited an 83% increase between 50 and 80 °C. At 80 °C, stress and total energy values remained constant after 20 and 40 min respectively. LL muscle collagen content was 16.4±2.3 mg/g of dried muscle. Collagen solubility at 77 °C for 1 h was high: 75.3±8.1%.

Electrical impedance and tenderisation in bovine meat.

The electrical properties and the muscle fibre mechanical resistance of different beef muscles were measured between 1 and 14 days post-mortem. Electrical measurements used different electrode types working by contact with or penetration of muscle. Measurements from the different electrode types were strongly correlated (0.83< r(2) <0.96). Both impedance (Z) and the ratio Z(1 kHz)/ Z(100 kHz) decrease during ageing. These parameters show an almost linear relationship with muscle fibre mechanical resistance. However, the slope of these curves for one muscle changed among animals. Therefore, these electrical parameters are not useful for the prediction of the state of ageing. Meat electrical anisotropy decreases both when frequency increases and with ageing. Electrical anisotropy measured at 1 kHz is significantly (r=0.59, N=129 P<0.01) correlated to muscle fibre mechanical resistance. The mean anisotropy decreases linearly with muscle fibre mechanical resistance in the same way for Semimembranosus and Pectoralis Profundus muscles. When meat is fully aged it is almost electrically isotropic. Electrical anisotropy is, therefore a potential parameter to evaluate meat ageing, but improvements in accuracy are needed before it can be used.

Relationships between muscle characteristics and meat quality traits of young Charolais bulls.

Charolais bull calves (106) were used to study the variability in meat quality attributes in relation to the variability in muscle characteristics in the Longissimus thoracis (LT) muscle. The variability in traits was adjusted either to constant age or constant weight at slaughter and thus originated only from differences between animals born, reared and fattened in the same location. The following meat quality attributes were measured: the strength of the myofibrillar resistance to a 20% compression strain measured on the raw meat 2, 7 and 21 days post mortem; and taste panel scores of tenderness (initial and overall), flavour and juiciness of steaks grilled to a 55°C core temperature 6 or 15 days post-mortem. The following muscle characteristics were measured 24 h after slaughter: pH, dry matter, protein, lipid, heme iron and collagen contents, collagen solubility, LDH and ICDH activity, the proportion of slow twitch myosin heavy chain, the mean muscle fibre area and the mean sarcomere length. One fourth to one third of the variability of 2 day mechanical strength and 15 day tenderness or flavour scores were related to the variability in muscle characteristics. Tenderness and strength measurements were predominantly related to the muscle fibre area, collagen characteristics and energetic metabolic activity. Dry matter content was the principal muscle characteristic related to flavour.

Modelling the effect of sarcomere length on collagen thermal shortening in cooked meat: consequence on meat toughness.

Normal and contracted pieces of Semimembranosus and Longissimus Dorsi muscles from cull cows were cooked for 90 min at temperatures up to 80°C. For both muscles, at 50°C the normal samples have higher breaking stress than contracted samples. The breaking stress of normal samples decreases at 55°C. This decrease is not observed for contracted samples. The contracted samples become the tougher above 60°C. Drip and cooking losses are the highest in contracted samples. Sarcomere length decreases above 60°C whatever the raw sarcomere length. The amplitude of thermal shortening of perimysium collagen fibres in cooked meat has been calculated. This theoretical model takes into account the changes in the waviness of collagen fibres associated with changes in raw sarcomere length and the geometrical changes of fibre bundles due to drip, cooking losses and cooking shortening. The calculations lead to the conclusion that thermal shortening of collagen fibres at 60°C is lower in contracted samples than in normal samples. As the final modulus of collagen fibres decreases when their thermal shortening increases, this can explain part of the differences observed between the toughness of normal and contracted cooked meats. In particular, it can explain why contracted cooked meat becomes tougher than normal meat just above 60°C and why there is a decrease in normal meat toughness between 55 and 60°C. This work therefore emphasises the role of collagen in toughening associated with cold shortening.

Assessment of breed type and ageing time effects on beef meat quality using two different texture devices.

