**3. Discussion**

#### *3.1. Lipid Oxidation*

In the present study, the incorporation of OEO at levels of 0.2 and 0.3 g/kg DM or monensin in the lamb diet had a similar effect on the lipid oxidation of lamb. The control treatment was slightly higher in malonaldehyde formation in meat. However, this difference was not statistically different from the control treatment. Notwithstanding, the result is still promising as far as the application of OEO concerns, since replacing OEO for monensin in lamb diets shows to be a beneficial choice.

However, meat from male lambs fed with a high level of OEO had significantly higher malonaldehyde (MDA) formation when compared to the control treatment.

The effectiveness of essential oil in preventing oxidation in lamb meat has also been reported by Nieto et al. [33]. They tested distilled dietary rosemary leaf (DRL, 0%, 10% and 20%) to prevent lipid oxidation and the sensory deterioration of cooked lamb, under retail display conditions. Cooked lamb fillets were stored at 0, 2, or 4 d (4 ◦C) in a display cabinet and then reheated, simulating catering practices. The cooked lamb suffered rapid lipid oxidation and odour and flavour spoilage associated with slight rancidity and warmed-over flavour. DRL feeding delayed lipid oxidation (thiobarbituric acid reactive substances, or TBARS) and volatile compounds more effectively in the first two d of storage. Percentages of 10% and 20% of DRL provided equal antioxidant capacity.

These positive effects of essential oil have also been found in bovine meat. Rivaroli et al. [34] fed crossbred young bulls with different doses of an essential oil blend (oregano, garlic, lemon, rosemary, thyme, eucalyptus, and sweet orange). They found that a dose of 3.5 g/animal/d decreases lipid oxidation. However, higher doses could have a pro-oxidant e ffect, and they are not recommended in feedlot animals.

Antioxidants that interact with reactive oxidant species (ROS) might become pro-oxidants, causing lipid and protein oxidation [35,36]. Low concentrations of essential oils might prevent this, and antioxidant activity is kept as observed in the present study, where low and medium oregano oil doses (0.2 and 0.3 g/kg DM diet) resulted in lower TBARS values, and the high doses produce an increase of lipid oxidation.

It is important to mention that in the present study, the lipid oxidation is considered still low (TBARS values lower than 2.0), which is in agreemen<sup>t</sup> with the report of Campo et al. [37]. They revealed that the TBARS value of 2.0 (2 mg MDA/kg meat) could be considered the threshold where the rancid flavour overpowers beef flavour. Therefore, it is considered as the maximum level for the positive sensory perception of beef. These authors indicated that from that point onwards, it can expected for beef to be rejected due to a strong sensory perception of lipid oxidation.

In physiological conditions, mammals constantly produce reactive oxygen species (ROS). Low concentrations of ROS are essential for several physiological processes, including protein phosphorylation, apoptosis, and cellular defence against microorganisms [38]. Oxidative stress refers to a lack of balance between the production of ROS and the level of antioxidants. Domestic animals are frequently exposed to oxidative stress, especially under intensive breeding systems [39]. Oxidative stress is responsible for numerous disease processes in animals. Many secondary metabolites formed by plants serve as defence agents against physiological and environmental stressors, and pathogenic microorganisms [40]. The main molecules responsible for the antioxidative properties of herbs and spices are phenolic substances. In particular, *Origanum vulgare* is an herb rich in phenolics [41].

Essential oils are rich sources of natural antioxidants, such as the phenolic compounds, and due to their high redox properties and chemical structure, they a ffect lipid metabolism in animal tissues by exerting beneficial e ffects on the antioxidant enzyme activity. Furthermore, phenolic compounds also prevent the production of reactive oxygen species and the o ff-flavors that are formed from the oxidation of polyunsaturated fatty acids [42]. Dietary supplementation with EOs is a simple and convenient strategy to uniformly introduce natural antioxidants into phospholipid membranes, where they may e ffectively inhibit the oxidative reactions by preventing the formation of radicals, and it appears to be a more e ffective way of slowing down hte lipid oxidation of animal products compared to post-mortem addition [43–45].

Other benefits of OEO have been stated in the literature. OEO modifies ruminal microflora, which also modifies the concentration of ruminal volatile fatty acid. Fat deposition (mainly unsaturated fatty acids, UFAs) is promoted when the concentration of propionic acid decreases and the acetic acid increases. Under some circumstances, UFAs are more susceptible to oxidation [46–48], and they may promote the formation of MDA in absence of antioxidants, as observed in this study (Figure 1).

