**1. Introduction**

The utilisation of available resources for resilient animal production systems, food security and maintenance of the health and wellbeing of human–animal population are important for the sustainability of future agriculture and food production. The use of available feed resources from cultivated pasture and natural range lands in animal production systems may be beneficial for the wellbeing of livestock. The same is true for those humans who consume milk, meat and offal from those animals, especially when compared to products from livestock fed commercially formulated feeds or low-quality forages that are deficient in nutrients. In many livestock production systems, pasture and forage diets

**Citation:** Ponnampalam, E.N.; Sinclair, A.J.; Holman, B.W.B. The Sources, Synthesis and Biological Actions of Omega-3 and Omega-6 Fatty Acids in Red Meat: An Overview. *Foods* **2021**, *10*, 1358. https://doi.org/10.3390/ foods10061358

Academic Editor: Angel Cobos

Received: 19 May 2021 Accepted: 9 June 2021 Published: 11 June 2021

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are common sources of omega-3 (n-3) polyunsaturated fatty acids (PUFA). Grain-based and feedlot diets are, instead, common sources of omega-6 (n-6) PUFA.

It is well known that lipids (fats from animals and oils from plants) provide energy, nutrient mediation, signal transduction, disease prevention, insulation, cell membrane structure, and organ protection upon consumption. Lipids include triglyceride, phospholipid, cholesterol, cholesterol ester, free fatty acids (FFA), sphingomyelin subgroups and glycolipids. Not many animal or plant scientists understand the complexity of the lipid fraction and the differences between animal and plant tissues. For example, fat deposits in animal tissue are mainly triacylglycerols (TAG); muscle lipids contain TAG, cholesterol and phospholipids; plant leaf tissue lipids are mainly polar lipids (glycerophospholipids); oil seed lipids are mainly TAG. The different lipid fractions in plant and animal tissues can be separated and observed by using a thin layer chromatography (TLC) technology, an example is shown in Figure 1.

**Figure 1.** Lipid fractions of lyprinol (green-lipped mussel) separated by using a thin layer chromatography (TLC) technology. Lipid fractions were separated by thin layer chromatography (TLC) on silica gel plates (Silica gel 60H, Merck, Darmstadt, Germany). The solvent system for all TLC was petroleum spirit/diethyl ether/glacial acetic acid (85:15:2 by volume). Lyprinol (50 g) was made up in 1 mL of chloroform, and from this stock, 10 μL (sample 1), 20 μL (sample 2) and 30 μL (sample 3) were spotted as shown above. Lipid classes were visualised with fluorescein 5-isothiocyanate against TLC standard 18-5 (Nuchek Prep Inc, Elysian, MN). Lipid fractions identified from left top to bottom are cholesterol esters (CE), triacylglycerols (TAG), free fatty acids (FFA), cholesterols (CHOL) and phospholipids (PL).

Previous research of both humans and animals, including livestock and companion animals, demonstrate that dietary background plays a major role on lipid metabolism, fatty acid (FA) synthesis and fat accretion in the body—more so than genetic or gender associated factors alone. The effects of dietary fat are contributed by both their energy concentration and the types of lipids present. Genetic effects in FA synthesis and accretion in farm animals has been shown to be associated with desaturase and elongation activity [1]. Genetics are also known to influence desaturase and elongase activity in humans [2]. There are many studies which refer to the influence of gender on PUFA synthesis in human (higher activity in females than males).

Fatty acids are classified as saturated or unsaturated. Unsaturated FA can be further delineated as monounsaturated (MUFA) and PUFA. Common dietary sources of PUFA include leafy vegetables, oilseed, nuts, meat, eggs and seafood. Characteristics of PUFA are their low melting point and liquid state when held at room temperature. Hence, these are often referred to as oils. It is of interest, therefore, that fat melting point has been applied to estimate the unsaturated FA content of non-liquefied fat deposits from, for example, beef and sheep meat [3,4]. There is substantive evidence to support the regular consumption of n-3 PUFA, as these are beneficial for growth, development, health and the welfare of humans and animals [5–9]. The n-3 PUFA alpha-linolenic acid (ALA, C18:3n-3) and the long chain derivatives eicosapentaenoic acid (EPA, C20:5n-3), docosapentaenoic acid (DPA, C22:5n-3) and docosahexaenoic acid (DHA, C22:6n-3) have each been reported to play a role in the prevention of cardiovascular disease, diabetes, hypertension, inflammation, allergies, cancer, renal disorders, neural function and improve immune response [5,10,11].

