There is a general agreement in the literature that intramuscular fat content increases flavor and juiciness (Corbin et al., 2015; Emerson et al., 2013; O’Quinn et al., 2012; Savell et al., 1987; Smith et al., 1985; Dubost et al. 2013; and Hocquette et al., 2010). Du, Yin, and Zhu (2010) referred to the work of Hausman et al. (2009) and Tong et al. (2008) in saying that “intramuscular fat is crucial for meat palatability.” However, several studies have failed to show a relationship. Data extracted from the French INRA BIF-beef database (Integrated and Functional Biology of Beef) contained 331,153 measurements including more than 15,764 measurements related to animal growth of which 621 variables were observed across 5 muscle types from 5,197 animals that were 1–120 months of age belonging to 20 different breeds and from 43 different experiments. Chriki et al. (2012b) showed that the relationship between intramuscular fat content and flavor was low (partial correlation coefficient of 0.11) but statistically significant especially for Charolais and Limousin young bulls. The correlation was not significant for fatter animals such as steers or females or young bulls from lean breeds (such as Blonde d’Aquitaine) (Hocquette et al., 2011b).
Renand, Havy, and Turin (2002) reported similar findings showing that flavor was not correlated with an intramuscular fat level in young bulls from lean French breeds (1.2% intramuscular fat level) compared to fatter French breeds. The absence of relationship in these studies may be a result of low variation within each study for either IMF or flavor or from the fact that there is a curvilinear relationship between flavor and intramuscular fat level. Whereas Thompson (2004) found that 16% of the variation in flavor can be explained by differences in intramuscular fat in a large data set has many variations (from 0.3 to 15% IMF), Hocquette et al. (2011b) reported that only 3% of the variation in flavor could be explained by differences in IMF with the BIF dataset characterized by little variation and small absolute values. McKeith et al., 1985, and Legako et al. (2015) found that percent fat was correlated with overall palatability, tenderness, juiciness, and flavor, but that n-aldehydes were negatively correlated with palatability and percent fat possibly because of the retention of volatile compounds by fat, delaying flavor release (Chevance et al., 2000; and Chevance and Farmer, 1999).
A lower total fat content is associated with a more significant proportion of the fat that is polar (Wood et al., 2008) and more susceptible to oxidation (Mottram, 1998). Maillard products are closely associated with flavor development (Mottram, 1998; and Legako et al., 2015) especially the sulfur-compounds even though they are fat-soluble, and thus, their rate of release is delayed resulting in flavor being a latent sensation for fatty beef (Legako et al., 2015). Corbin et al. (2015) reported that consumer flavor liking scores increased with increased fat percentage. They found that grain-fed cattle grading USDA Select and Standard produced beef with lower consumer flavor liking scores than beef having higher levels of fat due largely to less robust desirable flavors, especially fat-like and umami flavors, and not to the presence of undesirable flavors. Corbin et al. (2015) concluded that the fat level was the primary driver of beef flavor acceptability when no undesirable off-flavors were present (for cattle finished on grain).
Duarte et al. (2013) reported that the IMF content and the numbers of preadipocytes and adipocytes were higher in Wagyu than Angus. Gotoh et al. (2009) reported the IMF content in the longissimus for 24 mo. old Wagyu to be 23.3%; for German Angus, 4.4%; for Belgian Blue, 0.6%; and for Holstein Friesian, 4.7%. Japanese Black and the European cattle breeds do not differ as to the mechanism of postnatal fat accretion [a combination of an increase in adipocyte number (hyperplasia) and adipocyte size (hypertrophy)]. Still, they differ in their efficiency of accumulation of IMF (Motoyama, Sasaki, and Watanabe, 2016). Every 1% increase of longissimus IMF required an increase of 3.0 kg of subcutaneous adipose tissue in Wagyu, 4.3 kg in Holstein Friesian, 7.9 kg in German Angus, and 10.7 kg in Belgian Blue (Gotoh et al., 2009). Shirouchi et al. (2014) reported that Perilipin 1 and adipose differentiation-related protein mRNA levels were higher in the IMF of Wagyu than for Holstein suggesting that Wagyu were more advanced in the maturity of IMF cells.
