*2.1. Experimental Design and Diets*

Meat samples (LTL) were obtained from yearling Angus steers that had been fed with finishing diets including increasing levels of wet DG with solubles. For this purpose, a total of thirty-six weaned steers (initial live weight, 191 ± 12 kg) were randomly allocated to 12 pens (three steers per pen) and fed with a high-concentrate diet for three weeks to adapt to the feeding system. Then, the pens (3 pens per treatment) were randomly assigned to one of the four dietary treatments. Steers were fed for 70 days. The control diet consisted of cracked corn grain, soybean meal, and alfalfa hay. The dietary treatments consisted of four levels of inclusion of DG, replacing the corn and soybean meal: 0% (0DG), 15% (15DG), 30% (30DG), or 45% (45DG) (dry matter (DM) basis; Table 1). The diets were formulated to be isoenergetic (2.95 Mcal/kg DM) and isoproteic (14.9%), except for 45DG which supplied 17.9%. Three samples of each diet were collected during the feeding, vacuum packed, and stored in darkness until analysis. No effect of feeding diets on animal performance during the trial was observed and animals showed an average daily gain of 1.586 ± 0.045 kg/d. Details of the feeding diets and cattle performance have been previously reported [20,21].


**Table 1.** Composition of the finishing diets on a dry matter (DM) basis (g/100 g DM).

<sup>1</sup> Dietary treatment: 0DG, control, 15DG 15% DG, 30DG 30% DG and 45DG 45% DG. <sup>2</sup> Nucleus composition (Vetifarma SA, La Plata, Argentina): vitamin A: 1,000,000 IU/kg; vitamin D3: 200,000 IU/kg; vitamin E: 6500 IU/kg; vitamin B1: 650 ppm; manganese: 12,000 ppm; zinc: 12,000 ppm; copper: 6000 ppm; cobalt: 40 ppm; selenium: 60 ppm; iodine: 200 ppm; with added calcium 0.05%.

When animals reached slaughter conditions (commercial endpoint based on visual appraisal and final body weight of 316 ± 14 kg on average), they were transported to a licensed commercial abattoir in a single group. Animals were slaughtered according to standard commercial procedures on the same day after mixing groups to avoid perislaughter effects. Carcasses were suspended through the Achilles tendon and were not electrically stimulated.

#### *2.2. Sample Preparation*

After slaughter, carcasses were individually graded and chilled at 4 ◦C. Forty-eight hours later, a section of the LTL from the 13th rib region was removed from the striploin of each carcass and transported under refrigeration to the Food Technology Institute of the National Institute of Agricultural Technology (INTA), Buenos Aires, Argentina. At 72 h post-mortem, two steaks (2.5 cm thick each) from each rib section were stored at one of the following retail display conditions that resembled commercial local practice:


Other steaks from each rib section were stored at −80 ◦C until further analysis.

## *2.3. α- and γ-Tocopherol, β-Carotene, Retinol and Lutein Content*

The contents of α-tocopherol, γ-tocopherol, β-carotene, α-carotene, retinol, and lutein from feedstuffs and meat samples were determined as described by Buttriss and Diplock [22] with modifications by Descalzo et al. [23]. Briefly, 5 g of lean tissue was placed in a plastic conical tube containing 10 mL of phosphate buffer (0.05 M; pH 7.7) and homogenized for 2 min at 3000 rpm with an Ultraturrax T25 homogenizer (IKA, Darmstadt, Germany). Aliquots of 1 g of homogenate were placed into a screwcap test tube with 3 mL of pyrogallol 1% in ethanol. Thereafter, 0.3 mL of KOH 12 N in water was added to each tube for saponification. The tube contents were mixed by vortexing for 30 s, and placed in a stirred water bath for 30 min at 70 ◦C. After cooling, 1 mL of water was added to each tube. Following the addition of 5 mL of n-hexane, the samples were mixed by vortexing for 2 min; the upper hexane layer was then transferred into a new screw cap tube

