Intramuscular fat (IMF), also known as marbling, is one of the most important meat quality traits affecting quality grading, carcass price, and sensory acceptance of meat (Corbin et al., 2015; Silva et al., 2019). Thus, several studies have been conducted to better understand the pathways involved in IMF deposition in order to understand how to manipulate this desirable trait (Campos et al., 2016). Feeding regime impacts animal growth rate and has been related to changes in lipid metabolism, which directly impacts subcutaneous fat and IMF composition (Mwangi et al., 2019; Wicks et al., 2019), and consequently impacts beef color and tenderness (Gagaoua et al., 2018; Hughes et al., 2020; Valenzuela et al., 2020).

Feedlot-fed animals are commonly subjected to a high-energy diet, which provides greater substrate availability that leads to increased propionate in the rumen and therefore glucose production, which is related to increased muscle and fat deposition (Ladeira et al., 2016; Wicks et al., 2019). In contrast, nutrient intakes are often below animal requirements for maximal growth in a pasture system, which affects animal growth and lean tissue and fat deposition (Mwangi et al., 2019; Wicks et al., 2019). McIntyre et al. (2009) observed that grain-fed animals that experienced a high growth rate (1.34 kg/d) had higher backfat thickness and greater beef marbling scores than grass-fed animals with a low growth rate (0.63 kg/d). Moreover, Koch et al. (2019) reported that lighter (higher L* values) steaks from grain-fed cattle result from higher marbling scores and lipid content of the muscle, which were also associated with more tender beef (Jung et al., 2015). Gómez et al. (2021) indicated that muscle and fat deposition is predominantly due to the finishing regime, and to a lesser extent to growth rate, although the effects of these factors on carcass and beef traits may be cumulative.

Finishing regime (grain vs. forage) and growth rate are often confounded because cattle fed in intensive feeding programs tend to grow at faster rates. Thus, muscle from cattle reared under different finishing systems may have divergent metabolic signatures that are reflective of their inherent differences in biochemical processes, which may impact their subsequent transformation into high-quality beef (Gagaoua et al., 2017). Therefore, in this research, the muscle lipid profiles from cattle differing in growth rate and feeding regime were compared using multiple reaction monitoring (MRM) profiling—a mass spectrometry–based method for small molecule profiling that allows for accelerated discovery of a large number of discriminating molecular features (Xie et al., 2021)—and correlated to beef color and tenderness.

All experimental procedures involving animal care were conducted in accordance with the Institutional Animal Care and Use Committee Guidelines of the College of Animal Science and Food Engineering at the University of Sao Paulo (protocol approval number 13.1.541.74.0).

Intramuscular fat mostly consists of structural lipids, phospholipids, and TG (Listrat et al., 2016), which is the result of the intake of fatty acids through the diet, de novo fatty acid biosynthesis, TG formation, and TG degradation (Nürnberg et al., 1999). IMF content is mainly affected by cattle breed, diet composition, and animal’s age (Nürnberg et al., 1999; Mwangi et al., 2019; Wicks et al., 2019) but also depends on the muscle growth rate (Hocquette et al., 2009). In the present study, feeding regime and growth rate impacted IMF deposition, mainly because F-H animals had higher IMF deposition than P-L animals (Table 1). However, under a similar growth rate, F-L and P-H animals had similar IMF deposition, suggesting that diet was the main driver of IMF deposition. Koch et al. (2019) also reported a higher IMF deposition in animals fed high-concentrate diets (higher ADG) when compared with those fed high-quality forages (lower ADG). This is most likely due to increases in propionate from starch-based diets, which is the primary lipid precursor in intramuscular adipose tissue, compared with acetate from a grass-based diet (Smith and Crouse, 1984). This may also help explain the higher IMF deposition in feedlot animals fed ad libitum because there would be even greater amounts of propionate compared with those fed in feedlots to attain slower growth rates comparable to P-L cattle. Therefore, feeding regime or dietary component (grain vs. grass) was the main factor altering IMF deposition.

