Combining label-free and label-based accurate quantifications with SWATH-MS: Comparison with SRM and PRM for the evaluation of bovine muscle type effects.

Mass spectrometry has proven to be a valuable tool for the accurate quantification of proteins. In this study, the performances of three targeted approaches, namely selected reaction monitoring (SRM), parallel reaction monitoring (PRM) and sequential windowed acquisition of all theoretical fragment ion mass spectra (SWATH-MS), to accurately quantify ten potential biomarkers of beef meat tenderness or marbling in a cohort of 64 muscle samples were evaluated. So as to get the most benefit out of the complete MS2 maps that are acquired in SWATH-MS, an original label-free quantification method to estimate protein amounts using an I-spline regression model was developed. Overall, SWATH-MS outperformed SRM in terms of sensitivity and dynamic range, while PRM still performed the best, and all three strategies showed similar quantification accuracies and precisions for the absolute quantification of targets of interest. This targeted picture was extended by 585 additional proteins for which amounts were estimated using the label-free approach on SWATH-MS; thus, offering a more global profiling of muscle proteomes and further insights into muscle type effect on candidate biomarkers of beef meat qualities as well as muscle metabolism.

Prediction of the Secretome and the Surfaceome: A Strategy to Decipher the Crosstalk between Adipose Tissue and Muscle during Fetal Growth.

Crosstalk between adipose and muscular tissues is hypothesized to regulate the number of muscular and adipose cells during fetal growth, with post-natal consequences on lean and fat masses. Such crosstalk largely remains, however, to be described. We hypothesized that a characterization of the proteomes of adipose and muscular tissues from bovine fetuses may enhance the understanding of the crosstalk between these tissues through the prediction of their secretomes and surfaceomes. Proteomic experiments have identified 751 and 514 proteins in fetal adipose tissue and muscle. These are mainly involved in the regulation of cell proliferation or differentiation, but also in pathways such as apoptosis, Wnt signalling, or cytokine-mediated signalling. Of the identified proteins, 51 adipokines, 11 myokines, and 37 adipomyokines were predicted, together with 26 adipose and 13 muscular cell surface proteins. Analysis of protein-protein interactions suggested 13 links between secreted and cell surface proteins that may contribute to the adipose-muscular crosstalk. Of these, an interaction between the adipokine plasminogen and the muscular cell surface alpha-enolase may regulate the fetal myogenesis. The in silico secretome and surfaceome analyzed herein exemplify a powerful strategy to enhance the elucidation of the crosstalk between cell types or tissues.

Contribution of connective tissue components, muscle fibres and marbling to beef tenderness variability in longissimus thoracis, rectus abdominis, semimembranosus and semitendinosus muscles.

BACKGROUND: The present study aimed to identify relationships between components of intramuscular connective tissue, proportions of the different fiber types, intramuscular fat and sensory tenderness of beef cooked at 55 °C. Accordingly, four muscles differing in their metabolic and contractile properties, as well as in their collagen content and butcher value, were obtained from dairy and beef cattle of several ages and sexes and were then used to create variability. RESULTS: Correlation analyses and/or stepwise regressions were applied on Z-scores to identify the existing and robust associations. Tenderness scores were further categorized into tender, medium and tough classes using unsupervised learning methods. The findings revealed a muscle-dependant role with respect to tenderness of total and insoluble collagen, cross-links, and type IIB + X and IIA muscle fibers. The longissimus thoracis and semitendinosus muscles that, in the present study, were found to be extreme in their tenderness potential were also very different from each other and from the rectus abdominis (RA) and semimembranosus (SM). RA and SM muscles were very similar regarding their relationship for muscle components and tenderness. A relationship between marbling and tenderness was only present when the results were analysed irrespective of all factors of variation of the experimental model relating to muscle and animal type. CONCLUSION: The statistical approaches applied in the present study using Z-scores allowed identification of the robust associations between muscle components and sensory beef tenderness and also identified discriminatory variables of beef tenderness classes. © 2020 Society of Chemical Industry.