All fish are rich in long chain n-3 PUFA, especially EPA and DHA, but this is especially true for oily fish such as salmon and mackerel. The levels of these same PUFA are comparatively moderate in red meat sourced from pasture grazed ruminants, these having levels similar to many white fish which are low in fat such as snapper, leatherjacket, flounder [12,13]. The application of grains or some feedlot rations within livestock industries to hasten animal growth rates can diminishes the level of n-3 PUFA in red meat. In addition, recent climate variation has led to prolonged drought in some parts of the world, which diminishes the availability of n-3 PUFA rich feed sources to livestock and increases reliance on concentrate and commercial feeds that are rich in n-6 PUFA.

Collective research indicates that the evolutionary aspects of modern farming (agribusiness), selection of specialised pastures for high yield, commercially oriented crop and animal production systems, and food processing have contributed to alterations in the concentrations of n-3 and n-6 PUFA in pasture and field crops [9]. This is believed to be impacting the health and wellness of animals and humans. Indeed, the ratio of n-6 and n-3 PUFA (n-6/n-3 ratio) in human and animal diets is proposed to have been nearly 1:1 during evolutionary time, but direct interventions and climate variation has led to a shift towards a ratio closer to 20:1. This is of concern because present recommendations advise that animal and human diets should have a n-6/n-3 ratio of 1–4:1 to help maintain a balanced and healthy life [14]. The nutritionally important n-3 PUFA found in meat and other products, such as milk and non-lean edible parts of a carcass, are summarised in Table 1.

**Table 1.** Common name, abbreviation and scientific name (IUPAC, International Union of Pure and Applied Chemistry) of omega-3 (n-3) and omega-6 (n-6) fatty acids found in dietary sources.


This overview aims to describe the biochemical basis of n-3 and n-6 PUFA and agricultural practices unique to the modern era that are applied for their enhancement in red meat. Special reference is made to their preservation, biological actions and recommended dosages within a human diet.

#### **2. Synthesis**

#### *2.1. Molecular Structure of Omega-3 and Omega-6 Fatty Acids*

PUFA can be classified by carbon chain length, where 20–24 carbon atoms are long chain and 26 or more carbon atoms are very long chain PUFA (FAO/WHO, 2008). Researchers (i.e., nutritionists, dietitians and biochemists) often use the 'n minus' term of notation to name the naturally occurring *cis* unsaturated FA, where the 'n minus' indicates the position of first double bond of the FA closest to the methyl end of the molecule. For example, ALA is designated as C18:3n-3 since the first double bond is present 3 carbon atoms from the methyl end, but this nomenclature does not specify the position and confirmation of remaining double bonds in the molecular structure [15]. In this system, the *cis* unsaturated FA are classified as n-3 (omega-3), n-6 (omega-6) and n-9 (omega-9). Chemical structure of n-3 and n-6 PUFA naturally available in meat and other dietary sources are shown in Table 2.

**Table 2.** Names and chemical structures of commonly available omega-3 and omega-6 fatty acids.

#### *2.2. Biosynthesis of Omega-3 and Omega-6 Fatty Acids*

ALA is the precursor (parent) FA of the n-3 family, whereas linoleic acid (LA) is the precursor FA of n-6 family. Among the four n-3 PUFA most commonly found in animal tissues (i.e., ALA, EPA, DPA and DHA), ALA cannot be synthesised by humans and animals and is therefore referred to as an essential FA [16]. Only plants can produce essential FA, and animals and humans must obtain these FA, through dietary means, for use in the synthesis of their longer chain n-3 and n-6 PUFA derivatives *viz*. EPA or DHA, or arachidonic acid (AA) [17]. In the body, the synthesis or conversion of ALA to its longer chain derivatives is controlled by many biological factors that, according to both animal and human studies, are slow and inefficient [18,19]. The conversion efficiency is not dependent on the metabolic demand of the body but is mainly determined by the amount of ALA and (interestingly) LA, present in the diet. This is because of their competitive nature whereby the same enzymes mediate ALA and LA desaturation and elongation processes.

