**1. Introduction**

Lamb meat is widely consumed in some geographical areas, for instance in Mediterranean countries, but has a non-healthy nutritional image, mostly due to the general idea of having high levels of saturated fatty acids (FA), variable contents of trans-fat, and low levels of omega-3 polyunsaturated FA [1]. However, the quality of lamb meat and its FA profile is closely related to the feeding conditions of the animals. The intramuscular fat (IMF) of lambs reared under intensive feeding conditions is characterized by high levels of saturated and omega-6 FA, and a low amount of omega-3 FA [2–5]. In contrast, meat from grass-fed lambs has shown a more desirable FA composition, with lower contents of saturated FA and higher levels of omega-3 FA [6].

In the last decade, there has been growing interest in finding appropriate and natural ways to manipulate IMF composition, with grazing being one of the best alternatives. However, in regions where fattening on pasture is not feasible for climatic reasons, other approaches to increase omega-3 FA have been evaluated, such as adding plant-derived

**Citation:** Núñez-Sánchez, N.; Avilés Ramírez, C.; Peña Blanco, F.; Gómez-Cortés, P.; de la Fuente, M.Á.; Vioque Amor, M.; Horcada Ibáñez, A.; Martínez Marín, A.L. Effects of Algae Meal Supplementation in Feedlot Lambs with Competent Reticular Groove Reflex on Growth Performance, Carcass Traits and Meat Characteristics. *Foods* **2021**, *10*, 857. https://doi.org/10.3390/ foods10040857

Academic Editors: Claudia Ruiz-Capillas, Ana Herrero

Received: 18 March 2021 Accepted: 13 April 2021 Published: 15 April 2021

Herranz and Mohammed Gagaoua

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

oils rich in omega-3 FA [7–9]. Dietary marine algae have been shown to improve meat nutritional value in lambs [8,10–13] due to their high eicosapentaenoic (EPA), docosapentaenoic (DPA) and docosahexaenoic (DHA) FA contents. Very recently, our group has reported that the dietary inclusion of marine algae at the level of 2.5% enhanced omega-3 FA contents in lamb meat with competent reticular groove reflex (RGR), with the increase being significantly higher when the marine algae was bottle-fed in comparison to the same amount of algae meal mixed in the concentrate [14]. Fostering the RGR of the newborn animal into adulthood and using it to include emulsified lipid sources into the abomasum, bypassing the rumen, has been demonstrated as a fruitful strategy to enhance the healthy unsaturated FA presence in ruminant milk and meat [14,15].

Marine algae supplementation may have undesirable effects on growth performance, carcass traits and meat quality characteristics [16], such as a decrease in average daily gain and feed intake, and thus an extension of the fattening period [10,17]. Moreover, the high content of polyunsaturated FA in the IMF from lambs fed marine algae increases its peroxidability index [14], which could lead to meat quality degradation during storage [18]. Changes occurring in the meat micro- and ultra-structure during the aging process are associated with favorable modifications in its meat tenderization and water-holding capacity. However, as stated previously, the oxidation of lipid components, as well as the destabilization of meat color, can negatively affect the meat's final value [19]. Again, marine algae are a good source of antioxidants. Most researchers consider polyphenolic compounds (i.e., phenolic and cinnamic acids, phlorotannins, and bromophenols) among the main factors responsible for such antioxidant properties [20].

The farm to fork strategy includes hygienic, compositional, nutritional, sensory, and technological quality characteristics in the general concept of meat quality throughout the food chain, in order to obtain high-quality products [21].

The aim of this study was to assess the effects of supplementing lambs with competent RGR with marine algae as a source of omega-3 FA on growth performance, carcass traits and meat quality characteristics.

