The PCV and NLR analyses were performed using the heparinized samples in an automatic hematology analyzer (COULTER A^c·T diff; Beckman Coulter, Inc., Brea, CA, USA), following the Coulter principle (counting and sizing).

Serum cortisol concentration was determined through quantitative radioimmunoassay (RIA), using a commercial kit (RIA Izotop; Institute of Isotopes, Ltd., Budapest, Hungary) [14]. Each sample was determined in duplicate from 10 μL of plasma, with the corresponding controls. The interassay- and intra-assay coefficients of variance were 4.67% and 6.32%, respectively.

Blood serum glucose concentration was measured using a commercial kit (SPINREACT, glucose LQ, GOD-POD; SPINREACT, S.A.U., Girona, Spain) with a UV-visible spectrophotometer (Spectronic GENESYS 5; Thermo Scientific, Waltham, MA, USA) at 505 nm.

At 24 h postmortem, meat pH was determined in the (LTL) muscle of each carcass stored in the cold chamber at −1°C, using an insertion-type portable pH meter (model HI99163; Hanna Instruments, Woonsocket, RI, USA) with a meat puncture electrode, which was inserted into a small incision (4-cm depth) in the (LTL) muscle of the carcass (11th/12th ribs interface). The pH meter was recalibrated after every five samples. Carcasses showing pH values ≥5.8 were classified as DFD and meat was considered normal quality when pH was <5.8, according to the Mexican Ministry of Agriculture regulation [15].

Meat color of the raw (LTL) muscle samples was measured at room temperature (25°C) with a Konica Minolta Colorimeter (CR-200 Chroma meter, Osaka, Japan), equipped with pulsed xenon arc lamp with double-beam feedback system, a standard illuminant D₆₅, using a 2° position of the standard observer, and 8 mm of reading area. Color measurements were reported in terms of Luminosity (0–100) (L*), index of red (a*), index of yellow (b*), and Chroma (color saturation) (0 to ~85) and hue (color angle) values were determined in the CIELab color space model. Chroma (C*) and hue angle (h*) values were calculated as C* = (a*²+ b*²)^1/2 and h* = tan⁻¹ (b*/a*), respectively [16]. Each measurement for each sample was carried out in triplicate on five preselected locations at the cut surface of each sample.

Three 2.54-cm diameter raw (LTL) muscle sample cores were sheared longitudinally parallel to the muscle fiber, using a hand-held steel core borer. The cores were sheared perpendicular to the muscle fibers using and a TA.XT-plus Texture Analyzer (Stable Micro Systems, Surrey, UK) using a Warner-Bratzler shear device. Assay parameters were as follows: test speed 4.0 mm/seg, down stroke distance 35.00 mm. Shear force values (in Newton [N]) were considered as follows: <22.2 N = tender meat, 22.2 to 35.5 N = moderately tender meat, 35.6 to 53.2 N = tough meat, and >53.2 N = extremely tough meat [17]. Three replicates of WBSF values for each core were averaged for each raw meat sample and used in statistical analyses [15].

One 5-g slice (0.5×0.5×3.0 cm) of each raw (LTL) muscle sample was cut longitudinally to the muscle fiber. The slice was placed on a plastic rack in a glass container with absorbent paper at the bottom, covered with plastic film to prevent evaporation and kept at −1°C for 24 h. Then, the slice was removed from the container and reweighed. The amount of drip was expressed as percentage: % drip loss = ([initial weight – final weight]/initial weight) × 100 [18].

The incidence of meat pH<5.8 was 8.9% of the population. No statistical differences (p>0.05) between PCV values of pH groups were observed. Normal values of PCV in cattle are 24% to 46% [5,22]. The PCV increases as a result of stress by transport and handling. Some authors have reported increased PCV in steers after transport to the slaughterhouse. Stress causes a release of catecholamines, which in turn increase blood pressure and causes contraction of the spleen, increasing erythrocyte mobilization into the blood stream [23]. In our study, PCV was within normal values in 98% of the animals; however, as the period from transportation until slaughter was not evaluated, it was not possible to determine if these results were due to the lack of exposure to stress during transportation.

No significant difference (p>0.05) in NLRs between the two groups was detected. The normal value for NLR in cattle is 1.5. NLR values lower than 1.5 result from neutropenia and/or lymphocytosis, while values higher than 1.5 result from neutrophilia and/or lymphopenia, which suggests the presence of stressful, inflammatory or infectious events [5]. In our study, average NLR was 1.7, indicating that the animals may be subjected to stressful situations during slaughter. According to Tadich et al [24], adrenaline is responsible for the neutrophilia and monocytosis that are observed during stressful situations, provoking a decrease in neutrophil mobilization from the blood to the tissues and an increase in the migration of marginal neutrophils found in the blood vessel wall and tissues close to blood circulation.