Decision tree, a learning tool for the prediction of beef tenderness using rearing factors and carcass characteristics.

BACKGROUND: The present study explored the potential use of decision trees on rearing factors (q = 10) and carcass characteristics (q = 12) for the development of prediction model rules of beef tenderness prediction/categorization. Accordingly, 308 young bulls were used by a sensory panel to evaluate the tenderness potential of ribeye steaks grilled at 55 °C. A classification and regression tree method was implemented and allowed the prediction of tenderness using (i) rearing factors, (ii) carcass characteristics or (iii) both. RESULTS: The resultant tree models yielded predictive accuracies of 70.78% (with four rearing factors: concentrate percentage; fattening duration; initial body weight and dry matter intake); 67.21% (with four carcass characteristics: fatness carcass score; carcass weight; dressing percentage and muscle carcass percentage) and 84.41% (with six rearing factors and carcass characteristics) compared to the k-means clustering of tenderness. In the final and robust regression tree, from the 22 attribute information, two carcass characteristics (fatness carcass score and muscle carcass percentage) and four rearing factors (fattening duration; concentrate percentage; dry matter intake and initial body weight) were retained as predictors. The first splitter of the 308 ribeye steaks in accordance with their tenderness scores was fatness carcass score, followed by fattening duration and concentrate percentage. CONCLUSION: The trial in the preset study highlights the importance of thresholding approach for efficiently classifying ribeye steaks in accordance with their tenderness potential. The overall prediction model rule was: IF (fatness carcass score ≥ 2.88) AND (concentrate ≥ 82%) [AND (muscle carcass ≥ 71%)] THEN meat was [very] tender. © 2018 Society of Chemical Industry.

Assessment of cattle inter-individual cluster variability: the potential of continuum data from the farm-to-fork for ultimate beef tenderness management.

BACKGROUND: This study implemented a holistic approach based on the farm-to-fork data at the four levels of the continuum (farm - slaughterhouse - muscle - meat) to study the inter-individual cluster variability of beef tenderness. For that, 171 young bulls were selected on a large database of 480 animals according to the industrial expectations based on animal and carcass characteristics. The targeted factors were age at slaughter (14; 20 months), carcass weight (370; 470 kg), EUROP conformation (7; 15) and fatness (2.5; 5) scores of the carcasses. Multivariate analyses and unsupervised learning tools were performed. RESULTS: Principal component analysis combined to agglomerative hierarchical clustering allowed ten clusters to be identified that differed (P < 0.0001) for the four targeted factors. The clusters were further different for variables belonging to each level of the continuum. The results indicated an inter-individual cluster variability rising in tenderness in link with the continuum data grouped according to industrial expectations. The associations of the whole variables of the continuum with tenderness were very important, but farm-to-fork continuum-levels dependent. The findings showed that the variables contributing most to the inter-individual cluster variability of tenderness seemed to be more related to the rearing practices, mainly feeding, and their consequences on carcass properties rather than to the muscle characteristics evaluated by enzyme metabolism and connective tissue. CONCLUSION: It seems that considering the continuum data would allow possible trade-off managements of tenderness to identify levers at different levels from the farm-to-meat. © 2019 Society of Chemical Industry.

Serine Protease Inhibitors as Good Predictors of Meat Tenderness: Which Are They and What Are Their Functions?

