*3.2. Macronutrients*

### 3.2.1. Total Protein and Amino Acids

Proteins are essential macronutrients for human energy and nutrient requirements, as protein or amino acid deficiencies are known to cause severe health issues, especially in pregnan<sup>t</sup> women, children [94] and the elderly. The consumption of indispensable (i.e., essential) amino acids, which cannot be synthesized in the human body, highlights the importance of consuming balanced protein sources from food. The amino acid balance and digestibility have been used to define protein quality in different food sources. Animalderived products, including red meat, provide complete proteins. Animal protein is necessary, for instance, in situations where patients require high consumption of protein for tissue and musculoskeletal recovery [95,96]. Additionally, amino acid content plays an important role in the development of meat flavor compounds and sensory characteristics during cooking, which highlight the importance of amino acid composition for consumer acceptance of red meats.

With muscle protein being a functional tissue, studying the change in protein content of a muscle must consider changes in fat or moisture content which could therefore influence protein content. Thus, most of the studies reviewed have evaluated genetic and dietary approaches and their influence on changes in individual amino acid content. Descriptive studies have measured total protein of muscle tissue derived from red meat livestock species [3,97,98]; however, there is a lack of studies evaluating total protein or amino acid content in red meats and how these components can be modified to enhance its nutritional value using nutritional or genetic approaches [99]. Drazbo et al. [100] found that total protein in pork *longissimus dorsi* muscle was different when feeding a diet with protein and amino acid levels reduced by 15% relative to the standard levels. Specific pork loin essential amino acid composition was also different when supplemented with dietary additives like ginseng [101]. Muscle histidine and valine concentration have been observed to be lower when fed 5:1 and 10:1 PUFA ratio diets compared to 1:1 and 2.5:1 PUFA ratio (n-6:n-3) diets [102], suggesting lower concentrations of these amino acids are observed when fed a high saturated fatty acid profile. However, no effect on total protein was observed from diets with 2.0% supplemented palm oil and 0.5% or 1.0% CLA [103]. A study observed cattle fed grass silage had higher free amino acid levels compared to animals fed a concentrate diet, and many individual amino acid concentrations were also significantly different [104]. A study on lambs weaned at different ages revealed no difference in crude protein percent; however, looking at individual amino acids, essential amino acids were higher when weaning occurred at an earlier age [105], suggesting potential shifts in fiber type composition and therefore amino acid composition. Additionally, in the latter study, environmental factors such as production system or weaning system could have affected amino acid composition. A study using diet manipulation revealed protein percent in lamb leg was significantly impacted by feeding olive cake [106]; this suggests the increase in fat and decrease in moisture of the diet influences protein content.

Regarding genetic approaches to manipulate total protein or amino acid content in red meat, existing studies have revealed heritability of both total protein and individual amino acids ranges from low to high for beef and pork [107–109]. A prior study based on targeted single nucleotide polymorphisms (SNPs) and total significant SNPs revealed moderate to high heritability estimates of 0.42 and 0.26, respectively, for total protein content in pork [109]. In addition, pork breeds have also been compared to evaluate the different genetic backgrounds associated with total protein and amino acid profiles [110]. Studies have also estimated heritability of different amino acids in beef, revealing estimates of 0.34, 0.17, 0.66, 0.40 and 0.33 for alanine, glutamine, taurine, anserine and inosine, respectively [108]. Similarly, Ahlberg et al. [107] revealed high heritability estimates of total

protein content in beef *semitendinosus* and *longissimus* muscles of 0.75 and 0.70, respectively. One study revealed that the *longissimus dorsi* mean amino acid content was significantly different between lamb breeds for arginine, glutamine and tyrosine [111]. The studies to date sugges<sup>t</sup> a potential for manipulating individual amino acid concentrations content using diet, as well as genetic approaches, due to the moderate to high heritability of these components; however, further research is needed, specifically to evaluate the potential to manipulate total protein content.