Due to the low conversion efficiency, it is necessary to provide substantial amounts of dietary ALA to promote higher levels of required EPA and DHA in the circulatory and tissue systems. Therefore, it is suggested that animals and humans should be fed with edible wild plant leaves or vegetable oils rich in ALA or, alternatively, with marine-based diets which are rich sources of EPA and DHA and avoid the desaturation and elongation processes required by ALA [20,21]. There are studies that have also shown significant increases in EPA, DPA and DHA concentrations in the blood of humans or muscle tissues of ruminants when terrestrial-based diets rich in ALA, such as flax(seed) or canola, are fed for long durations and/or at high doses [3,5,6,13,18–22]. Some studies indicated that there are three desaturase enzymes involved in the formation of 22 carbon long chain n-3 (DHA) and n-6 (DPAn-6) PUFA from ALA and LA in human and animal tissues. The dietary sources and biosynthetic pathways of n-3 and n-6 PUFA in mammals involving delta-6, delta-5 and delta-4 desaturase enzymes are shown below in Figure 2. It should be noted that some research has found there to be no involvement of delta-4 desaturase enzyme activity in the conversion of ALA to DHA and LA to DPAn-6, respectively. Rather, there will be a further elongation through the second use of delta-6 desaturase enzymes and then beta-oxidation processes take place in the circulatory and peripheral tissue systems for the synthesis of DHA and DPAn-6 PUFA [22]. This is illustrated in Figure 3.

**Figure 2.** Dietary sources and biosynthesis of omega-3 and omega-6 fatty acids through enzymatic desaturation and elongation processes adapted from Ponnampalam et al. [23].

**Figure 3.** A diagram of omega-3 and omega-6 fatty acid elongation and desaturation to highlight the second use of delta-6 desaturase adapted from Gibson, Neumann, Lien, Boyd and Tu [22].

Long chain PUFA (LCPUFA) in animal and human muscle tissues are mainly found in phospholipids, where they play a major role in the metabolic, functional and physiological status of the body, organelles and tissues. In vivo studies conducted in animals have indicated that the relative levels of n-3 and n-6 LCPUFA in animal tissues can be regulated by altering the balance of ALA and LA in the diet. Gibson, Neumann, Lien, Boyd and Tu [22] used rats as a model species to show that feeding ALA at 1–3% and LA at 1–2% of dietary energy, while maintaining the intake of total PUFA less than 3% of dietary energy, DHA in plasma phospholipid can be positively and linearly increased. Mammals can convert ALA into LCPUFA such as EPA, DPA and DHA via a series of desaturase and elongase catalysed reactions [24]. Both the FA desaturase 1 (FADS1) and FA desaturase 2 (FADS2) prioritise ALA compared with LA. High LA intake, such as characterised by grain finishing or feedlot feeding of animals, can interfere with the desaturation of ALA and also of 24:5n-3, which is a precursor of 24:6n-3, the final precursor of DHA (Figure 3). The concentration of ALA present in the phospholipids of plasma and tissues is usually less than 0.5%. It is not known whether this level is sufficient for FADS2 to compete with LA, which is comparatively more abundant in animal tissues [25]. Past research has indicated that the conversion of ALA to DHA is not immediate, nor as effective as direct consumption of fish or a fish oil supplement [26–28].

Human studies conducted using isotope-labelled ALA have shown that males, when compared to females, are less efficient at synthesising EPA and DHA from ALA. The estimated net conversion rates of ALA to EPA is 21% for females and 8% for males, and of ALA to DHA is 9% for females and 0% for males. Sex differences in EPA and DPA content have been observed, with females having higher erythrocyte phospholipid EPA, lower adipose tissue EPA and lower plasma DPA content than males. There was a significant difference between sexes in terms of human response to increased dietary ALA, with females having a significantly greater increase in the EPA content of plasma phospholipids after six months of an ALA-rich diet compared to males [29]. A detailed study of genetically divergent sheep, raised in several disparate production regions, showed there to be a small gender effect on health claimable fatty acid content EPA and DHA such that females had higher levels than males. As female lambs approach their reproductive stage, it is possible that they synthesise more n-3 PUFA in the body for the production of series-3 eicosanoids, which is associated with the ovulation process, conception and pregnancy. Lambs from Merino dams had about 2 mg/100 g higher levels of EPA + DHA than lambs from crossbred dams when the sire breed was Poll Dorset. This is similar to Ponnampalam et al. [30], who found that the ratio of PUFA to saturated FA (SFA) in meat increased from second cross Poll Dorset to first cross Poll Dorset and from first cross Poll Dorset to purebred Merino. This same study also found this to be due to an increase in PUFA, and not due to a decrease in SFA [30].