Forty-two male yearlings were used to assess the influence of breed type and ageing time on beef texture. Samples of the M. longissimus dorsi of four breed types [double muscled (DM), dual purpose (Brown Swiss, BS), fast growth (FG) and unimproved type (UT)] were aged for 1, 3, 7, 10, 14 or 21 days at 4°C and frozen at -18°C until analysed. Cooked samples (to end-point of 70°C) were assessed using a Warner-Bratzler (WB) device. Raw samples were assessed using a compression device in which transverse elongation was prevented. There were no significant differences in WB values of cooked meat due to breed type, but ageing had a significant (P<0.05) on maximum load. Ageing, but not breed type, had a significant effect on the compression values of raw meat at low compression rates (P<0.001). Compression values, of raw samples, at 80% compression differed significantly (P<0.001) between breed types, but were not affected by ageing. Compression values of raw samples, at 80% compression, were affected by breed type, probably because of genotype differences in the contribution of connective tissue.

Correlations between successive measurements of myofibrillar resistance of raw Longissimus dorsi muscle during ageing.

The feasibility of meat classification soon after rigor, based on an ageing criterion, was investigated with a view to obtain sets of regularly aged meat as well as to optimize storage. Muscle fibre resistance was measured on the m. longissimus dorsi of 45 heifers on days 1, 2, 8 and 14, and the resistance values obtained on different days were correlated. Resistance on day 2 was predictive of about 65% of the variation observed on day 8, i.e. when the meat has reached approximately 92% of the tenderization recorded between days 1 and 14. In this study, the predictions based on day 1 resistance were less accurate because the baseline reference was too close to rigor. A class defined at day 2 as the meat which has a resistance lower than 20 N cm(-2), will give fully aged meat at day 8. This class represent 52% of the initial population The various classes of meat, which reach full ageing at different times, can be despatched through variable length marketing circuits so as to achieve full ageing before getting to the consumers.

Meat quality traits in the emu (Dromaius novaehollandiae) as affected by muscle type and animal age.

Meat quality traits were determined in the major muscles of the emu (Dromaius novaehollandiae) at different slaughter ages (6, 10, 14, 17 or ≥20 months). A mean ultimate pH value of 5.5 was reached within around 3 h post mortem, but this value was 6.1 in animals that had suffered a preslaughter stress (transportation and fasting). The collagen and pigment contents varied widely among the muscles. The protein and pigment contents increased with animal age, but this effect was perceptible only between 6 and 14 months. The other chemical constituents were little affected by muscle type or animal age. The intense red colour of emu meat, due to a high pigment content, was very sensitive to oxidation, thus limiting the storage of fresh meat under aerobic conditions to short periods of time. Despite a rapid post-mortem tenderization (≤24 h), the residual myofibrillar strength obtained after extended ageing remained intermediate between those reported for chicken and beef. The tenderness of meat, cooked to 60 °C, differed between muscles and decreased with increasing age, thus reflecting the changes occuring in the concentration and in the heat stability of the intramuscular connective tissue.

Predicting variability of ageing and toughness in beef M. Longissimus lumborum et thoracis.

The object of this study was to determine muscle characteristics which might predict meat toughness. Eleven Charolais cattle were slaughtered at approximately 26 months of age and the Longissimus lumborum et thoracis muscle was taken 1 hr post mortem and stored at 12 °C for 24 hr and then at 4 °C. The average half-life for ageing in these raw muscles was 4.6 days but the toughness varied widely between the animals. Toughness varied 3-fold and the rate of ageing varied 20-fold between animals. Correlations were done to determine which characteristics might explain this variability. Toughness was correlated positively with increase in oxidative status of muscle and the initial levels of calpastatin. Toughness was correlated negatively with the initial levels of µ- and m-calpains and cysteine and serine proteinase inhibitors, the initial pH values and the rates of their decline. The rates of ageing were highly correlated positively with the initial levels of proteinase inhibitors and the rates of decline of calpastatin and negatively with the ultimate amounts of expressible juice. There was a wide variability in tenderness in M. Longissimus lumborum et thoracis from similar animals. Variations in metabolism and enzyme activity controlled by inhibitors and calpains appear to be largely responsible for this variability.