and the aqueous phase was reextracted with 5 mL of n-hexane. The combined extracts were taken to dryness under a dry nitrogen gas stream, and the residue was dissolved in 500 μL of absolute ethanol (J.T. Baker, Mexico, HPLC grade) and filtered through a 0.45 μm-pore nylon membrane before injection of samples. All samples and standards (external standards for each vitamin) were analyzed by reverse-phase high-performance liquid chromatography (HPLC) using a Thermo Scientific Dionex UltiMate 3000 RS system consisting of a quaternary pump with a membrane vacuum degasser connected to an autosampler with an injection loop (10 to 100 μL) and a C18 column (250 × 4.6 mm i.d., Alltima, 5 μm particle size; Alltech, Argentina) fitted with a guard column (Security GuardAlltima C18, Alltech, Argentina). The mobile phase was ethanol:methanol (60:40, *v*/*v*) at a flow rate of 1 mL/min. The technique was optimized to determine tocopherols, carotenoids, and retinol within the same elution time of 25 min. Tocopherols were detected by fluorescence at λex = 296 and λem = 330 nm. A diode array detector was set at λ = 445 nm and λ = 325 nm for the detection of carotenoids and retinol, respectively. Calibration curves were performed with DL-α-tocopherol (Merck, Darmstadt, Germany), γ-tocopherol, β-carotene, lutein, and retinol standards (Sigma-Aldrich, St. Louis, MO, USA) diluted in ethanol. Chromatograms were recorded using the Chromeleon 6 software.

For feedstuff samples, the methodology was identical, except that the samples were diluted 1 to 10 with ethanol before injection.

#### *2.4. Muscle Ferric Reducing Antioxidant Power (FRAP)*

The FRAP assay applied to meat samples measures endogenous ions that could react with tripyridyltriazine (TPTZ) and develop blue color. Following the procedure described by Ou et al. [24] and modified by Descalzo et al. [25], 5-g chopped meat samples were disrupted for 2 min at 3000 rpm with an Ultraturrax homogenizer (IKA, Staufen, Germany) in 10 mL of potassium phosphate buffer (0.05 M, pH 7.7). Homogenates were centrifuged at 10,000× *g* for 30 min at 4 ◦C and the supernatant was collected. Then, 83-μL aliquots of supernatant were added to 2.5 mL of FRAP buffer containing 10 mM TPTZ, 40 mM HCl and 20 mM Fe2Cl3 (Sigma-Aldrich, St. Louis, MO, USA) added to 300 mM acetate buffer. The reaction mixture was incubated for 5 min at 37 ◦C in a water bath and then cooled in an ice-water bath for 10 min. Immediately after, samples were measured at λ = 593 nm (Spectrometer UV–vis-BIO Lambda 20, Perkin Elmer). Another 83-μL aliquots of supernatant were added to 2.5 mL of TPTZ/HCl solution, without the addition of Fe2Cl3, to determine endogenous Fe+2 content. The FRAP activity of the samples was measured against a calibration curve made with ferrous sulfate (Fe2SO4·7H2O, Sigma-Aldrich de Argentina SA) within a range from 100 to 1000 μM, and results were expressed as Fe+2 equivalent in μM.

#### *2.5. Thiobarbituric Acid Reactive Substances (TBARS)*

TBARS were analyzed by the steam distillation method, as described by Pensel [26] with modifications of Descalzo et al. [23], and expressed as mg of malonaldehyde (MDA) per kg of lean muscle. Briefly, triplicate aliquots (5 g) of meat were chopped and processed in a stomacher-type homogenizer for 2 min in bags containing 12.5 mL of trichloroacetic acid (Merck, Darmstadt, Germany) solution (20% *w*/*v*) in 1.6% metaphosphoric acid. Then, 12.5 mL of water was added, and the mixture was processed for another 30 s. Slurries were filtered and aliquots of 5 mL were separated. An equal volume (5 mL) of 0.02 M 2-thiobarbituric acid (Sigma-Aldrich, St. Louis, MO, USA) was added. Samples were incubated at 80 ◦C for 1 h until a pink color was developed. Color intensity was determined at maximum absorption, λ = 530 nm, and TBARS concentrations were calculated from a calibration curve by using 1,1,3,3-tetraethoxypropane (Sigma-Aldrich, St. Louis, MO, USA) as standard within a range from 0 to 0.5 μM.
