*3.3. Factors Affecting Omega-3 and Omega-6 Fatty Acid Deposition in Muscle Tissues (Meat) of Ruminants*

The modern era has brought advancement in agricultural practices and food processing. However, large scale commercially oriented crop and animal production has, in general, decreased n-3 PUFA concentrations, increased n-6 PUFA concentrations and increased n-6/n-3 ratios in meat, eggs and milk when sourced from intensively farmed animals compared to animals living in range lands. Within this context, there are three things to recognise. The amount of a specific n-3 PUFA deposited in the cells and tissues (1) are not directly related to the amounts of n-3 PUFA present in the animal diets; (2) are related to the amount of n-6 PUFA available in the feeds for consumption and amount already deposited in the peripheral tissues; and (3) are dependent on the interference of desaturation and elongation enzymes reactions of both FA families that generate their LCPUFA in tissues. Many different factors influence the n-3 and n-6 PUFA concentrations of meat in ruminants, which will be briefly discussed below.


consume these pastures, fodders and other by-products of field crops to support their good health and wellbeing. Phytonutrients are classified as alkaloids, polyphenols, organosulfur compounds and so on. It is likely that animal feeds containing some types of polyphenols, such as tannins, phenolic acids and flavonoids, can protect dietary PUFA from the hydrolysis and biohydrogenation in the rumen resulting in beneficial effects. Hence, increased n-3 LCPUFA would be available for absorption across enterocytes and, therefore, have increased deposition within tissue and meat. This is possibly due to these phytonutrients having low bioavailability and long retention times within the rumen, causing a slow degradation of fibrous diets by the microflora and allowing the PUFA and other nutrients present in the diet to bypass the rumen and be available for intestinal absorption by host animals.

F. Level of antioxidants and carotenoids in muscle tissues: Ruminants are specialised to consume 80–85% of diet as forage (fibrous materials) such as green pastures, fodders, silage and other forage materials. From these diets, they ingest adequate amounts of antioxidants, such as vitamins, minerals and carotenoids. Monogastric animals grown under intensive systems consume 80–85% concentrated diets and they receive carotenoids and antioxidants from the ingredients of cereal grains, protein meal and oilseeds. Carotenoids, such as carotenes and xanthophylls, are pigments present in leaves, seeds, fruits and animal products of blood, meat and milk. Carotenoids have the ability to act as antioxidants as they are quenchers of singlet oxygen (1O2) and other reactive oxygen species (ROS) or substances that causes oxidative damage in the body from cell to tissue level. The biological roles of carotenoids and polyphenols in the ruminant digestive system and their metabolism are not yet fully understood. It is speculated that increased level of antioxidant potential in the circulatory and tissue systems can protect the oxidation of n-3 LCPUFA from the tissues. This improves the health and wellbeing of individuals. For example, several studies [7,21,35–37] described the relationship among antioxidants, n-3 PUFA and lipid oxidation in muscle tissues in sheep and goats.

#### **4. Biological Actions**

In the context of human health and wellness, ALA, EPA, DPA, DHA and their secondary metabolites have been the focus of attention throughout the previous 50 years. It seems reasonable to suggest that the mode of actions and effects would be similar between farm and companion animals. The dramatic advancement in the analytical technologies of n-3 PUFA, identification of their intermediate metabolites and understanding of their important role in human growth, development and disease prevention has facilitated the introduction of new dietary regulations and recommendations for foods high in PUFA and *trans* FAs, particularly n-3 PUFA and vaccenic acid. In this context, long chain PUFA are considered essential and/or health enhancing nutrients that impact on growth and development in early life as well as metabolic disorders and chronic diseases in later life.

DHA is a major constituent of cardiomyocytes, sperm, grey matter of the brain and the retina. Several studies have indicated DHA is necessary for central nervous system functionality as well as the visual activity of infants. The 20 and 22 carbon chain-length PUFA (i.e., EPA, DHA, and AA) can be converted to a series of hormone-like substances called eicosanoids and docosanoids, respectively, including prostaglandins (PGs), thromboxanes (TXs), prostacyclin (PGI2), leukotrienes (LTs), resolvins (RVD) and other lipid mediators (Figure 5). These eicosanoids and docosanoids contain many intermediary metabolites and isoforms. These agents play major roles in the regulation of diverse pathophysiological functions, including blood pressure, platelet aggregation, blood clotting, blood lipid profiles, immune response, the inflammation response to injury and infections and the resolution of inflammation [15,39]. A large proportion of research conducted in laboratory animals and humans has been devoted to the pathophysiological functions and properties of EPA, DPA and DHA and the roles of the derived lipid mediators.

**Figure 5.** The formation of lipid mediators (intermediary metabolites and isoforms) from eicosanoids and docosanoids derived from long chain omega-3 (EPA, DPA, DHA) and omega-6 (AA) fatty acids in animals and human tissues or body.