Evolution of Marbling in Wagyu
It is instructive to study the Wagyu breed to understand marbling—its evolution, history, and utility as a basis for branding. Wagyu (“Wa,” meaning “Japanese” and “gyu” meaning “cattle”) originated from native Japanese breeds which evolved by adapting to the Japanese climate and environment (Motoyama, Sasaki, and Watanabe, 2016). This climate is characterized by harsh winters when forage is withered or covered with deep snow, creating opportunities for vitamin A deficiencies. Since vitamin A is fat-soluble, it is stored in adipose tissues. Therefore, the need to store large quantities of vitamin A to prevent winter-time deficiency may have contributed to the need for large depots of fat (Motoyama, Sasaki, and Watanabe, 2016). Since the climate also is characterized by long, warm, humid summers during which the animals were used to pull plows in rice patties, there was an adaptive advantage for small subcutaneous fat depots to allow for body heat dissipation. So, large intramuscular fat depots were evolutionarily significant to provide readily accessible sources of quick energy required to pull plows through water and storage reservoirs for vitamin A that did not impede heat dissipation (Hirooka, 2014).
At one time, it was believed that marbling could be enhanced only by extending the grain feeding period (Hausman et al., 2009), Now, however, it is believed that intramuscular fat can be enhanced through either the enlargement of existing adipocytes (hypertrophy) or by increasing the number of adipocytes (hyperplasia) (Du et al., 2010b). Most research on intramuscular fat has focused on the conversion of preadipocytes to adipocytes, adipocyte lipid metabolism, and hypertrophy through nutritional management (Hausman et al., 2009; and Smith et al., 2009a,b). There is less information about mechanisms regulating the initial stage of adipogenesis and the conversion from a multipotent mesenchymal stem cell to preadipocytes (Du, Yin and Zhu, 2010). An understanding of these mechanisms is required for the development of production systems designed to enhance (or inhibit) marbling. Both muscle cells and adipocytes (fat cells) are derived from mesenchymal stem cells (MSCs) (Du, Yin, and Zhu, 2010).
MSCs are abundant in the skeletal muscle at early developmental stages, especially during the fetal and neonatal stages, but are less prevalent in older animals. Most MSCs develop into myogenic cells, but a small portion of MSCs morph into adipocytes which mature into intramuscular fat deposits called marbling (Du et al., in 2010b). Adipogenesis is regulated by genetics, nutrition, and certain environmental factors, each having roles dictating key signaling pathways regulating adipogenesis in skeletal muscle and, thus, marbling (Harper and Pethick, 2004). As compared to European cattle, Japanese black (and red) cattle have high levels of marbling, which appears to result primarily from an increase in the number of intramuscular adipocytes, though an increase in adipocyte size has also been detected (Gotoh et al., 2009). The relationship between utero adipogenesis and marbling has been the subject of several reviews (Fernyhough et al. 2007; Hausman et al., 2009; Hocquette et al. 2010; Smith et al., 2009; and Du, Yin and Zhu, 2010).
For bovine, primary muscle fibers form within the first two months of gestation (Russell and Oteruelo, 1981). Most muscle fibers are formed during the secondary myogenesis between 2 and 7 months of gestation (Russell and Oteruelo, 1981). Maternal nutrient fluctuation during late gestation reduces adipogenesis and reduces muscle fiber size although not fiber number (Du et al., 2010a; and Greenwood et al., 1999). Late gestation is the critical period for intramuscular adipogenesis. Adipogenesis begins during mid-gestation (Feve, 2005; Gnanalingham et al., 2005; and Muhlhausler, Duffield, and McMillen, 2006) partially overlapping the period of secondary myogenesis (Du and Zhu, 2009). Maternal nutritional management during mid- to late gestation can enhance the number of MSCs committed to adipogenesis, increasing the number of intramuscular adipocytes and enhancing the potential for marbling. Matrix metalloproteinases (MMPs) have important cell-signaling and gene regulatory roles during animal growth that impacts meat quality (Christensen and Purslow, 2016).