### 3.2.2. Total Fat and Fatty Acid Composition

Meat lipids continue to remain as the nutrient component with the highest potential for modification, in both content and composition, presenting opportunities for value added production and health promotion [112]. For instance, low-fat of n-3 enriched meats are considered functional foods for overweight individuals, since their consumption improves the body fat index, n-3 levels and the n-6:n-3 ratio, without impacting the Healthy Eating Index or intake levels of energy or other macronutrients [10].

Numerous studies have reported values for total fat, as well as different groups of fatty acids in meat. This area of research has been highly researched for the last 20 years and continues to attract attention from researchers in the area of animal and meat sciences (Table 3). The potential for manipulation of lipids is clear when the ranges in the literature were considered, with clear differences between monogastrics and ruminants. In monogastrics such as swine and poultry, meat fatty acid composition is reflective of their diets, whereas, in ruminants, dietary unsaturated fatty acids undergo extensive biohydrogenation by the rumen bacteria and are transformed into saturated fatty acids [113]. This phenomenon limits the ability to increase the content of these fatty acids in ruminant meats through feeding polyunsaturated fatty acids (PUFA) sources such as oilseeds and fish oil [114]. Conversely, during ruminal biohydrogenation of PUFA, several intermediates are produced, and a portion of them passes from the rumen and subsequently finds its way into meat after post-ruminal absorption. Specific biohydrogenation intermediates such as conjugated linoleic acids (CLA) and vaccenic acid (VA, *trans*-11 18:1) have been associated with several health benefits including anti-inflammatory and anti-diabetic effects [115].

**Table 3.** Nutrient range of fatty acids in red meats.


Key targets for manipulation include increasing n-3 PUFA across species, and specifically in ruminants increasing contents of "healthy" PUFA biohydrogenation intermediates including CLA and VA [112,114]. In addition, a primary target has been to reduce saturated fatty acid (SFA) content as well as increasing levels of oleic acid (*cis* 9-18:1) [116]. Feeding grains, oilseeds, forages, grass or DDGS, among other feedstuffs, has a large impact on intramuscular fat (IMF) and the proportions of the different fatty acid groups. For example, meat from grass-fed ruminants tend to present lower IMF and higher proportions of n-3 PUFA, CLA and VA compared to concentrate-fed ruminants [27]. Feeding concentrate based diets, however, have been associated with decreased PUFA/SFA ratios, but, over the finishing period, there is increased conversion of SFA to monounsaturated fatty acids (MUFA), and relative rates are influenced by breed [117]. On the other hand, manipulation

of PUFA biohydrogenation intermediates may have more to do with interactions between diet, rumen microbiology, and managemen<sup>t</sup> than host genetics. Indeed, large increases in VA and CLA in steers has been linked to feeding management, for example, feeding a PUFA rich supplement (flaxseed co-extruded with peas) before feeding hay, instead of feeding a hay and supplement mix, has led to a substantial increase in VA and CLA [118,119]; these differences are related to shifts in the rumen microbial population [120].

While many studies have reported the potential for manipulation of VA in beef and lamb, it is important to look at the total amount of *trans* fatty acids and the relative proportions of *trans*-10-18:1 and *trans*-11-18:1, as *trans*-10 has been associated with detrimental effects on blood lipid profiles through upregulation of hepatic triacylglycerol and cholesterol synthesis [115,121]. Again, host genetics are not considered as a primary factor in the accumulation and proportions of *trans* fatty acids, but different forages, grass or vitamin supplementations can lead to shifts in *trans* fatty acid amounts and isomer proportions [27]. In beef and lamb, feeding sources of PUFA leads to a large number of biohydrogenation intermediates including several conjugated and non-conjugated 18:2 and 18:3 isomers for which the roles in the human body are still unclear [115]. CLA has also received more attention recently due to the potential health benefits of nitro-fatty acids in humans [122], but the role of host genetics in these processes is not ye<sup>t</sup> clear. In fact, in ruminants, dietary ingredients and additives that modify the rumen microbiome may have a larger effect than direct supplementation of fatty acid supplements. On the other hand, dietary supplementation of PUFA has a large impact on pork fat composition, leading to large increases in PUFA and n-3, even long chain n-3, especially when using marine sources, such as fish oil and algae [28]. In cattle, however, some limitations exist regarding long chain PUFA deposition due to their preferential incorporation into phospholipids [123]. Further manipulation of pork IMF can also be achieved through altering lean deposition by reducing the protein or lysine content in diets (i.e., causing lean to fat repartitioning), or by adding CLA into pig diets [124].