The effect of categorical pH group was significant for glucose (p<0.05). Glucose levels in the pH<5.8 group (6.82 mmol/L) were higher (p<0.05) than those in the pH≥5.8 group (5.64 mmol/L). Considering that normal values of glucose in cattle are 3.3 to 4.6 mmol/L [22], 69.6% of the 448 carcasses evaluated showed hyperglycemia, 21% had normal glucose values, and 9.4% showed hypoglycemia. Short fasting periods produce hypoglycemia; this triggers catecholamine release, which promotes glycolysis and gluconeogenesis, resulting in hyperglycemia [25]. Tadich et al [24] mentioned that fasting during transportation resulted in higher blood glucose and cortisol levels, PCV, and leukocytes than fasting during confinement.

No statistical differences (p> 0.05) between cortisol values of pH<5.8 group (75.33 ng/mL) than those observed in the pH≥5.8 group (63.89 ng/mL) were observed. Similar results were reported by Poleti et al [26] who found cortisol levels of 61.6 and 70.2 ng/mL between high (pH≥6.0) and normal (pH<5.8) ultimate pH groups, respectively, in bovine Longissimus thoracis muscle. In our study, the percentages of animals with normal (25 to 45 ng/mL), high (>45 – 90 ng/mL), or extremely high (>90 ng/mL) [3] cortisol levels were 23.2% (36.1 ng/mL), 44.3% (63.0 ng/mL), and 22.7% (108.8 ng/mL), respectively. This suggested that the animals faced stressful conditions. The percentages (44.3% and 22.7%) of the animals that showed high and extremely high levels of cortisol can be attributed to the inadequate handling of animals during preslaughter and slaughter. Moreover, animals had a rest period from 30 to 90 min, but 90.4% of the animals showed a pH≥5.8. Moreover, some animals were not properly stunned, because palpebral reflex and vocalizations were observed after stunning, and these animals were not stunned again. As this stressful situation was of short duration and happened immediately prior to death, it might not be reflected in the cortisol levels, which increase approximately 15 min after exposure to the stressful stimulus [25]. In contrast, when animals are exposed to stressful stimuli since the beginning of the fattening period, they manifest chronic stress, and thus, cortisol levels may be normal due to adaptation of the animal to the stressor. However, this effect may be reflected in the glucose levels, as glucose takes longer to increase, but also to decrease to normal levels after adaptation [24].

Of the 50 carcasses evaluated, 10 (20.0%) had pH<5.8 and 40 (80.0%) a pH≥5.8. The effect of categorical pH group was significant for pH (p<0.05), as the mean raw meat pH values (6.3) of the pH≥5.8 group were higher (p<0.05) than those of the pH<5.8 group (5.6). pH measurement is one of the most important quality parameters of meat quality as it is related to depletion of muscle glycogen reserves. After animal death by exsanguination, muscles become anoxic, triggering anaerobic glycolysis. High levels of stress hormones before or during slaughter decrease muscular glycogen reserves, as glycogen is hydrolyzed to lactic acid. Therefore, meat pH decreases from 7.0 to 5.5 are essential for reduction of bacterial growth [3,23]. Cortisol levels may play a role in correct acidification of meat. Therefore, to avoid adverse effects on the carcass, such as decreased shelf life, color alterations and decreased tenderness due to pH values >5.5, management of animal welfare during growth, transport, and slaughter are pivotal. According to Mexican regulation pH values ≥5.8 are related to DFD meat. This condition would result in poor-quality meat cuts and shorter shelf life [15].