MMPs are involved in the remodeling of intramuscular connective tissue increasing muscle fiber size during post-natal growth. MMP’s also increase the turnover rate of connective tissue which, in turn, suppresses the development of mature cross-links that are associated with tougher meat. MMPs influence adipocyte maturation and their lipid profiles and in conjunction with their influence on the development of intramuscular connective tissue, can result in lower intramuscular fat (Du et al., 2010a,b). MMP’s play an important role in the differentiation of mesenchymal progenitor cells toward myogenic, adipogenic, or fibrogenic cells (Christensen and Purslow, 2016). Thus, the manipulation of MMP’s provides the opportunity to alter progenitor cell differentiation and thus effect a fundamental change in the proportion of tissue that is a muscle, fat, and connective tissue with a profound effect on eating quality (Du et al., 2013).
Less fibrogenic differentiation may reduce intramuscular connective tissue deposition, thereby decreasing the background toughness of beef (Christensen and Purslow, 2016). Du et al. (2015) hypothesized that maternal nutrition during fetal development might be a form of “fetal programming” that can be altered to improve meat production (Du et al., 2015). Since single nucleotide polymorphisms for MMPs and tissue inhibitors of MMP’s are well-documented for beef cattle, these possibilities alter not only the connective tissue, muscle fiber, and intramuscular fat content of beef but also the fatty acid profile (Christensen and Purslow, 2016). Early research indicated that the fetus had precedence over the dam for nutrients (Hammond, 1943). Subsequently, however, it was shown that fetal development can be negatively impacted in the case of prolonged or severe restriction of maternal nutrition in that maternal undernutrition during pregnancy may result in offspring developing a “thrifty phenotype” that is more prepared to deal with sparse nutrient availability (Barker, 1995).
Animal Growth Rate
Intramuscular fat accretion can be altered through nutritional management throughout the growth phase of an animal (Faulkner et al., 1994; and Bruns et al., 2004), including the backgrounding period prior to finishing (Anderson and Gleghorn, 2007). Thus, nutritional management can alter the distribution of fat among the various depots. One reason for this is that intramuscular adipocytes preferentially use glucose as a substrate for fatty acid synthesis, whereas subcutaneous fat uses acetate (Smith and Crouse, 1984). Ruminant animals have long been known to exist in a chronic near-diabetic state since the products absorbed from rumen fermentation are primarily fatty acids and not sugars (Church, 1988). Ruminants, however, must have some metabolic glucose to function. Since little glucose is absorbed, most of the glucose is acquired by gluconeogenesis.
Starch fermentation, as compared to fiber fermentation, in the rumen increases propionate production while decreasing acetate production. Since propionate is gluconeogenic and acetate is not, glucose supply to the animal is increased (Church, 1988). Thus, high-starch corn supplements during growing periods when the animal is primarily consuming forage may enhance marbling development. Nascimento et al. (2016) reported that efficient [low residual feed intake (RFI)] Nellore steers had 11.8% less intramuscular fat than inefficient steers (high RFI), indicating that selection based on RFI may adversely impact beef quality by decreasing marbling. These authors also found that low RFI steers had greater WBSF at 0 d aging.
Some evidence exists that dietary intervention of young calves (prior to average weaning age) can increase marbling when animals are slaughtered at the usual weight and age (Scheffler et al., 2014) These researchers stratified fall-born, Angus-sired steer calves by sire and randomly assigned them to normal weaned or metabolic-imprinted treatments. At 105 ± 6d (135kg) of age, metabolic-imprinted calves were transitioned to a diet containing 20% CP and 1.26 Mcal/kg NEg fed ad libitum until they were 253 d of age. Normal weaned calves were weaned at 253 ± 6 d of age without supplement. At this time, all cattle were grazed together for 156 d on mixed summer pasture and then fed a corn silage-based feed-yard diet until harvested at 1.0 to 1.2 cm backfat. metabolic-imprinted calves were heavier when normal weaned calves were weaned (341 vs. 265 ± 4.2 kg).
