Of all the beef quality traits, tenderness is considered to be the most important. It appears that among the main determinants of tenderness are collagen characteristics and the muscle fiber type  and, in association with this, the extent of proteolysis during post-mortem ageing and also the amount of intramuscular fat which facilitates mastication of beef. Indeed, with the same animal groups, intramuscular fat content and collagen solubility were shown to be positively associated with tenderness, whereas muscle fiber cross-sectional area were shown to be negatively associated with tenderness . However, the contribution of collagen characteristics to tenderness may depend on the cooking temperature (55°C in our work, which is lower than in other studies) since heat-induced changes of intramuscular connective tissue were observed . Among the proteolytic systems which control post-mortem proteolysis , it has been recently proposed that the apoptotic pathway may be of great importance, since it is the first event occurring after the animal's death. Our results confirm these observations using a new DNA chip. The aim of this manuscript was hence to validate this tool by confirming two well-accepted or recently discovered biological determinants of beef tenderness: the relationships of beef tenderness scores (i) with expression levels of genes corresponding to heat shock proteins on one hand and (ii) with expression levels of genes related to fat and energy metabolism in muscle fibers on the other hand.
Relationships between beef tenderness scores and expression levels of genes encoding heat shock proteins
After slaughter of the animals, there is generalized cell death in all organs and tissues due to the lack of oxygen and hence anoxia of all cells. This stage precedes rigor-mortis and subsequently meat ageing during which protein degradation occurs thanks to the activity of different proteolytic systems (for a review, see ). More and more evidence is now described in the literature indicating that the muscle death process at the beginning of conversion of muscle into meat may play an important role in regulating meat quality . This cell death process, often referred to as apoptosis, is regulated by many proteolytic enzymes including caspases, the activity of these enzymes being itself regulated by many factors.
Cell death induces considerable cell stress and, in response to stress, cells rapidly produce heat shock proteins (HSP) that play a universal role in maintaining cellular homeostasis. HSP help in maintaining the integrity of the cell and have an anti-apoptotic activity . For instance, proteomic studies showed that Hsp27 in fresh Blonde d’Aquitaine muscle and levels of Hsp27 fragments in aged meat explained up to 91% of variation in sensory scores . It was hypothesized that higher levels of Hsp27 associated with limited aggregation of muscle proteins could facilitate the action of proteolytic enzymes during meat ageing. Therefore, the positive correlation between tenderness scores and levels of specific Hsp27 protein isoforms or fragments in Blonde d’Aquitaine  contrasts with the negative correlation we observed in this work between Hsp27 mRNA levels and tenderness scores. This may be explained by the different sources of variability between the two studies: ageing time  or animal variability in this study. However, our results fit well with the positive correlation of Hsp27 protein level and shear force value in Korean cattle . Indeed, a recent study with French breeds confirms that correlation of Hsp27 level may be positive or negative depending on the breed . Other authors have described the complex effects of meat pH upon the sub cellular distribution of muscle HSP during ageing of beef . In other experiments, HspB1 (encoding Hsp27) and its regulator genes were shown to be negatively correlated with intramuscular fat content while being associated with a high shear force  and hence low tenderness as in our work.
Our results with the GENOTEND chip also confirm the negative correlation between DNAJA1 expression and the tenderness score , but for young bulls and steers of year 1 only, which confirms our hypothesis that some markers of beef quality are highly dependent on rearing practices and environmental conditions. DNAJA1 encodes a member of the large 40 kDa heat shock protein family (Hsp40). This protein is a co-chaperone of the 70 kDa heat shock protein (Hsp70) and is believed to play a role in protein folding and mitochondrial protein import. The DNAJA1/Hsp70 complex also directly inhibits programmed cell death (or apoptosis), which supports the hypothesis of Ouali et al.  that apoptosis is important for the tenderization of beef during ageing. Depending on the animal group (young bulls or steers slaughtered year 1), DNAJB9
DNAJC3 or DNJAC10, which are also members of the Hsp40 family, were negatively correlated to tenderness scores (data not shown) just like DNAJA1, which supports the role of members of the Hsp40 family in tenderness. In humans, the DNAJ family has over 40 DNAJ members  and can be subdivided into three subfamilies: DNAJA proteins, DNAJB proteins and DNAJC proteins with different structural features. Our results also support the role of some HspA (Hsp70), and some HspB (small HSPs) including Hsp27, in addition to that of DNAJA1 (Hsp40). For instance, CRYAB, which encodes αB-crystallin and shares homology with Hsp27, was also negatively correlated with tenderness in steers. However, the precise genes of this family which are individually negatively correlated to tenderness scores depends on the animal group and hence on its characteristics (gender, breeding management, etc).