In this study, F-H steaks had greater total TG deposition than F-L and P-L animals, whereas P-H steaks had greater TG deposition than P-L cattle. In addition, F-H steaks had a higher amount of unsaturated TG, which was mainly due to TG with 3 double bonds compared with other treatments (Table 2). These differences in TG profile may be partially explained by the different requirements of TG mobilization in order to release fatty acids so that they are oxidized in the mitochondrial matrix as a source of energy for animal growth, which may be supported by the impact of treatments on mitochondrial beta-oxidation of the long-chain fatty acids pathway. Ladeira et al. (2016, 2018) reported that higher levels of glycerol in the muscle, as suggested in F-H animals in the present study, may be indicative of TG hydrolysis, which may support mitochondrial oxidation as lower levels of free carnitine may reflect utilization for long-chain fatty acid transport into the mitochondria. In the present work, F-H animals had higher hexadecanedioic acid mono-L-carnitine ester and stearoylcarnitine concentration than P-L animals (Supporting Information Table S2), which aid in the mechanism whereby long-chain fatty acids are transferred from the cytosol to the mitochondrial matrix to undergo beta-oxidation. Moreover, although F-L animals had greater concentrations of 9 different AC (including elaidic carnitine, O-oleoylcarnitine, palmitoylcarnitine, and cervonyl carnitine) than P-H animals, they had similar TG deposition and profile, except for TG with 1 and 3 unsaturations, which may be attributed to differences in the feeding regime. Therefore, these results may indicate that growth rate had the greatest impact at altering TG deposition and TG profile.

In addition to the neutral lipids primarily consisting of TG, IMF is also composed by polar lipids, containing mostly phospholipids (Legako et al., 2015). Scollan et al. (2014) reported that the overall fat content of the animal and muscle has an important impact on proportionate fatty acid composition because of the different fatty acid composition of TG and phospholipids. Wood et al. (2004) stated that cattle with lower levels of IMF are expected to produce meat with a higher amount of unsaturated fatty acids, which are restricted almost exclusively to the phospholipid fraction. Moreover, Bressan et al. (2016) reported that the IMF profile largely depends on the finishing system, in which grain-fed animals have more saturated fatty acids whereas grass-fed animals have more unsaturated fatty acids. In the present study, despite the higher deposition of IMF and unsaturated TG in F-H steaks compared with other treatments, feeding strategies did not change the concentration of phospholipids or total phospholipid deposited in the lean. However, phospholipid profiles within classification were impacted, in that P-L steaks had greater amounts of most PC, PE, and SM compared with F-H steaks whereas P-H beef had more PC, PE, and SM compared with F-L steaks (Table 3). These data may be explained by changes in the IMF composition caused by the impact of the treatments on the glycerolipid metabolism pathway (Figure 5). Moreover, PC, PE, and SM did not present a clear abundance direction when comparing growth rate within the feeding regime (F-H vs. F-L and P-H vs. P-L). Differences in phospholipid profiles across treatments may be partially explained by the impact of the treatments on the phospholipid biosynthesis and sphingolipid metabolism pathways (Figure 5). Therefore, these results may indicate that feeding regime (grain vs. pasture) seemed to be the main factor that altered phospholipid profile.

Some MRM from the PC, PE, SM, and AC classes were positively correlated with beef lightness (L*) (Supporting Information Table S6). Specifically, SM(d18:1/24:1(15Z)) was the most correlated MRM with L*, in addition to being one of the top 25 MRM clustered to distinguish F-H versus P-L and F-H versus F-L (Figure 1B and 3B, respectively). Interestingly, although SM(d18:1/24:1(15Z)) content was 1.07- and 1.14-fold higher in F-L and P-L when compared with F-H, respectively, lower L* values were observed in F-L and P-L steaks. The SM(d18:1/24:1(15Z)) is a type of sphingolipid found in animal cell membranes that helps prevent damage to the cell structure (Gault et al., 2010). Hughes et al. (2020) reported that the lipid component of cell membranous structures is believed to be partially responsible for light scattering; thus, microstructural components in muscle cells dictate light scattering and beef lightness. The results of this study suggest that modifications to the cell structure (such as the fluidity of membrane increasing the permeability), mainly in F-H steaks when compared with other treatments, contributes to the light scattering process, in which light is diffused or deflected by collisions with particles of the medium that it transverses. Similarly, Koch et al. (2019) observed lighter steaks from feedlot-fed cattle with higher marbling scores and lipid content within the muscle, because greater IMF deposition alters the muscle structure (Valenzuela et al., 2020). Moreover, Bate-Smith (1948) reported that light scattering is also partially dependent on the texture of the meat surface, which is in agreement with the WBSF data found in this study, where pasture-fed animals produced less tender meat and lower L* values than feedlot-fed animals.