#### **2. Materials and Methods**

#### *2.1. Experimental Design and Diets*

This experiment was carried out on the premises of the Animal Production building of the University of Córdoba (Spain). Details about the experimental design and diets have recently been published [14]. Briefly, a total of 48 male Manchega-breed lambs at 42 days of age were weighed (11.6 ± 1.67 kg) and assigned in pairs of similar body weights to 1 of 24 adjacent pens (1.40 m<sup>2</sup> raised slatted floor cages with individual troughs for feed and water, within an environmental controlled room). Pens were distributed in 8 blocks according to average body weight and allocated randomly to one of the three treatments (16 animals per treatment): (i) the control group, which consisted of a typical pelleted concentrate without algae meal supplementation (NOALG), (ii) the algae meal in concentrate group, which received the same concentrate as NOALG but mixed with 2.5 % algae meal (*Aurantiochytrium limacinum*; Forplus, Alltech Spain, Guadalajara, Spain) plus 250 mL daily of milk replacer in a single feeding (ALGCON), and (iii) the algae meal in milk replacer group, which received the same concentrate as NOALG plus 250 mL daily of milk replacer supplemented with algae meal in a single feeding (ALGMILK). The concentrate was composed by 70% cereals (barley, maize and wheat, 40:20:10), 20% soybean meal, 6% wheat bran and 4% minerals and vitamins, so its calculated composition was 16% crude protein and 11.0 MJ/kg metabolizable energy, as fed. In the ALGCON and ALGMILK diets, milk replacer provided an extra 0.89 MJ of metabolizable energy per day, and algae meal a total of 0.75 MJ/kg of concentrate. Besides this, these diets caused an increase in the crude protein intake of 10 g/d with the milk replacer and 3 g/kg of concentrate with the algae meal. The ALGCON and ALGMILK treatments provided ~584 mg of omega-3 FA per 100 g of concentrate consumed. Lambs in the ALGCON and ALGMILK groups had

competent RGR. All animals had free access to wheat straw and fresh water throughout the whole experimental period (49 days on fattening).

#### *2.2. Sampling and Analysis*

The average body weight per pen was recorded weekly during the experiment, in the morning before feeding. The average daily feed intake (ADFI, kg/day) per pen was calculated daily by weighing each day the amount of concentrate offered and subtracting the amount of feed refusals found in the feeders the following morning. Besides this, average daily gain (ADG, kg/day) and feed conversion ratio (FCR, feed to gain kg/kg) were calculated for each pen.

When the average body weight of the set of lambs reached ~25 kg (in the range of the commercial slaughter weight for this type of lambs), the animals were tagged to track their carcasses, and transported from the Animal Production facilities at the University of Cordoba to a commercial slaughterhouse (COVAP, Pozoblanco, Córdoba, Spain), located ~90 km (~1.0 h) away, in a vehicle adequately conditioned. Then, the animals were placed in pens (8 lambs per pen) and remained there for approximately 14 h, with free access to water but not feed. After lairage, lambs were stunned, slaughtered, and dressed.

The carcasses of the animals were weighed in the first 45 min after slaughter (hot carcass weight, HCW). The dressing was calculated as the ratio of HCW to final body weight and expressed as a percentage. Lamb carcasses were classified according to their weight as class A (<7 kg), class B (7.1 to 10 kg), and class C (10.1 to 13 kg). A trained and experienced technician from the slaughterhouse visually graded carcass fatness considering the size of the kidney and pelvic fat deposits (types 1, low fat; 2, medium fat; 3, high fat) and muscle color (pale pink, pink, red) [22].

After 2 h at room temperature, all carcasses were chilled at 4.0 ◦C for 24 h in a commercial chiller and transferred to the Animal Production laboratory without disrupting the cold chain. The left shoulders were weighed and dissected into muscle, bone and fat (subcutaneous, pre-scapular and intermuscular) and the remaining tissues (major blood vessels, ligaments, tendons and fascias). Each fraction was weighed, and the results were expressed as the percentage of total shoulder weight to provide an estimate of the carcass composition. At 24 h post-mortem, the *Longissimus thoracis* muscle (T6 to T13 vertebrae) from each left carcasses was removed and divided into three pieces (T6, T7 to T12, and T13 vertebrae), which were further used for the assessment of meat quality (pH, color, drip and cooking losses, and Warner–Bratzler shear force) and parameters related to meat oxidative stability. The *Longissimus thoracis* muscle from the right carcass was vacuum-packed, aged in a refrigerated chamber at 2–4 ◦C in the dark for 6 days (7 d postmortem), and further used for the same determinations.

