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 effect, 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
]. 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 agreement 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 affect lipid metabolism in animal tissues by exerting beneficial effects on the antioxidant enzyme activity. Furthermore, phenolic compounds also prevent the production of reactive oxygen species and the off-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 effectively inhibit the oxidative reactions by preventing the formation of radicals, and it appears to be a more effective way of slowing down hte lipid oxidation of animal products compared to post-mortem addition [43
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
], 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
]. In the present study, monensin has a similar effect 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
]. 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 differences 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 effect of oregano oil was not significant on carcass and lamb meat quality attributes.
Contrasting effects 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 effect 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 affect the meat quality characteristics.
The addition of the OEO and/or monensin in the lamb diet influences the colour (L*, a*, b*, and C*) parameters of the meat. According to recent reports by Payne et al. [66
], the colour values in finishing lambs (240 d old) are L* = 34.3, a* = 5.7 and b* = 16.9. The L* values in the present study are higher (L* = 40), meaning a lighter meat. The high L* value could be attractive to consumers that prefer lighter meat [34
]. A positive result could be that yellowness (b*) was relatively lower compared to Payne et al. [66
], since consumers do not expect to find high b* in fresh meat. Lightness (L*) was higher in the meat from oregano and monensin treatments compared to the control. As noted by Rivaroli et al. [34
], in feedlot-finished young bulls that were fed with essential oils, L* values were superior to the other literature data of cattle finished in feedlot.
Colour is one of the most important quality characteristics to determine the consumer decision for purchasing meat. The natural colour of meat is produced by the myoglobin and hemoglobin pigments. These three components that define the colour of meat are all highly susceptible to oxidation [67
]. An unattractive brown colour can result from the oxidation of red oxymyoglobin to metmyoglobin. The mechanisms that modify pigment distribution in animal tissues could be activated by lowering hemoglobin oxidation by dietary OEO supplementation [57
]. Antioxidants have the ability to retard meat colour deterioration by extending the red colour and delaying metmyoblobin formation. Simitzis et al. [57
] included 1 mL OEO/kg in lambs diet and found higher a* and b* values. In lamb meat, Nieto et al. [33
] indicates that lambs fed with 3.7% and 7.5% of oregano leaves produced significant differences regarding the colour values. In this study, as the storage period was prolonged, the L* and b* values increased and the a* value decreased. Similarly, Simitzis et al. [29
] pointed out that supplementing lamb diets with OEO resulted in significant effects on meat colour (L*, a*, and b*).
Different results have been found in other animal species. The colour of pork patties was investigated by Carpenter et al. [69
]. They did not find significant changes in colour parameters by the addition of grape seed and bearberry extracts to the diet. Similar results were obtained for fresh chicken breast meat [25
], whereas the incorporation of rosemary and oregano extracts in pig rations resulted in significant differences in the luminosity of meat.
Similar results have been reported by Camo et al. [24
], who reported that the packaging of lamb meat using rosemary and oregano extracts resulted in the difference in meat redness of the treated animals compared to the controls. Intrinsic characteristics of the animals have also an effect on meat colour. Lamb meat colour changes by body weight, sex, and breed [70
]. In this way, Hopkins and Fogatry [71
] found that the colour of the m. Longissimus thoracis
varied with breed. Based on the findings obtained in this study, the effect of OEO on meat colour parameters was found to be in agreement with the literature and within the reference ranges.
Possibly, components from OEO accumulate on the meat, as it has been reported in non-ruminants [72
]. Essential oils have an antioxidant activity when used directly on the meat or supplemented ante-mortem [46
], which may protect meat pigment from oxidation throughout storage. If dietary OEO are accumulated in meat, it might mean that they passed the rumen without being degraded. Alternatively, colour might remain stable, as carvacrol supports the activity of glutathione peroxidase and superoxide dismutase, which are two of the most important antioxidant enzymatic complexes in mammals [75
It is important to highlight that dietary MO not only maintained a higher and more stable redness, yellowness, and saturation during storage, but it also reduced the compression force, and, although not significant (p
> 0.05), lower TBARS were observed. Improvement of the oxidative stability of MO meat was shown by the stable colour during storage. Dietary antioxidants such as tocopherol deposited in meat may avoid rancidity or the oxidation of tissue components [76
]. Carvacrol has a high antioxidant activity [65
]. It is possible that the antioxidant activity of OEO is more related to protein protection (pigments) than to lipid components. Some spices and their extracts such us oregano have a high antioxidant activity due to their phenolic compound content, which improves the nutritive value and the quality of meat, because they prevent meat oxidation [65
Moura et al. [77
] evaluated dietary monensin (SM) and incrementing levels of copaiba (Copaifera
spp.) essential oil (CO) on nutrient intake, time spent eating and ruminating, performance, carcass traits, and the meat quality of feedlot lambs. They observed that the addition of CO at 1.5 g/kg increased Warner Bratzler shear force and decreased L* intensity in Semimembranosus
meat in comparison to SM.
