Meat quality traits in Brazilian animal breeding programs have not been fully explored because of the late expression of these attributes and the complex evaluation that can only be made after slaughter. Furthermore, on the domestic market, producers are generally not paid for meat quality, a fact that diminishes interest in improving meat quality traits and has hindered their inclusion in traditional selection objectives. In contrast, on international markets, meat tenderness is one of the most valued traits [39], a fact that highlights the importance towards improving this trait since Brazil is one of the world’s largest beef exporters.

Meat tenderness is the preferred sensory attribute of consumers [7]. According to Scollan et al. [46], the European food industry has sought to improve this trait to gain market share over other types of food. In Brazil, about 80% of the cattle herd consists of Zebu animals or their crossbreeds [1]. In this respect, the improvement of meat tenderness becomes important since Ferguson et al. [17] have shown that the higher the proportion of Zebu genes in a population, mainly Nellore cattle, the less tender the meat. Meat tenderness can only be measured after slaughter making this trait more complex to select animals. Thus, alternative tools are useful to include meat tenderness in animal breeding programs [9].

Modern recently developed large-scale RNA sequencing technologies (RNA-Seq) have been useful in understanding the genetic and physiological processes that regulate the phenotype of quantitative traits in a certain situation [34]. RNA-Seq permits analysis of the transcriptional profiles of cells, tissues or organs in a certain situation and the discovery of known and unknown genes involved in a given cellular process [57]. This new technique can be used to identify novel potential molecular markers that permit more accurate and early genetic predictions [51], with a consequent reduction in the generation interval that would contribute to the improvement of difficult-to-measure traits such as meat tenderness.

RNA-Seq has been widely used in recent studies to investigate differentially expressed genes related to meat tenderness in different species. For example, genes related to the degradation of filamins, lipogenesis and collagen synthesis have been identified in a study on meat tenderness in broiler chickens [40]. Gonçalves [20] found genes related to metabolic pathways involved in apoptosis, calcium transport, proteolysis and ribosome synthesis in castrated Nellore cattle, classified as extreme for tenderness based on estimated breeding values for shear force measured after 14 days of aging. Bongiorni et al. [8], who studied gene expression in longissimus dorsi muscle of animals of two Italian beef breeds (Maremmana and Chianina) representing the extremes for meat tenderness, detected differentially expressed genes related to growth and sodium-potassium pumps, among others.

Despite the above-mentioned publications, studies investigating differentially expressed genes related to meat tenderness in cattle are rare. In this respect, the better understanding and identification of the transcripts and biological processes, associated with this complex and economically important trait, will permit to highlight genes that could contain potential biomarkers involved in meat tenderness.

The objective of this study was to identify genes differentially expressed in muscle tissue (longissimus dorsi) of Nellore cattle with divergent meat tenderness using RNA-Seq in order to obtain data that increase our understanding of the genetic and metabolic mechanisms underlying this trait.

A higher proportion of Zebu genes in cattle herds considerably reduces meat tenderness when compared to taurine breeds. In Brazil, the herd mostly consists in Zebu cattle, mainly Nellore, then improve meat tenderness is very important, because for the beef export market, in which Brazil plays an important role, tenderness is paramount in determining the value of the product.

Gene expression studies have been used as a tool to identify gene candidates and metabolic pathways related to traits of economic interest. In the present study, the USP32 (ubiquitin specific peptidase 32) transcript was expressed more in tender meat. Members of the ubiquitin-proteasome system are important during the transformation of muscle into meat. These proteins are involved in proteolysis, causing the degradation of myofibrillar proteins in muscle cells [47].

In a genome-wide association study (GWAS) of Nellore cattle using different meat tenderness measures, Tizioto et al. [52] identified genes of the USP family, including USP32. Another study on cattle also associated genes of the USP family with meat tenderness. In Wagiu cattle, the USP2 gene was strongly associated with meat tenderness [12] and gene expression analysis in Nellore cattle showed that the USP2 gene was expressed more in tender meat samples [20].

The functional categories cell junction, regulation of cell communication and intrinsic component of membrane are related to the binding, communication and transport of molecules between cells [10]. Among the transcripts related to these categories, CTNNB1 (catenin - cadherin-associated protein beta 1), which was expressed more in tender meat, is involved in the same metabolic pathway as actin and myosin. Actin and myosin are the proteins found in thin and thick myofilaments, respectively, which form the myofibril that is responsible for muscle contraction. These proteins are the most abundant in the mechanism of muscle contraction, accounting for 52 to 56% of all muscle proteins [48].

