2.3. Measurements, Sample Collection and Laboratory Analyses
Samples of the diets were taken from each batch mix, and aliquots were pooled in one sample per concentrate for chemical analyses [19
Average BW per pen was recorded 2 h before morning feed delivery at the beginning of the trial and every 30 days thereafter. Total feed intake in each pen was calculated as the sum of feed consumed throughout the experimental period. Following, average daily gain (ADG, kg/day), average feed intake (FI, kg/day) and feed conversion ratio (FCR, feed to gain kg/kg) were calculated for each pen.
On day 91 of the trial, all heifers were weighed, and three heifers per pen (12 heifers from each treatment) having the final BW closest to the median weight of the pen were tagged to track their carcasses. Then, the heifers were transported from the farm to a commercial abattoir located approximately 145 km (~1 h 40 min), in an adequately conditioned vehicle. After a 10-h rest period, the heifers were slaughtered by captive-bolt pistol, exsanguinated and dressed using standard commercial procedures. Thus, the carcasses were not electrically stimulated.
The carcasses were weighed (hot carcass weight (HCW)) and graded for conformation (E, excellent; U, very good, R, good; O, fair; P, poor) and fatness (1, low; 2, slight; 3, average; 4, high; 5, very high) by trained personnel at the abattoir, according to the EU official classification system for beef carcasses [21
]. Afterwards, in the first 45 min after slaughter, temperature (Tª0
), pH (pH0
) and lean and subcutaneous fat colour of the carcasses were registered. Temperature and pH were monitored in the longissimus muscle by using a portable pH-meter (Crison PH25, Hach Lange, Barcelona, Spain) equipped with a glass electrode suitable for meat penetration and automatic temperature compensator. The probe was inserted in a scalpel incision approximately 1 cm into its geometrical centre at right angles to the sagittal plane surface. The pH electrode was recalibrated at room temperature every five samples, using two standard buffer solutions at pH 4.0 and 7.0, and rinsed between measurements. Three measurements for lean (rectus abdominis muscle) and caudal subcutaneous fat colours were taken with a hand-held spectrophotometer (CM-2600d, Minolta Co., Osaka, Japan; λ: 360–740, Δλ: 10 nm, specular component excluded mode, D65 standard illuminant, 10° visual angle and 8 mm measurement aperture), standardised against a white tile (L* = 97.78, a* = 0.19, b* = 1.84) and light trap supplied by the manufacturer. Colour coordinates were expressed as L* (lightness), a* (redness) and b* (yellowness), and the average value for each of them was reported and used for calculation of C* (chroma or vividness of h*) and h* (hue angle or the degree to which a colour stimulus can be described as red, green, yellow, blue or the combinations between them) as C* = (a*2
and h* = arctangent (b*/a*) × 360°/(2 × π) [22
]. For fat colour, readings were taken on subcutaneous fat covered with plastic food wrap (calibration was performed using the food wrap to maintain the integrity of the results). The carcasses were then split along the spine into two halves and chilled at 4 °C for 24 h in a commercial chiller. After, the half carcasses were reweighed to obtain the cold carcass weight (CCW), and temperature (Tª24
) and pH (pH24
) were measured as indicated above. The dressing was calculated as the ratio of cold carcass weight (CCW) to final BW at the farm, while chilling losses were calculated as the ratio of the difference between HCW and CCW to HCW. Both data were expressed as percentages.
The longissimus muscle was cut out from the fifth to the last thoracic vertebrae of the chilled left half carcasses and cut into three portions that were individually packed in sealed vacuum bags to get contact between the bag and the meat. The bags were identified and delivered via refrigerated transport (4 °C) to the laboratory. At the laboratory, dorsal fat thickness was measured with a stainless steel calliper at the 12th to 13th rib interfaces, over the longissimus muscle at a point three-quarters the ventral length of the ribeye (FT1) and over the latissimus dorsi muscle (FT2). The portion in each bag within each animal of origin was considered a sample that was randomly assigned to one of three different ageing times (7, 21 and 28 days) at 2–4 °C in darkness on stainless steel gratings. The bags were turned over and rotated among shelf positions every day to minimise location effects. After each ageing time, meat samples were unpacked, and pH was immediately determined. Meat samples were then subsampled as needed for determinations of drip loss (DL), cooking loss (CL), Warner–Bratzler shear force (WBSF), colour, pigment contents and oxidative stability.