[Anticardiolipin antibodies, cerebral ischemia and adrenal hemorrhage in a newborn infant]. / Anticorps anticardiolipine, ischémie cérébrale et hémorragie surrénalienne chez un nouveau-né.

BACKGROUND: Anticardiolipin antibodies, usually associated with vascular thrombosis in systemic lupus erythematosus, have been recently found associated with strokes in childhood, and acute adrenal hemorrhage in the adult. CASE REPORT: A 3 day-old fullterm newborn suffering from cerebrovascular ischemia and bilateral massive hemorrhage was found to have anticardiolipin antibodies detected during the neonatal period and 7 months later. There was no evidence of pathology in the mother or perinatal asphyxia. However, anticardiolipin antibodies were present in the mother 5 and 15 weeks after delivery. CONCLUSION: Association of anticardiolipin antibodies and vascular thrombosis has never been reported in the neonate. Persistence of such antibodies favor the hypothesis that they are not transmitted by the mother; they could represent an early manifestation of primary antiphospholipid syndrome.

Mechanical properties of meat.

Determination of meat mechanical properties is still usually performed under empirical conditions, using tests which have been developed many years ago. This paper discusses the limitations of these methods. It also reviews recently developed research which has been focused on a more analytical approach to mechanical tests performed in closely controlled conditions. The objectives of these investigations are on the one hand to obtain specific properties of the myofibre and connective tissue structures involved in meat toughness and on the other hand to understand the fracture mechanisms involved in the perception of texture. Due to the anisotropic and composite nature of meat, attention has been focused on the influence of the direction and amplitude of strains to be used for the characterisation of myofibres and connective tissue, respectively. These strain conditions are linked to the contraction and stretching state of each of these structures. Indeed, whatever the type of deformation (shear, compression or tension), myofibre resistance can be determined under low strain conditions as long as collagen fibres in the connective tissue remain in a more crimped state than myofibres, i.e. in raw or slightly cooked meat of normal sarcomere length. On the contrary, determination of connective tissue resistance has, up to now, only been obtained using large strain tests. Contraction of collagen fibres induced by cooking makes the separate analysis of meat structures more difficult, but this can partially be overcome by applying mechanical tests in different strain directions. However, the role of the spatial distribution of connective network on the mechanical properties of meat still remains largely unknown. Image analysis and ultrasonic methods, currently developed to study this problem, are briefly presented.

Cryopreserved arterial homografts: preliminary study.

The use of arterial or venous allografts for vascular reconstruction was first reported in 1951, but long-term results have been disappointing. Rejection and inappropriate methods of preservation are the main reasons for failure. A successful solution to this problem could be achieved by programmed cryopreservation with cryoprotectant. Our study had two aims: to define the biomechanical properties of cryopreserved arterial allografts and to study their histologic appearance. Arteries were removed as part of a protocol for multiorgan harvesting for transplantation. Cryopreservation was performed within the first 24 hours after harvesting. Programmed cryopreservation with 15% dimethyl sulfoxide (cryoprotectant) was used. Mechanical testing was done immediately after thawing. Two groups were tested: a control group of fresh aortas and a group of cryopreserved aortas. Axial and circumferential strips were tested. High strain modulus and stress and strain characteristics were calculated for each strip. There was no statistically significant difference between the mechanical properties of fresh and cryopreserved human descending thoracic aortas. Biochemical tests were performed in the preservative solution at 1 and 7 days in both groups. There was no statistically significant difference between the two groups at day 1 or day 7 (p > 0.05). Histologic studies before and after arterial cryopreservation included standard and electron microscopy and showed that arteries had normal structure after cryopreservation. These results confirm that programmed cryopreservation with cryoprotectant does not alter the molecular or geometric configuration of collagen or elastic fibers. Endothelial cells were still present, however, their viability and function were not assessed.(ABSTRACT TRUNCATED AT 250 WORDS)