Since years, serine proteases and their inhibitors were an enigma to meat scientists. They were indeed considered to be extracellular and to play no role in postmortem muscle proteolysis. In the 1990's, we observed that protease inhibitors levels in muscles are a better predictor of meat tenderness than their target enzymes. From a practical point of view, we therefore choose to look for serine protease inhibitors rather than their target enzymes, i.e. serine proteases and the purpose of this report was to overview the findings obtained. Fractionation of a muscle crude extract by gel filtration revealed three major trypsin inhibitory fractions designed as F1 (Mr:50-70 kDa), F2 (Mr:40-60 kDa) and F3 (Mr:10-15kD) which were analyzed separately. Besides antithrombin III, an heparin dependent thrombin inhibitor, F1 and F2 comprised a large set of closely related trypsin inhibitors encoded by at least 8 genes bovSERPINA3-1 to A3-8 and able to inhibit also strongly initiator and effector caspases. They all belong to the serpin superfamily, known to form covalent complexes with their target enzymes, were located within muscle cells and found in all tissues and fluids examined irrespective of the animal species. Potential biological functions in living and postmortem muscle were proposed for all of them. In contrast to F1 and F2 which have been more extensively investigated only preliminary findings were provided for F3. Taken together, these results tend to ascertain the onset of apoptosis in postmortem muscle. However, the exact mechanisms driving the cell towards apoptosis and how apoptosis, an energy dependent process, can be completed postmortem remain still unclear.

Expression Marker-Based Strategy to Improve Beef Quality.

For beef cattle research, a main objective is to control concomitantly the development of muscles and the qualities of beef cuts. Beef quality is a complex phenotype that is only detectable after slaughter and is highly variable. The beef industry is in need of tools to estimate beef quality of live cattle or online in abattoirs, with specific attention towards sensory attributes (tenderness, juiciness, flavour, and colour). Identification of relevant genetic and genomic markers is ongoing, especially for tenderness--a top priority quality attribute. In this paper, we describe the steps of an expression marker-based strategy to improve beef sensory quality, from the discovery of biomarkers that identify consistent beef and the biological functions governing beef tenderness to the integration of the knowledge into detection tests for desirable animals. These tools should soon be available for the management of sensory quality in the beef production chain for meeting market's demands and assuring good quality standards.

How Muscle Structure and Composition Influence Meat and Flesh Quality.

Skeletal muscle consists of several tissues, such as muscle fibers and connective and adipose tissues. This review aims to describe the features of these various muscle components and their relationships with the technological, nutritional, and sensory properties of meat/flesh from different livestock and fish species. Thus, the contractile and metabolic types, size and number of muscle fibers, the content, composition and distribution of the connective tissue, and the content and lipid composition of intramuscular fat play a role in the determination of meat/flesh appearance, color, tenderness, juiciness, flavor, and technological value. Interestingly, the biochemical and structural characteristics of muscle fibers, intramuscular connective tissue, and intramuscular fat appear to play independent role, which suggests that the properties of these various muscle components can be independently modulated by genetics or environmental factors to achieve production efficiency and improve meat/flesh quality.

Relationships between the abundance of 29 proteins and several meat or carcass quality traits in two bovine muscles revealed by a combination of univariate and multivariate analyses.

We aimed to evaluate the relationships between meat or carcass properties and the abundance of 29 proteins quantified in two muscles, Longissimus thoracis and Rectus abdominis, of Rouge des Prés cows. The relative abundance of the proteins was evaluated using a high throughput immunological method: the Reverse Phase Protein array. A combination of univariate and multivariate analyses has shown that small HSPs (CRYAB, HSPB6), fast glycolytic metabolic and structural proteins (MYH1, ENO3, ENO1, TPI1) when assayed both in RA and LT, were related to meat tenderness, marbling, ultimate pH, as well as carcass fat-to-lean ratio or conformation score. In addition to some small HSP, ALDH1A1 and TRIM72 contributed to the molecular signature of muscular and carcass adiposity. MYH1 and HSPA1A were among the top proteins related to carcass traits. We thus shortened the list to 10 putative biomarkers to be considered in future tools to manage both meat and carcass properties. SIGNIFICANCE: In three aspects this manuscript is notable. First, this is the first proteomics study that aims to evaluate putative biomarkers of both meat and carcass qualities that are of economic importance for the beef industry. Second, the relationship between the abundance of proteins and the carcass or meat traits were evaluated by a combination of univariate and multivariate analyses on 48 cows that are representative of the biological variability of the traits. Third, we provide a short list of ten proteins to be tested in a larger population to feed the pipeline of biomarker discovery.