3.4. Fatty Acid Profile
The supplementation treatments, SM and MO, modified the fatty acid profile compared to the other treatments, whereas HO treatment modified the fatty acid profile undesirably. The similarity between SM and MO might imply that as they modify the rumen environment, the growth rate of rumen microflora changes, resulting in changes in the fermentation profile [49
]. These changes impact the fatty acid profile [79
], as it has been reported that monensin was at least partially effective to inhibit the biohydrogenation of unsaturated FAs in the rumen. This consequently increased the percentage of n-6 and n-3 PUFAs and conjugated linoleic acid in milk.
) in ruminant diets [55
] and essential oils have bactericidal or bacteriostatic effects [13
]. The antibacterial effect is more evident in Gram-positive bacteria, where the cell membrane acts directly with hydrophobic components [80
]. SM and some compounds in essential oils are lipophilic; hence, they do not penetrate the membrane of Gram-negative bacteria [81
]. However, Gram-negative bacteria are not completely resistant to the lipophilic compounds in essential oils, because low molecular weight molecules can interact with the cellular lipid bilayer [82
]. Thymol and carvacrol can also disintegrate the external membrane of Gram-negative bacteria [83
]. Hence, SM and essential oils affect equally Gram-positive and Gram-negative bacteria, but they use different pathways. The levels of essential oil inclusion are fundamental, because it has been reported that low levels are not enough to modify the ruminal microflora and high levels reduced significantly the bacterial counts, while neither of them change the ruminal fermentation rate [78
In this regard, several authors have already shown the mechanism of SM inducing ruminal environmental changes. It has been pointed out that SM modifies the ruminal and intestinal microflora, which causes a higher nitrogen and carbon retention in the animal [3
]. Additionally, SM promotes the growth of propionic acid-producing microorganisms. Therefore, the concentration of propionic and butyric acids increase, while acetate decreases in ruminal fluid. This leads to an acetate:propionate ratio decline [3
], which in turn favours the recovery of energy used by the animal [79
]. Additionally, SM reduces the formation of methane and lactic acid produced by other microorganisms [87
In the present study, most of the FAs that were statistically different are saturated or monounsaturated. This might indicate that triglycerides are accumulating in the intramuscular adipocytes within the neutral lipid fraction. Nevertheless, phospholipidic variations may take place, considering that this fraction is easily altered with the diet [89
]. An advantage of monensin is that it does not only change the microbial populations in the rumen to such levels that the fatty acid profile is modified, but it also changes the digestibility of nutrients and the utilisation of proteins [3
]. Ionophores such as SM alter the fat deposition in beef, particularly arachidonic (C20:4) and linolenic (C18:3n3) acids [91
]. Furthermore, in bovine milk, SM also changes the amount of fat and increases C18:2 [93
]. However, an outstanding characteristic of OEO is that its active components (carvacrol and thymol) of OEO are potent antimicrobials affecting populations such as E. coli
, Staphylococcus aureus
, Salmonella typhimurium
, protozoa, fungi, Ruminococcus fibrisolvens
and Fibrobacter succinogenes
, which modifies ruminal fermentation and is fundamental in the conversion of dietary nutrients to muscle tissue [11
]. Specifically in sheep, there is evidence that carvacrol decreases acetate concentrations and increases propionate and butyrate. Both are volatile fatty acid precursors of muscle and fat components in the animal [14
Other essential oils have also been studied in lamb nutrition, and their results are promising. Parvar et al. [94
] investigated the effects of Ferulago angulata
(chavil) essential oil (FAE) dietary supplementation on growth performance, meat quality characteristics, and the fatty acid composition of longissimus
muscle (LM) in fattening lambs. They found that the supplements increased the concentrations of PUFA and decreased SFA contents in meat. Lambs that used diets containing FAE had a lower n-6:n-3 fatty acid ratio compared to the control treatment. They concluded that FAE (up to 750 mL/kg DM) can be used in diets without adverse effects on physical parameters or the chemical composition of meat, and it enhanced the anti-oxidative status of lamb’s meat. On the other hand, negative effects of monensin in sheep have been observed. A study of lamb supplementation with monensin (zilpaterol hydrochloride, ZH; 0 or 10 mg/lamb daily) showed a decrease in the content of C20:5n3 (eicosapentaenoic acid), C22:6n-3 (docosahexaenoic acid), and total omega-3 fatty acids, compared with the zero ZH group [95
In monogastric animals such as chickens, the inclusion of carvacrol and thymol fat increases PUFA and decreases SFA in breasts [75
]. In this study, the PUFAS concentration was not different (p
> 0.05) between the control and monensin treatment. However, the PUFAs concentration was higher in MO. Promising results of OEO have also been reported in pork. Cheng et al. [59
] reported that dietary OEO enhanced the sensory attributes and anti-oxidative status of pork meat by improving IMF and n-3 PUFA proportion and antioxidant capacity.