For DL measurement, a block of meat trimmed of external fat and connective tissue and measuring 20 × 20 × 25 mm, was sliced so that the fibres ran across the longer axis of the sample. The slices were weighed and suspended on metal hooks placed on the inner side of the lid of a plastic airtight container so that the sample did not touch the container walls. After display for 24, 48 and 72 h at 4 °C, the slices were carefully dabbed and weighed again. The DL of each slice was calculated as the percent weight difference between the initial and final weight relative to the initial weight.
To assess CL and WBSF, a steak of 5 cm length was cut from the cranial end of each sample. Steaks were trimmed of external fat and epimysium and weighed prior to cooking. The steaks were individually placed inside plastic bags and boiled using a water bath (Precisterm 6000388, J.P. Selecta Co., Barcelona, Spain), which was preheated to 75 °C, to a final internal temperature of 71 °C [23
]. Internal temperature was monitored by an iron/constant thermocouple wire connected to a thermometer (HI 98,509 Checktemp Pocket Thermometer, Hanna Instruments, Guipúzcoa, Spain) inserted into the geometric centre of the steak. After cooking, the steaks were cooled at room temperature for 30 min, gently blotted dry by using paper towels and weighed again. Cooking loss (CL) was determined by calculating the weight difference of the steaks before and after cooking, expressed as percentage of initial weight. Then, five 1.27 cm diameter cores were removed parallel to the muscle fibre orientation from the lateral end of the cooked steaks. Peak WBSF (kg/cm2
) was measured perpendicular to the muscle fibres using a TA.XT-2 texture analyser (Texture Analyser, Stable Micro Systems, Surrey, UK) equipped with a Warner–Bratzler shear device (25 kg load cell) and a crosshead speed of 200 mm/min. The down stroke distance was 3 cm (the probe should cut the meat completely). Each core was assessed two times, and the 10 peak shear forces recorded per sample were averaged for statistical analysis.
For meat colour analysis, the steaks were placed on styrofoam trays, overwrapped with oxygen-permeable PVC film (O2
permeability = 15,500–16,200 cm3
/24 h at 23 °C) and placed in a dark chill room at 4 °C for 30 min to permit blooming before L*, a* and b* values were recorded (day 0 of colour measurement). Three measurements, changing the spectrophotometer orientation at each time, were made directly on the meat surface immediately after removing the film at non-overlapped zones of each steak, which avoided areas of connective tissue and intramuscular fat. The three values of L*, a* and b* were averaged for statistical analysis and C* and h* values were derived. Then, the samples were overwrapped again and kept in the dark at 4 °C for further colour analysis after 24 h of aerobic display. Overall colour variation between the initial measurement when removed from the vacuum package (0) and the next measurement at display (24) was determined using the colour difference coefficient (ΔE), which was calculated as ΔE = ((L*0
. Additionally, spectral data were used to estimate the surface colour stability (SCE) as R630/R580 [24
]. The reflectance values at four wavelengths (473, 525, 572 and 700 nm) were used to calculate the proportions of metmyoglobin as MMb = 2.375 × [1 − ((A473 − A700)/(A525 − A700))] × 100, deoxymyoglobin as DMb = 1.395 − ((A572 − A700)/(A525 − A700)) × 100 and oxymyoglobin as OMb = 100 − (AD + AM), where A473, A525, A572 and A700 are the common logarithms of the reciprocals of reflectance values at 473, 525, 572 and 700 nm, respectively [25
The extent of lipid oxidation at each ageing time was assessed by measuring thiobarbituric acid reactive substances (TBARS), expressed as mg of malondialdehyde (MDA)/kg of meat [26