Cellular and molecular large-scale features of fetal adipose tissue: is bovine perirenal adipose tissue brown?.

Epidemiological and fetal programming studies point to the role of fetal growth in adult adipose tissue (AT) mass in large mammals. Despite the incidence of fetal AT growth for human health and animal production outcomes, there is still a lack of relevant studies. We determined the cellular and large-scale-molecular features of bovine fetal perirenal AT sampled at 110, 180, 210, and 260 days post-conception (dpc) with the aim of identifying key cellular and molecular events in AT growth. The increase in AT weight from 110 to 260 dpc resulted from an increase in adipocyte volume and particularly adipocyte number that were concomitant with temporal changes in the abundance of 142 proteins revealed by proteomics. At 110 and 180 dpc, we identified proteins such as TCP1, FKBP4, or HSPD1 that may regulate adipocyte precursor proliferation by controlling cell-cycle progression and/or apoptosis or delaying PPARγ-induced differentiation. From 180 dpc, the up-regulation of PPARγ-induced proteins, lipogenic and lipolytic enzymes, and adipokine expression may underpin the differentiation and increase in adipocyte volume. Also from 180 dpc, we unexpectedly observed up-regulations in the ß-subunit of ATP synthase, which is normally bypassed in brown AT, as well as in aldehyde dehydrogenases ALDH2 and ALDH9A1, which were predominantly expressed in mouse white AT. These results, together with the observed abundant unilocular adipocytes at 180 and 260 dpc, strongly suggest that fetal bovine perirenal AT has much more in common with white than with brown AT.

The worsening of tibialis anterior muscle atrophy during recovery post-immobilization correlates with enhanced connective tissue area, proteolysis, and apoptosis.

Sustained muscle wasting due to immobilization leads to weakening and severe metabolic consequences. The mechanisms responsible for muscle recovery after immobilization are poorly defined. Muscle atrophy induced by immobilization worsened in the lengthened tibialis anterior (TA) muscle but not in the shortened gastrocnemius muscle. Here, we investigated some mechanisms responsible for this differential response. Adult rats were subjected to unilateral hindlimb casting for 8 days (I8). Casts were removed at I8, and animals were allowed to recover for 10 days (R1 to R10). The worsening of TA atrophy following immobilization occurred immediately after cast removal at R1 and was sustained until R10. This atrophy correlated with a decrease in type IIb myosin heavy chain (MyHC) isoform and an increase in type IIx, IIa, and I isoforms, with muscle connective tissue thickening, and with increased collagen (Col) I mRNA levels. Increased Col XII, Col IV, and Col XVIII mRNA levels during TA immobilization normalized at R6. Sustained enhanced peptidase activities of the proteasome and apoptosome activity contributed to the catabolic response during the studied recovery period. Finally, increased nuclear apoptosis prevailed only in the connective tissue compartment of the TA. Altogether, the worsening of the TA atrophy pending immediate reloading reflects a major remodeling of its fiber type properties and alterations in the structure/composition of the extracellular compartment that may influence its elasticity/stiffness. The data suggest that sustained enhanced ubiquitin-proteasome-dependent proteolysis and apoptosis are important for these adaptations and provide some rationale for explaining the atrophy of reloaded muscles pending immobilization in a lengthened position.

Cluster analysis application identifies muscle characteristics of importance for beef tenderness.