During the grazing phase, usually weaned steers gained more weight than metabolic-imprinted steers (0.69 vs. 0.35 ± 0.03 kg/d) although the two groups were similar in feed-yard performance and USDA yield grade. Metabolic-imprinted steers produced heavier carcasses (564 vs. 524 ± 5.6 kg) with higher marbling scores (645 vs. 517 ± 23) (Scheffler et al., 2014). Because there were only 12 animals/treatment, this is considered poor evidence. Yet, it provides some indication that short-term feeding of propiogenic (high grain feed) before standard weaning time can increase marbling in beef.
In contrast to corn, by-product feeds such as distiller’s grains plus solubles and soybean hulls are low-starch energy supplements that provide the animal energy by providing highly digestible fiber and fat. Lake et al. (1974) and Lomas et al. (2009) reported that increasing starch supplementation to grazing stocker cattle increased the ultimate marbling score. The effects of grain content of growing diets and grain supplementation to postweaning grazing cattle on intramuscular fat development have received considerable attention; however, results are variable (Choat et al., 2003; Bumpus et al., 2005). This variation in results may be due to the level of total energy intake or the stage of maturity of animals during the stocker phase. Lake et al. (1974), Greenquist et al. (2009), and Lomas et al. (2009) reported that energy supplementation adequate to increase the postweaning gain from 0.60 to 0.83, 0.67 to 0.92, and 0.74 to 0.88 kg/d, respectively, ultimately improved the finished animal’s USDA marbling scores. However, energy supplementation that increased ADG of pastured cattle from 0.20 to 0.54 kg/d has been reported not to affect final USDA marbling scores (Sharman et al., 2013a,b).
Horn et al. (1995), Griffin et al. (2010), and Islas et al. (2010) also reported that the final USDA marbling score was not altered when supplementation increased gain for grazing animals from 0.88 to 1.07, 0.89 to 1.18, and 1.09 to 1.25 kg/d, respectively. These results suggest that energy supplementation prior to fattening may influence IMF development only when supplementation increases the rate of gain above a certain threshold, possibly between 0.6 and 0.9 kg/d (Sharman et al., 2013a,b). Thus, if the gain of supplemented animals is less than this (Sharman et al., 2013a,b), or if the gain of non-supplemented steers is more significant than this (Horn et al., 1995), then energy supplementation has little effect on intramuscular fat accretion. However, as indicated by the results of Sainz et al. (1995) and Hersom et al. (2004a), who reported no difference in final marbling score, even though gain ranged from 0.69 to 1.96 kg/d and 0.15 to 1.31 kg/d, respectively, the system is complicated by other factors that have not yet been elucidated.
At least one other factor may involve being the relative level of maturity of the animal. Carter et al. (2002) reported that adipocyte development begins to accelerate at about 64% of mature weight. Perhaps the provision of an energy supplement during grazing to more mature animals results in a more significant impact on adipocyte development. Greenquist et al. (2009) and Lomas et al. (2009) reported that energy supplemented steers with a final grazing weight of 477 and 417 kg had more outstanding final marbling scores than non-supplemented steers with a final grazing bodyweight of 437 and 386 kg, respectively. In contrast, Elizalde et al. (1998), Gunter and Phillips (2001), Bumpus et al. (2005), and Griffin et al. (2010) observed no difference in final marbling scores when final grazing bodyweight of supplemented and non-supplemented steers was less than this. Coleman et al. (1995), Sainz et al. (1995), and McCurdy et al. (2010a, b) also observed that grain content in growing diets had no influence on final USDA marbling scores for steers less than 380 kg.
Sharman et al. (2013a,b) found that the amount and/or type of supplement had little impact on marbling accretion for steers wintered on dormant native range, even though energy supplementation increased gain during winter grazing. Energy supplemented steers did have greater kidney, heart, and pelvic fat and greater mesenteric and omental fat levels, possibly due to the increased expression of lipogenic and adipogenic genes in perirenal adipose tissue. Even though marbling is the last fat depot to mature in the growing beef animal (McPhee et al., 2008), pre-feed-yard nutritional management has been reported to influence intramuscular fat deposition (Anderson and Gleghorn, 2007). As stated above, the primary substrate for fatty acid synthesis in intramuscular adipocytes is glucose, whereas the primary substrate for fatty acid synthesis in subcutaneous fat is acetate (Smith and Crouse, 1984).