As it has been previously pointed out, the structure of some lipid components from the essential oils changes as they transit through the digestive tract, and if they are absorbed in the intestine, the lipid profile and the oxidative stability of the meat might be modified [46–49]. In the present study, monensin has a similar e ffect to that of OEO in terms of lipid oxidation. This indicates that OEO could safely replace monensin in lamb diets, with the advantage of being a natural additive that promotes other positive changes in lamb, such as colour and shelf life preservation. OEO supplementation demonstrated lipid antioxidant activity in fresh lamb meat. OEO improves the antioxidant activity, which has an influence on retarding the lipid meat oxidation during refrigerated and long-term frozen storage. This process could be explained by carvacrol and thymol action on the permeability of cell membranes, and by the transformation of lipid and hydroxyl radicals into stable products [29]. This effect was supported in the present study.

The antioxidant effect of dietary OEO supplementation has also been demonstrated in poultry [44,50,51]. Moreover, OEO has been studied as an ingredient in meat formulations. In lamb burgers, the addition of 24 mL/kg of oregano extract is recommended as a natural antioxidant in replacement of sodium erythorbate, and the product has good acceptability [52].

#### *3.2. Compression Strength*

The tenderness of meat has been associated with intramuscular fat (IMF) content [53], and the increase of monounsaturated fatty acids (MUFAs) and PUFAs concentration in IMF could reduce the compression force of meat, thus producing more tender meat and, in this way, improving the quality.

Some of the intrinsec main factors that influence meat texture are the content and solubility of collagen, sarcomere diameter, intramuscular fat content, and proteolysis by calpains during ageing, among others [54]. The dietary inclusion of OEO decreases the concentration of acetic acid and increases propionic acid in rumen, which favours fat deposition [55] and improves meat tenderness. An increased quantity of subcutaneous fat and intramuscular fat decreases the rate of temperature decline, enhances the activity of autolytic enzymes in the muscle, lessens the myofibrillar shortening, and thereby increases the tenderness of cooked meat [56]. In the present study, it can be assumed that di fferences in tenderness between CON and MO are related to intramuscular fat deposition, since MUFA and PUFA are oilier in texture than saturated fatty acids. Apparently, the MO inclusion promoted a greater amount of MUFA and PUFA in the meat.

There are no other studies showing an improvement of lamb tenderness when animals were fed OEO. In the study of Simitzis et al. [57], the dietary oregano essential oil supplementation on lamb did not influence the tenderness of *Longissimus thoracis* muscle. Demirel et al. [58] reported that the e ffect of oregano oil was not significant on carcass and lamb meat quality attributes.

Contrasting e ffects of OEO on the tenderness and shear force of meat from other species are reported. Cheng et al. [59] observed that dietary OEO enhanced the tenderness and overall acceptance of pork. Forte et al. [60] showed that dietary oregano essential oil increased the meat tenderness, but it did not modify other pork quality traits, such as the pH, colour, drip loss, and cooking loss. However, OEO improved consumer perceptions of the meat quality, such as consistency and overall liking. In contrast, Ranucci et al. [61] evaluated a plant extract mix (chestnut and oregano essential oil) in a pig diet and evaluated the pig performance and meat quality. The fresh meat colour, pH, and WB shear force was not affected by OEO supplementation. Simitzis et al. [29] did not find any change in the meat shear force and sensory traits of meat from pigs supplemented OEO. As well, Rossi et al. [62] reported an enhancement of sensory attributes in meat from pigs supplemented plant extract (*Lippia* spp.) but did not find any tenderness improvement in the meat.

When adding essential oils to meat products, it has been pointed out that protein oxidation reduces meat tenderness, but the essential oils of oregano and rosemary can protect the thiols in pork patties and reduce the disulphide crosslinks of the myosin heavy chains, avoiding the tenderness reduction of meat [63].

Finally, Lei et al. [64] demonstrated that the addition of essential oil-cobalt had a significant e ffect on the meat quality of tested goats. Similarly, Velasco et al. [65] found that the incorporation of dietary dry oregano at 1% and 5% in the diet of Boer goats did not a ffect the meat quality characteristics.