BACKGROUND: An important controversy in the relationship between beef tenderness and muscle characteristics including biochemical traits exists among meat researchers. The aim of this study is to explain variability in meat tenderness using muscle characteristics and biochemical traits available in the Integrated and Functional Biology of Beef (BIF-Beef) database. The BIF-Beef data warehouse contains characteristic measurements from animal, muscle, carcass, and meat quality derived from numerous experiments. We created three classes for tenderness (high, medium, and low) based on trained taste panel tenderness scores of all meat samples consumed (4,366 observations from 40 different experiments). For each tenderness class, the corresponding means for the mechanical characteristics, muscle fibre type, collagen content, and biochemical traits which may influence tenderness of the muscles were calculated. RESULTS: Our results indicated that lower shear force values were associated with more tender meat. In addition, muscles in the highest tenderness cluster had the lowest total and insoluble collagen contents, the highest mitochondrial enzyme activity (isocitrate dehydrogenase), the highest proportion of slow oxidative muscle fibres, the lowest proportion of fast-glycolytic muscle fibres, and the lowest average muscle fibre cross-sectional area. Results were confirmed by correlation analyses, and differences between muscle types in terms of biochemical characteristics and tenderness score were evidenced by Principal Component Analysis (PCA). When the cluster analysis was repeated using only muscle samples from m. Longissimus thoracis (LT), the results were similar; only contrasting previous results by maintaining a relatively constant fibre-type composition between all three tenderness classes. CONCLUSION: Our results show that increased meat tenderness is related to lower shear forces, lower insoluble collagen and total collagen content, lower cross-sectional area of fibres, and an overall fibre type composition displaying more oxidative fibres than glycolytic fibres.

Characterization of Four Rearing Managements and Their Influence on Carcass and Meat Qualities in Charolais Heifers.

The study aim was to identify the effects of the rearing management applied throughout the heifers' life on the carcass (e.g., conformation, marbling, fat) and meat (color, texture, and sensory profiles) properties. From the individual data of 171 heifers from 25 commercial farms, a typology of four rearing managements was established from 50 rearing factors. The managements had an effect on the conformation, the color (fat and muscle), and the rhomboideus grain meat, for the carcass, and the lightness, the atypical flavor, and the overall acceptability for the longissimus (LM) meat. The carcass traits compared to the meat were more sensitive to a change of rearing management. Our results confirmed that it was possible to target the same carcass or meat quality from different managements. Moreover, according to the aims of the targeted carcass and LM meat quality, management 3 could be an interesting trade-off to jointly manage the quality of both products. For example, the carcasses that were produced had a high conformation, smooth meat grain and the LM meat was more liked. This management was intermediate compared to the other rearing managements and had a long fattening period with a diet mainly based on conserved grass and a high concentrate quantity.

Effects of Rearing Management Applied throughout the Charolais Young Bulls' Life on Carcass and Meat Quality.

The aim of this work was to study, for the first time, the effects of the rearing management (from birth to slaughter) applied throughout the life of young bulls on carcass and meat quality. Five rearing managements were defined statistically, from a combination of 30 rearing factors, using a hierarchical clustering on principal components. This study considered the individual data of 179 Charolais young bulls from commercial farms. The carcass traits were more sensitive to rearing management than the meat traits. Rearing management had an effect mainly on fat and overall meat grain for the carcass, and on color and tenderness for the longissimus meat. However, it was possible to produce carcass and/or meat with similar properties from different rearing managements. Among the five rearing managements defined in this study, two were identified as allowing the best trade-off to produce simultaneously high carcass and meat quality. The first management was characterized by absence of growth period and a short fattening duration, with a wrapped haylage or corn silage-based diet. The second management was characterized by short pre-weaning and growth periods, and a long fattening period.

Does the Rearing Management following by Charolais Cull Cows Influence the Qualities of Carcass and Beef Meat?

This study characterized, for the first time, the rearing managements (from birth to slaughter) applied throughout the cull cows' life and observed the effect of these managements on the carcass and meat properties. From the individual data of 371 Charolais cull cows, three rearing managements were defined and characterized with 60 rearing factors. The results showed that the rearing managements had low effects on the carcass and meat properties. For the carcass traits, only the carcass weight, and fat and longissimus (LM) colors at the level of the sixth rib were impacted. Concerning the meat, only the red color intensity, the fat aroma, the flavor intensity and persistence were affected. According to our results, this study confirmed that it is possible to produce carcass or meat with similar properties; consequently, it is difficult to favor a rearing management. However, to manage jointly both carcass and meat qualities, trade-offs are needed.

Does Finishing at Pasture Influence the Colour of Muscle from Suckler Bulls and Can Colour Be Used to Authenticate Their Pre-Slaughter Diet?