The relationship between total fat content and relative proportions of fatty acids is important to consider, as higher IMF corresponds to lower relative PUFA content, due to the smaller contribution of membrane phospholipids [125]. For this reason, when reporting fatty acid profiles in meat, it is important to provide either total IMF or use mg per 100 g of meat as the unit, instead of the percentage of fatty acids in total fat. Similarly, consumers do not eat denuded muscles, but commercial cuts, which combine lean with seam fat and subcutaneous fat. Thus, while the manipulation of fatty acid profile in IMF may be more limited, it is possible to obtain a larger impact when considering changes in the whole primal, including all fat depots [126]. In general, dietary effects observed in IMF tend to have a larger impact on larger fat depots. This is important when trying to enhance the lipid profile in order to reach certain health claims. In fact, an alternative for fresh meat products, such as ground meat, could be accomplished by either supplementing the diets of a small percentage of the animals or selecting carcasses with a naturally higher concentration of certain beneficial fatty acids, and then mixing the fat from those carcasses with lean from the regular population. Manipulation of fatty acid profiles also has to take into consideration effects on meat and fat quality (taste, oxidative stability, fat softness, etc.), as enhancing the healthfulness of the fatty acid profile will be of limited value if overall quality is negatively affected. Thus, studies investigating manipulation of fatty acid profiles need to be linked with complimentary studies on meat or meat product quality, including sensory evaluation [112].

Inter- and intra-breed differences are well known in terms of total IMF variability, with very obvious cases of genetic groups with higher marbling [127,128], while populations selected for other traits correlated to total fat, such as lean meat yield, have seen a decrease in IMF as a negative side effect [129–131] Studies show a medium to high heritability not only for total IMF, but also for the majority of fatty acid groups which can be endogenously synthesized in both ruminants and monogastrics [132,133]. According to GWAS studies in different species, both total IMF and fatty acid composition in

meat are influenced by key regulatory genes with major effects and multiple genes with smaller effects, and have shown moderate to high heritability estimates for IMF and low to medium heritability for specific fatty acids [134–139]. However, despite the potential to include IMF fatty acids in breeding programs, antagonistic genetic relationships with performance have usually minimized the emphasis on selection for these traits. A recent study [140] reported a series of genetic markers that could be used to manipulate IMF without impacting backfat thickness, opening new opportunities for animal selection. In terms of manipulating meat fatty acid composition, the influence of fatness on the lipid profile (decrease of relative proportion of PUFA with higher levels of IMF) must be taken into consideration [133]. Nevertheless, multiple studies have found SNPs for a number of candidate genes regulating intramuscular fatty acid metabolism [141]. Within the last few years, numerous studies have focused on alternatives to traditional genetic selection. This includes the use of transgenic animals (by nuclear transfer of modified DNA to an embryo) which can increase the endogenous production of certain beneficial fatty acids, such as omega-3 fatty acids [142–146]. This approach is also possible for feedstuffs, with crops being genetically modified to produce long-chain omega-3 fatty acids usually only available from marine sources [147]. Although these strategies present grea<sup>t</sup> potential for IMF and fatty acid manipulation, ethical and safety concerns still need to be addressed [148,149]. Moreover, consumer perception of genetically modified organisms and animal welfare could limit the large-scale implementation of these strategies [150].