Cattle grazing wheat pasture have 70% lower acetate/propionate ratio compared with cattle grazing dormant native tallgrass range and, thus, have greater substrate availability for gluconeogenesis resulting in increased energy available for growth, and fat accretion (Choat et al., 2003; and Hersom et al., 2004a,b) including both accretion intramuscularly and subcutaneously (Sainz et al., 1995). Marbling score, however, increases linearly with body weight, whereas 12th-rib fat thickness increases curvilinearly (Sainz et al., 1995; Bruns et al., 2004; and Sharman et al. 2013a,b). Sharman et al. (2013a,b) inferred from this that only a moderate level of energy intake is required for lipid filling of intramuscular adipocytes and that subcutaneous fat is strongly influenced by energy intake and carcass weight, but unlike marbling score, increasingly greater energy intake will increase subcutaneous fatness even when the animals are harvested at similar carcass weight. However, changes in the partitioning of fat among depots during the stocker phase of the production system may not be fully reflected in the carcass after finishing when cattle are slaughtered at similar levels of subcutaneous fat thickness (Sharman et al., 2013a,b).
The current method used by the industry to increase marbling is to extend feeding periods, which is detrimental in terms of decreasing feed efficiency and rising cost of gain. Wagyu steers fed a high-concentrate diet for 20 months in Japan showed a higher expression of adipogenic transcription factors in the subcutaneous and intramuscular adipocytes than those fed on a high-roughage diet (Yamada and Nakanishi, 2012). The saturated fatty acids palmitic (16:0) and stearic acid (18:0) strongly promotes adipogenic gene expression in intramuscular preadipocytes (Choi et al., 2013). Conversely, the monounsaturated fatty acid oleic acid (18:1cis-9), the polyunsaturated fatty acid linoleic acid (18:2n-6), and α-linolenic acid (18:3n-3) depress adipogenic gene expression. Choi et al. (2013) concluded that palm oil supplementation in finishing diets promotes lipid synthesis in adipose tissue. Therefore, intramuscular fat levels without depressing feed efficiency or increasing the palmitic acid content of beef.
The ethanol industry coproducts, such as dried distillers grains with solubles (DDGS) are often used as feedstuffs in feed-yard cattle diets. However, DDGS often contain a high level of sulfur, potentially limiting the inclusion rate in cattle diets (Kwiatkowski et al., 2006). High dietary sulfur (>0.4%) has been documented to have a detrimental effect on DM intake, gain, carcass weight, and IMF (Gibson et al., 1988; and Richter et al., 2012), and may lead to the development of polioencephalomalacia. A high sulfur diet may impact IMF through its effect on Vitamin C; conversely, vitamin C supplementation of cattle on high sulfur diets may offset the depressing effect of sulfur on marbling. Vitamin C has many roles in cellular metabolism but, as concerning red meat quality, is directly involved in oxidation-reduction reactions as an enzyme cofactor and in the synthesis of collagen (Rebouche, 1991). Since cattle synthesize ascorbate from glucose, the American National Research Council (NRC, 1996) does not specify a dietary requirement for vitamin C.
A decrease in serum ascorbate has been reported to occur throughout the finishing period of Japanese Black cattle (Takahashi et al., 1999) and the inclusion of a rumen-protected vitamin C to the diet has been reported to improve marbling scores of Japanese Black cattle (Yano, 2001). Vitamin C enhanced the differentiation of preadipocytes to adipocytes in vitro (Torii et al., 1998) and should aid in marbling (Lee et al., 2000). Supplementation of vitamin C to cattle consuming high sulfur diets has been shown to improve marbling scores. In contrast, vitamin C, including low and medium sulfur treatments, had no impact on marbling (Pogge and Hansen, 2013). Koutsidis et al. (2008a,b) reported that reducing sugar concentration of beef (e.g., ribose) is increased for high concentrate finished cattle (Koutsidis et al., 2008a,b). These sugars then react with free amino acids to produce flavor through Maillard’s reaction. Elmore et al. (2000, 2005) also reported that the production of volatile flavor precursors could be enhanced through supplementation of fats in the animal diet during finishing which alters the fatty acid profile of animal fat (Elmore et al., 2000, 2005).