Metabolism studies using stable isotope labelling, candidate gene single nucleotide polymorphisms (SNP), genome-wide association studies (GWAS) and metabolomics show interindividual variation in the conversion of LCPUFA precursors to LCPUFA products depends on genetic factors [31]. The FA desaturase genes (FADS1 and FADS2) code for enzymes that catalyse the introduction of double bonds at specific positions in a FA chain. FADS1 (D5-desaturase) and FADS2 (D6/D8/D4-desaturase) have specificity for several FA substrates [32]. Minor allele homozygotes (D/D) had significantly lower expression of FADS1 than the I/I major allele homozygotes. ARA is the immediate product of FADS1, leading directly to the hypothesis that individuals carrying D/D genotype have lower metabolic capacity to produce LCPUFA from precursors than I/I individuals. It was reported that individuals with I/I genotype having higher metabolic capacity to convert precursors to longer chain PUFA may be at increased risk for proinflammatory disease states as they efficiently convert LA to ARA [2] as FADS SNP was found to influence synthesis of ARA and synthesis of pro-inflammatory lipoxygenase products.

#### **3. Sources**

Twenty and 22 carbon LCPUFA, especially ARA, EPA and DHA, are ubiquitous in mammalian tissue, are bioactive components of membrane phospholipids and serve as precursors to cell signalling eicosanoids and docosanoids that are major drug targets (e.g., COX-1, COX-2 inhibitors, leukotriene receptor antagonists). LCPUFA can be obtained directly from animal foods or endogenously synthesised from 18 carbon essential FA precursors LA and ALA and their metabolites by an alternating series of desaturation and elongation reactions [32]. Vegans rely on this biochemical pathway to generate all LCPUFA from precursors. Classic carnivores (e.g., cats and most marine fish) have lost the metabolic ability to make LCPUFA and rely on consumption of animal tissue or fish to supply all their LCPUFA requirements.

#### *3.1. Feeding Type and Digestive System of Ruminants*

Cattle, sheep, goats, buffalo, yak, alpacas and deer are categorised as ruminants and are unable to digest plant material directly because they lack the enzymes needed to break down cell walls (cellulose and hemicellulose). Ruminants have a complex four-chambered stomach, comprising rumen, reticulum, omasum and abomasum, due to the nature of the high roughage feedstuffs they consume. Ruminant animals support a large population of bacteria, protozoans and fungi in their four-chambered stomach because they consume a large proportion (80–85%) of highly fibrous plant materials (roughage diets). Ruminant microorganisms play a major role in the degradation of undigestible fibrous materials, thereby making use of the dietary energy and nutrients by themselves as well as providing a medium for digestion and absorption in the small (duodenum, jejunum and ileum) and large (cecum, colon and rectum) intestines of the host animals. The important function of the salivary gland is adding saliva to the feeds to form bolus and to buffer pH levels in the rumen and reticulum so that the microbial activity and degradation process is optimised. The rumen and reticulum are home for the population of microorganisms that ferment and break down plant materials and produce volatile organic compounds and release other nutrients—both microbes and host animals use these volatile organic compounds for energy. The anatomical and functional attributes of small intestine of ruminants is similar to non-ruminants and ranges in length between approximately 12–30 times the body length of the animal [33].

#### *3.2. Digestion, Absorption and Deposition of Dietary Lipids in Tissue of Livestock*

Lipids are either consumed or synthesised de novo to contribute structure, integrity, recognition systems and energy to cells of most tissues. Not many researchers realise that the digestion and absorption of lipids (or fats) in ruminant and monogastric animals are different. This is due to their feeding nature and structure of digestive systems. In general, diets consumed by ruminants consist of 80–85% roughage and 15–20% concentrate while the diets consumed by monogastric animals are the opposite. More details on digestion, absorption and metabolism of dietary lipids can be found elsewhere [33,34]. Ruminant diets generally consist of 1–4% fat, and lipid supplements fed to ruminants above 5–6% on a dry matter basis have negative effects on rumen microbial activity, mainly on carbohydrate (fibre as cellulose and hemicellulose) degradation, particularly when PUFA are included in the diet. Supplementation of lipids in ruminant diets have some benefits to livestock industry in the following aspects: (1) it helps reducing the methane emission to environment from degradation of high fibrous diets; (2) it helps bypassing the dietary lipids (PUFA) from rumen to small intestine for absorption avoiding biohydrogenation; and (3) saving the dietary energy captured from methane emission for assimilation of tissue growth. With monogastric animals having a stomach as one organ for temporary storage of diet (fats) in the absence of rumen microbial activity, they can handle greater amounts of lipids in their diet for digestion and absorption process.

