*3.3. Colour*

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 [67,68]. 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 agreemen<sup>t</sup> 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 [46,73,74], 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 [49,55,78]. These changes

impact the fatty acid profile [79], as it has been reported that monensin was at least partially e ffective 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.

SM (Rumensin ®) in ruminant diets [55] and essential oils have bactericidal or bacteriostatic effects [13]. The antibacterial e ffect 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 [81,82]. 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 a ffect equally Gram-positive and Gram-negative bacteria, but they use di fferent 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 [3,84–86], 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 [87,88].

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 [89,90]. 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 [91,92]. 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 a ffecting 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 [11–13]. 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 e ffects 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 e ffects 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 e ffects 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 di fferent (*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.

#### **4. Materials and Methods**

#### *4.1. Animal Handling and Treatment Description*

Twenty male lambs (Dorper x Pelibuey. Initial body weight, 26.2 ± 3.9 kg) were randomly assigned to one of five treatments (*n* = 4 per treatment); CON: control, basal diet; SM: basal diet + 33 mg/kg monensin sodium (Rumensin 200®); LO: basal diet + 0.2 g/kg DM (dry matter) of OEO; MO: basal diet + 0.3 g/kg DM of OEO, and HO: basal diet + 0.4 g/kg DM of OEO. The basal diet was formulated for 27 kg-lambs to gain 250 g daily [96], and it consisted of 20% alfalfa hay and 80% concentrate DM basis (Table 3). OEO (62.7% carvacrol concentration) was extracted from the leaves (*Lippia* S. *berlandieri*) by steam distillation and obtained from Natural SolutionTM Jimenez, México. The diet adaptation period lasted 15 d, and the experimental period was 70 d.

**Table 3.** Ingredients and chemical composition (DM basis) of diets of finishing hair lambs supplemented with Carvacrol.


CON: 0 g/Kg MS Carvacrol; M: 33 ppm/Kg MS Monensin; Low: 0.2 g/Kg MS Carvacrol; Medium: 0.3 g/Kg MS Carvacrol; High: 0.4 g/Kg MS Carvacrol. 1 P 12%; Ca 11.5%; Mg 0.6%; Mn 2160 ppm; Zn 2850 ppm; Fe 580 ppm; Cu 1100 ppm; I 102 ppm; Co 13 ppm; Se 9 ppm; Vitamins: A 22,000 UI/Kg; E 24,500 UI/Kg.

Animals were housed in individual pens (equipped with feeding and drinking troughs) and fed twice daily (8:00 and 16:00 h). All experimental procedures with the animals complied with the institutional Bioethics code and Animal Welfare Guidelines, fulfilling the Official Mexican Norms. The protocol was approved by the Institutional Bioethics and Animal Welfare Committee on September 21, 2007, with official number P/302/2017. Description and declarations in this document also followed the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines.

At day 71 (average body weight 45.9 ± 2.82 kg) and after a 12-h fasting period, the animals were electrically stunned and slaughtered by exsanguination using conventional methods in the Meat Science Complex, Department of Animal Science and Ecology (UACH). The dressed carcasses were hung by Achilles tendon suspension in a chiller at 2 ◦C. At 48 h post-mortem, a portion of *Longissimus lumborum* (LL, 15 cm approx.) was excised longitudinally (after the 12th rib) from the right half of the carcass. The portions of *LL* were vacuum packed (Easy-pack, Rhino, Germany) in 7 μm thickness pouches and fast-frozen (−20 ◦C) until further analyses.

After two weeks, frozen muscles were sliced transversally to obtain 16 steaks (2 cm thickness) transversal to the muscle fibre direction. Steaks were analysed as follows: the two cranial steaks were

tested for lipid oxidation, the next 2 were tested for colour, the following three were tested for texture, and the last 2 were tested for the fatty acid profile.

Samples for lipid oxidation, colour, and texture were thawed at 3 ◦C for 24 h. Then, to simulate commercial retail display, samples were packed under a modified atmosphere (MAP, 75% O2-25% CO2, PVC Cryovac ® trays, PET-PVDC-PE top lidding. Rhino 4 sealed packer. USA) and placed in a chilled storage (3 ◦C, artificial led light, 12 h/d, 700 lx). Samples for fatty acids were re-packed in vacuum bags and kept frozen for 7 d more until fatty acid analysis. All measurements were performed in triplicate except for the compression test, for which measurements were taken at least six times.

## *4.2. Lipid Oxidation*

Lipid oxidation was determined after 7 d of simulated commercial retail display by TBARS (thiobarbituric acid reactive substances), and the values were expressed as mg malonaldehyde (MDA) per kg of meat, according to the distillation method [97].

#### *4.3. Compression Strenght Analysis*

To determine the compression strength of the samples, the methodology of the American Meat Science Association Guidelines [98] was followed. MAP chilled stored samples were placed in sealed plastic bags and cooked in a water bath (Fisher Scientific ® mod. Isotemp 215, Waltham, MA, USA) until an internal temperature of 72 ± 1 ◦C was reached at the geometric centre. Temperature was monitored with a thermocouple wire, which was attached to an infrared digital thermometer (Fisherbrand ™ Traceable ™ Infrared Thermometer with Trigger Grip). Subsequently, cooked steaks (2 cm thickness) were stored at 1 ◦C for 24 h. Compression strengt was determined by the "punch and die" method [99], which was modified by Jones et al. [100]. Steaks (2 cm thickness) were perforated transversally (parallel to muscle fibers) at least 6 times, and compression values were averaged per every steak (3 steaks per animal). Compression strength analysis was performed with a TA.XT2*i* texture analyser (Stable Micro Systems, Surrey, UK), attached to a 30 kg load cell, and set with a 20 mm cylindrical probe (Crosshead speed of 100 mm min−<sup>1</sup> at 3 cm of distance). Data are expressed in Newtons/cm3.