In the present study, several TG, AC, and PG were correlated with a* (redness) (Supporting Information Table S7), in addition to being among the top 25 MRM clustered, in their subset lipid, to distinguish between F-H and P-L (Figure 1). The direction of the correlation was observed to be conditional to the MRM concentration in the F-H and P-L groups but not in the F-L and P-H groups, in which the higher concentration in the F-H group compared with the P-L group contributed to a greater correlation. Overall, most of the TG and AC were negatively correlated with a*, which may suggest that the accumulation of compounds by oxidation of unsaturated fatty acids and meat phospholipids is correlated with myoglobin oxidation in fresh beef (Faustman et al., 2010). Ramanathan et al. (2020) reported that changes in oxymyoglobin and a* values appeared to be driven by the oxidation of unsaturated fatty acids in phospholipids and TG. Specifically, in this study, PG(34:1) and PG(36:2) were the most positively correlated MRM with redness, which is in good agreement with their greater abundance in F-H steaks compared with P-L steaks (Supporting Information Table S2). PG(34:1) and PG(36:2) are precursors of cardiolipins, which are plentiful in the intermembrane of mitochondria and suggestive of increased mitochondrial activity (Chen et al., 2018) and presumably critical in achieving and maintaining a bright cherry-red in fresh beef (Ramanathan and Mancini, 2018). Therefore, changes in TG, AC, and PG profiles promoted by increased growth rate may contribute to increased redness in F-H steaks mainly through greater unsaturated fatty acid oxidation and a corresponding increase in mitochondrial activity.

Several TG, PC, and PE were positively correlated with WBSF (Supporting Information Table S8), in addition to being among the top 25 MRM clustered, in their subset lipid, to distinguish between F-H and P-L and between F-L and P-H (Figures 1 and 2, respectively). The WBSF correlation was observed to be conditional to feeding regime, in that the higher the TG, PC, and PE concentration in pasture-fed animals compared with feedlot-fed animals, the higher the correlation observed. Specifically, PE(18:2) was the most positively correlated MRM with WBSF, which belongs to the PE class that is implicated in cellular apoptosis via mitochondrial permeability transition that was initiated by reactive oxygen species (Kaku et al., 2015). Oak et al. (2000) reported that PE degradation generates products that accelerate membrane lipid peroxidation, causing oxidative stress to cells, which positively affects the development of tenderness (Gagaoua et al., 2015). These authors hypothesized that PC hydrolysis at the first stage of postmortem apoptosis may invert membrane polarity (which will induce changes in membrane fluidity, thus increasing the permeability to ions such as Ca²⁺ and increasing the μ-calpain activity) and increase of beef tenderness. The results of the present study may suggest that steaks from feedlot-fed animals underwent higher postmortem degradation of PC and PE than pasture-fed animals. Therefore, although PC and PE concentration were not altered (Table 3), their profiles were modified by feeding regime and contributed to the tenderization of beef.

There are several possible explanations for the positive effect of lipids on tenderness, including the presence of TG within the perimysium in fat cells, which might have a physical effect in the process of tenderization by separating muscle fiber bundles and opening the muscle structure (Wood et al., 2004). Ouali et al. (2013) suggested more active participation of lipids in the tenderizing process through contributing to energy production in the first hours after slaughter, mainly via AMP-activated protein kinase (Scheffler and Gerrard, 2007). Polati et al. (2012) reported an increase of β-hydroxyacyl CoA-dehydrogenase, a member of the beta-oxidation of lipids, which is indicative of TG degradation and may be used as a biomarker of beef tenderness. Therefore, the main reason for the changes in TG and phospholipid profiles (which were caused by the feeding regime) contributing to beef tenderness is not completely clear, but results suggest that TG and phospholipid profiles—and their degradation and signaling—contribute to the development of tender beef.

This study elucidated the effects of feeding regime and growth rate on beef color and tenderness, as well as on the profile of muscle lipids, using a lipidomics approach based on MRM profiling. Lipid content and profile differed from feeding strategies, which were related to L*, a*, and tenderness. Overall, results indicate that feeding regime is the main factor that is responsible for altering IMF deposition and phospholipid profiles, which contribute to the development of beef lightness and tenderness. Moreover, growth rate was the main factor that affected TG deposition and profile, which was positively correlated with the redness of the beef. New insights were elucidated on the effects of feeding strategies on lipid profile in beef at class and molecular levels. These findings provide a comprehensive and in-depth understanding of lipidomic profiling of beef cattle finished under different feeding strategies and provide a basis for the relationship between lipid content and profiles and beef quality development.