The methodology of the measurements is described in detail in Avilés et al. [2]. Briefly, the drip loss (DL) of each sample was expressed as the percentage of weight loss of a sample hanging at 4◦ for 24 h, related to the initial weight. The pH was measured by inserting the glass electrode of a portable pH-meter (Crison® PH25, Hach Lange, Barcelona, Spain) approximately 1 cm into the *Longissimus thoracis* muscle between the T11 and T12 junction sites. Meat color was measured on the freshly cut surface of the middle section of the T12 vertebra, after 30 min of blooming at room temperature in the dark. A CM-2600d hand-held spectrophotometer (D65 illuminant, 8 mm diameter aperture, 10◦ standard observer, 8◦ viewing angle; Minolta Inc., Osaka, Japan) was used for color determination, according to the CIE system (CIE, 1986). Three measurements of color coordinates, expressed as L\* (lightness), a\* (redness) and b\* (yellowness), were performed, and the average was used for the calculation of color saturation (C\*) and hue (h◦). For the determination of cooking loss (CL) and Warner–Braztler shear force (WBSF), the samples were weighed, cooked in a plastic bag in a water bath at 75 ◦C until the temperature in the center of the sample reached 70 ◦C (monitored by a HI 98509 Checktemp® Pocket Thermometer, Hanna Instruments, Guipuzcoa, Spain), and then cooled at room temperature for 30 min, blotted dry, and weighed again. CL was expressed as the percentage of weight loss related to

the initial weight. WBSF values, expressed in kg/cm2, were taken for muscle cores of 1 cm<sup>2</sup> cross-sections using a texture analyzer (TA.TXT-2, Stable Micro Systems, Surrey, UK) equipped with a Warner–Bratzler shearing device (Mitutoyo series 500, Mitutoyo Corporation, Aurora, IL, USA). A total of 10 measurements was performed in each meat sample, and the average was provided as the WBSF value of the sample. The extent of lipid oxidation was assessed by measuring the thiobarbituric acid reacting substances (TBARS) using the method of Tarladgis et al. [23], as described in Avilés et al. [2]. The 1,1,3,3-tetraethoxypropane (TEP) standard curve was used for calculating the TBARS concentration and the results were expressed as mg of MDA kg−<sup>1</sup> of meat. The antioxidant capacity of the meat was determined through the 2,2-diphenylpicrylhydrazyl (DPPH) radical scavenging capacity assay. The meat hydrophilic and lipophilic extracts were obtained following the method described by Folch et al. [24]. The determination of the free radical scavenging activity of the hydrophilic and lipophilic extracts of the meat samples (DPPH water and DPPH fat, respectively) was carried out following the procedure described by Brand-Williams et al. [25]. The absorbance values in both hydrophilic and lipophilic extracts were expressed as % radical scavenging activity (RSA). Total polyphenols content was determined with the Folin–Ciocalteu reagen<sup>t</sup> according to the procedure described by Singleton [26]. Total phenolic compounds were quantified using a gallic acid reference standard, and the results were expressed as mg of gallic acid equivalents (GAE)/100 g of meat.

### *2.3. Statistical Analysis*

SAS UE 3.8 software (SAS Institute Inc., Cary, NC, USA) was used to perform the statistical analyses. Statistical significance was declared at *p* < 0.05. Productive and carcass traits, except for grading, were analyzed with the MIXED procedure. The statistical model included the treatment as fixed effect and the pen nested within the treatment as the random effect. When the fixed effect was significant, differences between least squares means were assessed by Tukeys's test. A repeated measurements analysis of meat data was carried out with the MIXED procedure. The statistical model included the fixed effects of treatment, ageing time, and their interaction; the repeated effect was ageing time; the subject of the repeated measurements was the animal nested within the treatment and the pen. When the fixed effects of the repeated measurement model were significant, differences between least squares means were assessed by paired *t*-test.