Each actin filament binds to the plasma membrane of the cell through a structure, called focal contact. This structure consists of binding proteins and of a transmembrane protein that are products of the “focal adhesion” pathway to which the CTNNB1 and TCF7L1 (transcription factor 7 like 1) genes belong. On the outer side of the cell, in the extracellular matrix, the transmembrane protein binds to a collagen fiber [14, 23]. According to Bailey [2], a direct association exists between collagen content and the toughening of meat. However, in the present study, the CTNNB1 and TCF7L1 transcripts were expressed more in tender meat.

The SYP (synaptophysin) transcript, which was expressed more in tender meat, encodes an integral membrane protein found in small synaptic vesicles. In a study on rats, [44] showed that the phosphorylation of synaptophysin is calcium dependent. The authors observed a four-fold increase in serine phosphorylation of synaptophysin in the presence of the calmodulin-calcium complex. According to Bailey et al. [2], serine proteases are responsible for the degradation of collagen, which, in turn, directly influences meat tenderness. In addition, calcium is essential for muscle contraction by acting as a catalyst of enzymatic proteolytic activity, which is directly related to the process of meat tenderization [37].

The AT2 transcript, which encodes angiotensin II, was expressed more in tender meat. This protein is involved in vasoconstriction and regulates the secretion of aldosterone, which, in turn, stimulates the reabsorption of sodium by the kidneys. In this respect, after slaughter and during bleeding, angiotensin is activated to restore blood pressure. The result of these stimuli is the depolarization of the cell membrane, altering the distribution of sodium and potassium, in addition to permitting the flow of calcium ions [43]. In a study on crossbred cattle (Luxi-Simmental), Zhong-Liang et al. [60] observed a decline in shear force after the injection of angiotensin II into the carcass for 7 days after slaughter. Bongiorni et al. [8], studying gene expression in longissimus dorsi muscle of Italian Maremmana and Chianina breeds, also found the differential expression of genes to be related to sodium and potassium flow.

The functional category “catalytic activity” is related to increases in the velocity of a biochemical reaction at physiological temperatures [10]. Some reactions that occur during the postmortem period depend on calcium and cellular pH, which decrease in the first 24 h after slaughter [25]. A member of this functional category is ASAH1 (N-acylsphingosine amidohydrolase (acid ceramidase) 1), which belongs to a family of hydrolases that catalyze the synthesis and degradation of ceramide into sphingolipid and free fatty acid and are acid pH dependent [32]. A genetic deficiency in ASAH1 that reduces its catalytic activity causes a lysosomal sphingolipid storage disorder characterized by the accumulation of lipids in cells and tissues throughout the organism [38]. ASAH1 also belongs to the “sphingolipid signaling pathway” and “sphingolipid metabolism” categories in which serine is also involved, with serine protease degrading collagen [2]. Thus, ASAH1, which was expressed more in tender meat, may be related to the process of meat tenderization.

Another member of the “catalytic activity” category is HMOX1 (heme oxygenase 1), which was expressed more in tough meat. This gene encodes a protein involved in the metabolism of porphyrins, molecules whose catalytic activity is activated by iron [35]. Porphyrins are precursors of hemes, the main components of hemoglobin, myoglobin and cytochromes which are responsible for the transport of oxygen and electrons in tissues [36].

The C-type lectin (CLEC) family comprises calcium-dependent carbohydrate-binding protein domains that are involved in cell-cell adhesion [15]. In the present study, the CLEC4G and CLEC12A transcripts were expressed more in tender meat. GWAS in Nellore cattle demonstrated an association of the CLEC12A gene with different meat tenderness measures [52].

The IQCG transcript (IQ motif containing G), which was expressed more in tender meat, encodes a protein that functions as a binding site for different proteins, including myosin light chains and calmodulins. Calmodulin phosphorylates myosin, a process that permits the sliding of fibers and muscle contraction. In this case, calcium present in the reaction, binds to calmodulin, attached to IQ motif, and stimulates the ATPase activity of myosin [42]. According to Duston [16], in addition to factors such as collagen content, the structure and state of contraction of myofibrils (which mainly consists of myosin and actin) directly affect meat tenderness.