Animal Growth Path
Vestergaard et al. (2000) found that beef from young bulls reared on pasture (restrictive fed) was inferior in the robustness of beef flavor and eating quality to beef from ad libitum fed young bulls mostly if the restrictively fed bulls had not been finished on a high energy diet for ten weeks (in a compensatory growth system) prior to slaughter. They also found that sensory evaluation revealed that meat from restrictively fed young bulls had an undefined off-flavor characterized as metallic, liver, cod-oil, and grassy. Bowling et al. (1977, 1978) found that feeding for at least 100 d increased the robustness of beef flavor.
Duckett and Pratt (2014) reviewed the literature concerning the effect of anabolic implants on marbling and reported that a single estrogenic implant reduced the marbling score by about 4%. In contrast, re-implanting resulted in reductions in the order of 7.4% for estrogenic implants and 11.5% for estrogenic implants, followed by combination re-implant. A single combination implant reduced the marbling score by about 5% whereas two combination implants reduced the marbling score by 9%. Reductions in the marbling score corresponded with increases in the longissimus muscle area. Duckett and Andrae (2001) quantified this relationship: marbling score = –0.796 × percentage increase in longissimus area – 1.99; r2 = 0.68. Duckett et al. (1999) reported that implantation didn’t reduce the amount of marbling, just diluted it in more muscle resulting in lower marbling scores.
Anabolic implants of ewes have been reported to have little impact on:
1) in vitro lipolysis rates of adipocytes (Green et al., 1992);
2) the steady-state of mRNA of lipoprotein lipase (a lipogenic enzyme instrumental in catalyzing the fatty acid uptake from by tissues) in longissimus (Waylan et al., 2004);
3) subcutaneous or intramuscular adipocyte size or volume or;
4) mRNA levels of key lipogenic enzymes (i.e., acetyl-CoA carboxylase, stearoyl-CoA desaturase, or lipoprotein) in intramuscular fat (Smith et al., 2007).
Anabolic implants, however, have been reported to down-regulate genes coding for lipogenic enzymes (i.e., stearoyl-CoA desaturase, fatty acid synthase, and fatty acid elongase 6) in the fat of steers with low levels of fat having low-quality grades (USDA Select) but not in the fat of steers exhibiting higher levels of fat having higher quality grades (USDA Choice) (Duckett et al., 2011). Examination of the adipocyte transcriptome revealed that 36 genes were differentially expressed resulting from anabolic implantation (Duckett et al., 2012). However, over 95 genes were differentially expressed between subcutaneous and mesenteric depots. Duckett et al. (2012) concluded that, for anabolic implanted animals, the genes differentially expressed in the transcriptome were not lipogenic genes; instead, they were genes related to insulin resistance, cell cycle regulation, and adipogenic differentiation.
As the animal grows, marbling deposition proceeds in a nonlinear manner so that most deposition occurs after lean growth has matured (Duckett et al., 1993). At that time, dietary energy is no longer needed for skeletal and muscular development and becomes available for adipocyte deposition. Therefore, implant schemes that stimulate lean growth in the middle to end of the finishing period appear to have the most significant influence on reducing marbling scores, possibly limiting the available energy for marbling deposition (Duckett and Pratt, 2014).
Given a variety of human health problems, the availability of high quality protein and other nutrients ensures that beef is now one of the most prevalent meat products in a wide section of society. The intramuscular fat levels and structure differ according to diet and breeding. Marbling is not only more complex than the production of subcutaneous fat and marbling, but it also contributes to a better quality of beef consumption. It is important to remind the key role of meat in nutritious dietary practices; high quality marbled beef is not only fine in health but also includes more advantageous fatty acids.