The primary objective of this study was to compare the colour of muscle from bulls finished at pasture or indoors on a high concentrate diet. The ancillary objectives were to identify possible explanations for any differences in the colour observed and the potential of muscle colour to discriminate between bull beef from different production systems. Growth, longissimus muscle colour, fibre type composition and metabolic profile were measured in late-maturing breed sired suckler bulls slaughtered at 19 months of age after 199 days at pasture (G0), 100 days indoors after 98 days at pasture (G0AL) and indoors for 199 days (AL). When compared to bulls finished indoors and offered a high concentrate ration, the carcass weight of G0 bulls was lower, their carcasses were leaner, and their longissimus muscle was similar in lightness but less red and had a lower glycolytic metabolism. The temperature at which the longissimus muscle reached pH 6.0 was lower (19.7 °C) for G0 than for G0AL (29.9 °C) and AL (31.6 °C), which did not differ. Co-variate adjustment for this variable removed the differences in redness. Adjusting the chill settings appears to be a practical strategy for abattoirs to minimise early post-mortem differences in muscle colour between lighter grass-fed and heavier concentrate-fed carcasses. The preliminary results demonstrate the potential of both L*, a*, b* values and the visible reflectance spectra of muscle to discriminate between grass- finished and concentrate-finished bull beef, but further refinement and validation of the models is required.

Myostatin gene inactivation increases post-mortem calpain-dependent muscle proteolysis in mice.

Myostatin deficiency leads to extensive skeletal muscle hypertrophy, but its consequence on post-mortem muscle proteolysis is unknown. Here, we compared muscle myofibrillar protein degradation, and autophagy, ubiquitin-proteasome and Ca2+-dependent proteolysis relative to the energetic and redox status in wild-type (WT) and myostatin knock-out mice (KO) during early post-mortem storage. KO muscles showed higher degradation of myofibrillar proteins in the first 24 h after death, associated with preserved antioxidant status, compared with WT muscles. Analysis of key autophagy and ubiquitin-proteasome system markers indicated that these two pathways were not upregulated in post-mortem muscle (both genotypes), but basal autophagic flux and ATP content were lower in KO muscles. Proteasome and caspase activities were not different between WT and KO mice. Conversely, calpain activity was higher in KO muscles, concomitantly with higher troponin T and desmin degradation. Altogether, these results suggest that calpains but not the autophagy, proteasome and caspase systems, explain the difference in post-mortem muscle protein proteolysis between both genotypes.

Protocol for high-resolution electrophoresis separation of myosin heavy chain isoforms in bovine skeletal muscle.

In this short communication we describe a specific protocol for SDS-PAGE separation of adult bovine myosin heavy-chain (MyHC) isoforms. The conditions defined in this protocol allow a good separation with a good reproducibility of the four MyHC isoforms (MyHC I, IIa, IIx, IIb) identified in adult skeletal muscle of this species. This procedure uses mini-gel electrophoresis system and does not involve preparation of gradient separating gels. In addition, this protocol can also be applied to the electrophoretic separation of ovine and camel MyHC isoforms.

Understanding the Determination of Meat Quality Using Biochemical Characteristics of the Muscle: Stress at Slaughter and Other Missing Keys.

Despite increasingly detailed knowledge of the biochemical processes involved in the determination of meat quality traits, robust models, using biochemical characteristics of the muscle to predict future meat quality, lack. The neglecting of various aspects of the model paradigm may explain this. First, preslaughter stress has a major impact on meat quality and varies according to slaughter context and individuals. Yet, it is rarely taken into account in meat quality models. Second, phenotypic similarity does not imply similarity in the underlying biological causes, and several models may be needed to explain a given phenotype. Finally, the implications of the complexity of biological systems are discussed: a homeostatic equilibrium can be reached in countless ways, involving thousands of interacting processes and molecules at different levels of the organism, changing over time and differing between animals. Consequently, even a robust model may explain a significant part, but not all of the variability between individuals.