The protein encoded by the PNP transcript (purine nucleoside phosphorylase), which was expressed more in tough meat, plays a role in nicotinate and nicotinamide metabolism. Nicotinate (niacin or vitamin B3) is a precursor of NAD⁺ and NADP⁺ coenzymes, which are essential for the production of ATP in the cell [28].

Numerous structural changes and biochemical events occur in the first 24 h after slaughter of the animal, which are responsible for the conversion of muscle into meat [25]. In the early postmortem stages, ATP levels are maintained constant by the conversion of ADP plus phosphocreatine into ATP and oxygen supply ceases because of the cessation of blood circulation. At this stage, slow production of lactate is observed and the onset of rigor mortis occurs (slow phase). The decrease in phosphocreatine levels characterizes the rapid phase, which consists of a rapid decline in available ATP that is used as an energy reserve after the consumption of glycogen and other carbohydrates and is therefore hydrolyzed again to ADP. The scarcity of ATP during this phase is accompanied by the release of calcium ions into the myofibrillar space, which causes muscle shortening with a direct influence on meat tenderness [5].

Another event that occurs during this phase is the anaerobic conversion of glycogen into glucose, producing lactate and reducing the pH of the medium. In addition, the transport of sodium and potassium across the cell membrane, which uses the energy released by the hydrolysis of ATP into ADP, is impaired because it occurs against the concentration gradient. The protons generated during the hydrolysis of ATP into ADP cause a significant decline in intracellular pH [3]. According to Darrel et al. [13], this drop in pH directly influences the final tenderness of meat, especially during the process of aging.

According to Koohmaraie et al. [26], calcium is responsible for the activation of calpains and calpastatins (calcium-dependent cysteine proteases) and calpain I has been shown to be the main enzyme responsible for postmortem tenderization of meat by degrading cytoskeletal proteins that confer the structural integrity of the myofibrillar matrix. Nevertheless, in the present study, the calpain and calpastatin genes were not differentially expressed in the tender and tough meat groups. This finding might be explained by the fact that the amount of calpastatin in cells is higher 24 h after slaughter [43] and in this study the samples were collected immediately after cleaning the carcasses. Other GWAS and gene expression studies of muscle tissue in Nellore cattle also found no relationship between meat tenderness and calpain or calpastatin [20, 52].

The EXOSC2 transcript, which encodes exosome component 2, was expressed more in tender meat. According to Jong et al. [22], this gene is related with collagen activity in humans. This found could indicated a relationship between this genes and collagen activity in bovines, because there is a direct association exists between collagen content and the toughening of meat [2].

The ZKSCAN2 transcript (zinc finger with KRAB and SCAN domains 2), which was expressed more in tender meat, is vertebrate specific and synthesizes zinc finger proteins that bind through an N-terminus to the SCAN domain (dimerization motif). The function of this gene is not well known, but zinc finger proteins have been associated with the regulation of growth factor transcription and lipid metabolism [45].

In cattle, the main histocompatibility complex class II is called BoLA-DQB (bovine leukocyte antigen) [24]. In the present study, the BoLA-DQB transcript was expressed more in tough meat. We found no studies investigating the association of this gene with meat tenderness. However, this gene has been associated with growth traits in Holstein and beef cattle (Angus, Charolais, Hereford, Limousin, Simmental); [4, 49] and, according to Koohmaraie et al. [27], animals with higher growth rates have more palatable and more tender meat.

When we compared this study with a GWAS study for meat tenderness using the same Nellore population, we do not found common genes between them, but there were some shared functions related to phosphorylation and catalytic activity [33]. These functions are related with oxygen and calcium transport, and collagen degradation, important processes for the the toughening of meat, especially after slaughter. In a GWAS study using another Nellore cattle population, Tizioto et al. [52] identified regions that influence tenderness at three different time points (24 h and 7 and 14 days after slaughter). Some of the genes reported by these authors were also identified in the present study, such as CLDN19, CLEC12A and USP32. In addition to these genes, the authors reported an association of genes belonging to the family of BoLA-BQD, CTNNB1, EXOSC2 and IQCG transcripts and meat tenderness.

Global gene expression analysis in animals phenotypically divergent for meat tenderness identified genes related to ubiquitin metabolism, transport of molecules such as calcium and oxygen, acid-base balance, collagen synthesis, actin and myosin, and fat accumulation. These results contribute to the understanding of the molecular mechanisms involved in the meat tenderization process, at the time of slaughter, and to the development of strategies to select animals with more tender meat.