In any species, acetate (mainly cattle and sheep as ruminants) or glucose (mainly swine and poultry as monogastric animals) is absorbed in the intestine to enter FA biosynthesis via malonyl-CoA production through the acetyl-CoA carboxylase reaction and then palmitate production through FA synthase. Once palmitate is synthesised, other medium to long chain SFA and MUFA are generated by desaturation and elongation process. Since animals cannot synthesise essential PUFA (ALA and LA), these lipids have to come from consumed feeds. In monogastric animals, dietary fats are unchanged by digestion in the intestine so that tissue FA more directly reflect their present in the diet. Several steps are involved in resynthesis and transport of lipids in ruminants from the enterocyte where FA are absorbed, until they reach the peripheral tissues such as adipose and muscle tissues. The FA, monoglycerides and diglycerides reaching the jejunum from micelles are absorbed into the epithelial cells of small intestine. These FA are esterified, and triglycerides and phospholipids are assembled into lipoprotein particles (chylomicrons, very low-density lipoproteins, etc.) in the enterocyte, which are then secreted into lymph vessels and enter the bloodstream.

In monogastric animals, the liver plays a major role in FA synthesis. In ruminant animals, the contribution of liver is minimal and, instead, FA synthesis is very extensive in adipose tissue. Upon entry to the blood, chylomicrons and very low-density lipoproteins acquire apoproteins apo-C and apo-E provided by high-density lipoprotein. Apo-C inhibits liver removal of chylomicrons and very low-density lipoproteins and this enhances the extent of diversion of these entities to other tissues. One of the apo-C components activates the lipoprotein lipase enzyme, which is situated primarily on the surface of the endothelium of skeletal muscle, adipose and mammary tissue sites. FAs and partial glycerides are apportioned to triglycerides, phospholipids and other lipids in the organs or oxidation for energy according to the metabolic demands of the body either in skeletal muscles, adipose and/or mammary tissues. The state of dietary lipids rich in n-3 PUFA from digestion in the intestine to deposition in the peripheral tissues through the circulatory systems is shown in Figure 4.

In the circulatory or tissue systems, diacylglycerol (DAG) is produced from phosphatidylcholine or from other phospholipids. Phospholipase C (PLC) cleaves the membrane phospholipid phosphatidylinositol-4,5-bisphosphate (PIP2) to generate inositol-1,4,5 trisphosphate (IP3) and DAG. Another type of phospholipase, phospholipase D (PLD), is activated by various stimuli in the cell. PLD hydrolyses phosphatidylcholine, which is abundant in the cell plasma membrane, producing phosphatidic acid and choline. Phosphatidic acid is hydrolysed by phosphatidic acid hydrolase to release DAG and phosphate. This is a second pathway that generates DAG. While this intermediate is the product of the action of both PLC and PLD, cellular responses in both cases are usually not identical due to differences in the cellular localisation of enzymes or the fatty acid composition of the DAG produced. The stimulation of specific cell-surface receptors activates phospholipase A2, leading to the release of arachidonic acid from the cell membrane.

**Figure 4.** The mechanisms of dietary omega-3 fatty acid digestion and metabolism in ruminants, adapted from Ponnampalam [34]. Abbreviations include fatty acid (FA); very low-density lipoproteins (VLDL); eicosapentaenoic acid (EPA); docosahexaenoic acid (DHA); high-density lipoproteins (HDL); lipoprotein lipase enzyme (LLE); intermediate fibres (IF); red fibres (RF); white fibres (WF); phosphatidylcholine (PC); phosphatidylethanolamine (PE); phosphatidylinositol (PI); Inositol triphosphate (IP3); diacylglyceride (DAG); and triglyceride (TG or triacylglycerol (TAG)).