Nevertheless, a major finding of our study is that a subset of genes belonging to the heat shock protein family may be suitable to better explain than muscle biochemical characteristics the variability of tenderness scores for both two groups of Charolais animals of the same gender or reared in similar environmental conditions. However, when the animals differ from the reference population by more than one factor (gender, or environmental conditions, etc), the expression levels of these genes cannot explain anymore the variability of tenderness scores. Further work is thus needed to extent the list of gene markers to have a more generic prediction of tenderness in other conditions of rearing and/or slaughtering and, if possible, in other breeds.
Relationships between beef tenderness scores and expression levels of genes related to muscle metabolism
As previously demonstrated recently by Zhao et al. , we observed that some genes known to be expressed only in adipocytes (FABP4), or involved in lipid deposition (PPARG, DGAT2, FASN) were characterized by expression levels positively associated with tenderness in young bulls slaughtered year 1. This strongly suggests that intramuscular fat level contributes to tenderness in agreement with previous research. Indeed, whereas it is well-known that intramuscular fat level affects mainly juiciness and flavor of beef, it also affects indirectly tenderness . In addition, with the same samples of this study, Guillemin et al  observed that intramuscular fat content was 45% higher in the most tender meat samples (determined from the shearforce measurement) compared to the least tender meat samples. Intramuscular fat is mainly stored within intramuscular adipocytes embedded in a connective tissue matrix in close proximity to a blood capillary. So, in fat-rich beef, intramuscular fat deposited between fibers fascicules may disrupt the structure of muscle connective tissue, thus contributing to increase meat tenderness . This is important in beef meat, in which the abundance and solubility of collagen (the main component of connective tissue) are a source of variation in tenderness especially in young bulls. Generally, intramuscular fat has a diluting effect on connective tissue (known to increase toughness) and consequently a tenderizing effect on meat. Meat from animals with a high intramuscular fat level often has a low shear force , and hence high tenderness. Therefore, it is not surprising to observe that FABP4 expression level, known to be a marker of intramuscular adipocyte number , was positively correlated with tenderness. Similarly, the more fatty acids are stored in these adipocytes, the bigger the adipocytes are, which favors tenderness by diluting connective tissue. Storage of fatty acids is controlled by two mechanisms. The first is due to the action of LPL which hydrolyses circulating triglycerides and hence favors uptake of resulting fatty acids. The second is associated with i) the activity of FASN which is a key enzyme involved in the de novo fatty acid synthesis and more precisely the elongation of fatty acids and (ii) the activity of DGAT which catalyses the formation of triglycerides from diacylglycerol and Acyl-CoA.
It is also well-known that muscles composed of fast fibers which low intramuscular fat content are more susceptible to early post-mortem proteolytic degradation than muscles mainly composed of slow fibers. Fast fibers tend to have higher levels of stored glycogen (which favors pH decline during ageing) and higher amounts of Ca2 + -activated myosin ATPase (which favors the speed of ageing). Therefore, after a short period of ageing, we expect fast muscles to generate tender beef compared to slow muscles due to a faster rate of ageing (for a review, see ). However, this may be not true after 14 days of ageing when a significant part of differences in ageing speed have disappeared. This may explain why the proportion of slow fibers has been reported to improve tenderness in cattle, especially for Longissimus muscle although this relationship may be not true for other muscle types or not always valid with data across muscle types (for a review, see ). However, the observation that oxidative fibers are associated with tender beef has been confirmed in our study by a higher expression of the HADHB known to be mainly expressed in oxidative fibers and also in tender beef in our case.
In contrast to the situation with HSP genes, we found no subset of genes related to fat metabolism or muscle fiber metabolism which were significant across environmental conditions and genders to predict beef tenderness. In some ways, this observation confirms results recently obtained by de Jager et al.  who observed differences in expression of the lipid storage genes between tough and tender genotypes, but also observed large differences in the expression of genes involved in fat and energy metabolism across two different experimental sites in Australia independently of genotype. A likely explanation is that these gene expression changes are reflecting differences in metabolic status of the animals such as turnover rates of nutrients (fatty-acid precursors such as glucose, triglycerides and fatty acids) or mitochondrial activity, especially considering nutritional regimens, feed density, ambient temperature, slaughtering conditions, etc between the different experimental sites or the different years of slaughtering.