The ARA is then rapidly converted into two major classes of enzymes, called cyclooxygenases (COX) and lipoxygenases (LOX). COX enhance the production of prostaglandins, prostacyclin and thromboxanes, while lipoxygenase enhance the production of leukotrienes. The physiological actions of these metabolites are widespread and diverse. Briefly, prostaglandins and prostacyclin are potent vasodilators whilst thromboxanes are potent vasoconstrictors, whereas leukotrienes produce bronchoconstriction. Lipoxygenases in plants and animals are heme-containing dioxygenases that oxidise PUFA at specific carbon sites to give enantiomers of hydroperoxide derivatives with conjugated double bonds. The number in specific enzyme names such as 5-LOX, 12-LOX, or 15-LOX refers to the ARA site that is predominantly oxidised. Of these, 5-LOX is best known for its role in the biosynthesis of leukotrienes A4, B4, C4, D4 and E4. The oxidised metabolites generated by 5-LOX were found to alter the intracellular redox balance and to induce signal transduction pathways and gene expression. The enzyme 5-LOX has been identified as an inducible source of ROS production in lymphocytes [40]. Cyclooxygenase-1 has been implicated in ROS production through formation of endoperoxides, which are susceptible to scavenging by some antioxidants in cells stimulated with TNF-α, interleukin-1, bacterial lipopolysaccharide, or the tumour promoter 4-otetradecanoylphorbol-13-acetate [41].

DPA is another n-3 LCPUFA which has potential in maintaining health and wellness of animals and humans. Its applicability and efficacy in terms of metabolic activity and disease prevention have not been fully investigated. However, it deserves attention for various reasons, including that it is the intermediate substrate of EPA conversion to DHA within the cell or tissue systems of animals and humans, and its tissue concentration is dependent on the balance between EPA and DHA. There is emerging evidence that DPA levels are positively correlated with the expression of certain enzymes involved in inflammatory processes of the cardiovascular system [42]. Research indicates that DHA and its metabolites are used for tissue-based metabolic activities such as insulin-stimulated energy disposal, phospholipid-induced signal transduction towards gene expression, active

autoimmune systems towards cell defence, etc. Within these contexts, the overall n-3 PUFA metabolic process must be efficient in converting EPA to DHA via DPA and the reverse reaction of DPA to EPA; and the availability of DPA in the tissue system is important for transitional processes to maintain both EPA and DHA levels. It is noteworthy that the concentration of DPA in red meat is equal or greater than that of EPA and DHA. Therefore, the contribution of DPA from red meat should not be neglected or ignored in terms of its role in the maintenance of health and wellness of people who regularly consume more red meat than fish or vegetables.

#### **5. Dietary Recommendation**

Past research has determined adults (aged 18 years and older) to have no upper intake limit for n-3 PUFA to ensure their safety, *viz.* ALA, EPA, DHA and DPA [43,44]. Instead, it is apparent that diets which fail to provide the minimum requirements of these FA are a greater health concern. In response, many authorities and organisations from around the world have proposed guidelines that define the daily recommendable intakes for n-3 PUFA, n-6 PUFA and total LCPUFA (Table 3). From these, we can observe that male and female adults have different requirements and, furthermore, the requirement for females will depend on their physiological status (e.g., every day, during pregnancy, during lactation). This complements previous knowledge that age will impact on dietary requirements for these PUFA, with children aged less than 18 years proposed to require a diet that contains more n-3 PUFA and n-6 PUFA than necessary for an adult [45].

The examples included in Table 3 also show that dietary FA guidelines differ between organisations and, sometimes, these differences are substantial. A possible basis for this disparity could be the basal diets typical to the populations represented by these organisations. For example, MHLW [49] identify the diet of the Japanese population to be comparatively lower in n-6 PUFA and, therefore, a lower requirement for n-3 PUFA is necessary to achieve an acceptable n-6/n-3 ratio. This is reliant on the n-6/n-3 ratio's importance to human health, an observation previously made and resulting in ratio recommendations that range from 5:1–10:1 for adults [46]. That said, the FAO [54] report that if n-3 PUFA and n-6 PUFA intakes adhere to their individual guidelines then there is no rationale to support a recommendation for n-6/n-3 ratio intake. This premise does depend on their being no biochemical competition or inhibition between the functionalities of n-6 PUFA and n-3 PUFA that affects their bioavailability.

**Table 3.** Examples of omega-3 (n-3), omega-6 (n-6), and total long chain polyunsaturated fatty acid (LCPUFA) dietary recommendations for a healthy adult (18 years and older). Abbreviations include male (M); female (F); alpha-linolenic acid (ALA); linoleic acid (LA); percentage total energy (%E); eicosapentaenoic acid (EPA); docosahexaenoic acid (DHA); and acceptable macronutrient distribution range (AMDR). Please note that for female adults, recommendations are categorised using physiological status (as everyday, during pregnancy and during lactation).