According to Table 3, hue* and chroma* variables were affected by the categorical pH group (p< 0.05); samples of pH<5.8 meat had higher hue* and chroma* mean values than those in pH≥5.8 meat. The mean value of complementary colors red green (a*) coordinate of 13.55 units of pH<5.8 meat was higher (p<0.05) than 11.05 units of pH ≥5.8 meat. Similarly, the mean calculated for complementary colors yellow green coordinate (b*) of pH<5.8 meat was 9.3 units, which was higher (p<0.05) than the 5.5 units of pH≥5.8 meat. According to Abril et al [27], pH≥6.1 meat from male cattle had lower L*, a*, b*, C*, and h* values than pH<6.1 meat at the most measuring times; this is characteristic of the darker and redder color of DFD meats. Poleti et al [26] observed that meat with high pH≥6.0 had lower L*, a*, and b* values within all postmortem times, indicating that muscle with high pH was darker, less red, and less yellow. During rigor onset, the increase in the overlapping myofilaments and shortening of the sarcomere is associated with higher reflectance. As pH falls, there is an increase in birefringence, more light scattering, reduced transmittance and a proposed increase in lightness value. Thus, in DFD, the muscles absorb light, making the meat appear darker. A darker meat of high pH could be attributed to a lower amount of free water at its surface and a lower oxygenation of myoglobin [28]. When animals are exposed to chronic or long-term stress before slaughtering, DFD meats can occur. The study of Loredo-Osti et al [10] revealed that the probability of risk for DFD meat increased 5.3% for each our spent in the waiting pens prior to slaughter, and after slaughter, a ΔpH value of 0.5 units represented a probability of observing dark-cutting carcasses equal to 79.7%. As previously mentioned, the animals in our study showed high and extremely high levels of cortisol and had a rest period from 30 to 90 min. According to the model, a significant (p< 0.05) increase of pH in meat was associated with a decrease in b* (r = −0.546) and C* (r = −0.480) values. In our study, 81.6% of the animals had L* values within a range of 39.5 to 40.3. According to Muchenje et al [29], L* values of 37.0 to 40.4 can be considered DFD in beef. It has been suggested that L*, which denotes lightness, could be used as an indicator as it has shown to be the most important parameter related to DFD. Thus, meat that is dark may cause substantial economic loss to the meat industry worldwide. Color is the most important sensory attribute of meat that influences consumer purchasing decisions because consumers associate the bright cherry red color with freshness and quality, and any deviation from this is perceived as degradation in quality. Holman et al [30] found that an L* value between 14.5 and 44.0, a* between 3.3 and 14.8 (which increases as L* increases), and an optimal b* value of 19 in beef are critical to consumer acceptability. Thus, consumers would probably consider the beef samples of our study acceptable according to L* and a* values, but not for b* values.

As shown in Table 3, no significant difference (p>0.05) between pH groups was observed. The precise shear force value at which a carcass is deemed DFD meat at ultimate pH is >54.3 N, which corresponds to a tough meat [31]. Of the samples analyzed, 92% showed tenderness values >54.3 N. Thus, tenderness found in both pH groups corresponded to DFD meat. According to the model, the interaction of shear force and L* values of the pH≥5.8 group was correlated (r = 0.928) (p>0.05). There is an increased incidence of tough meat and decreased lightness between pH values of 5.8 and 6.3 [32]. In the pH range of 5.8 to 6.2 less tender meat is generated; this has been attributed to low titin and nebulin degradation rates. According to Purchas [33], DFD beef with intermediate pH values (5.8 to 6.2) had the highest shear force values due to decreased sarcomere length. Reduced sarcomere length is recognized as an important cause of increased toughness in meat, and it appears that sarcomeres increase in length as pH decreases below 6.2. Furthermore, tenderness is affected by temperament ranking as increasing excitability is associated with higher (p<0.05) serum cortisol concentrations The B. indicus genotype is commonly responsible for an important variation in tenderness, because the meat from B. indicus breeds is tougher than meat from B. taurus breeds [34]. Animals that had very excitable temperaments, such as B. indicus, produced steaks with shear force values >104.9311 N when compared with calm temperaments with shear force values <50.9946 N. The threshold value for acceptability in food service establishments is 38.2459 N as beef tenderness is a primary factor determining customer satisfaction [35]. It is important to note that the animals in our study were B. indicus × B. taurus and Warner-Bratzler shear force values for both pH groups were higher than these thresholds. Furthermore, these shear force values were higher than those recommended by Mexican regulations [15]: <31.3813 N = tender meat, 31.3813 to 38.2459 N = moderately tender meat, and >39.2266 N = tough meat, and thus meat would be considered tough or DFD.

As shown in Table 3, no significant difference (p>0.05) between pH groups was observed. A mean moisture loss from dripping of 2.4% is indicative of DFD meat [15]. According to the model, drip loss values of the pH≥5.8 group were related (r = 0.990) to L* values (p>0.05), possibly associated to shortening of the sarcomere and associated with higher reflectance. Ultimate meat pH and rate of pH decline are influenced by biochemical events premortem- and postmortem, which act on structural components in muscle cells [36]. As 85% of the water in a muscle cell is held in the myofibrils, drip loss is influenced by interfilament spacing and the extent of lateral and transverse shrinkage of myofibrils at rigor, postmortem cytoskeletal protein degradation, the development of drip channels and extracellular space, and the permeability of the cell membrane to water [36]. In our study, the carcasses were stored at temperature below 10°C when their pH was still >6.0; cooling could have caused muscle fiber to shorten, although it may also have been caused by previous electrostimulation during the slaughter process. Hargreaves et al [37] mentioned that the cooling rate of the carcass influences pH and, therefore, the occurrence of DFD meat.

Principal component analysis indicated that two principal factors described 97.49% of the variation between variables (Figure 2). The first factor accounted for 94.07% of the total variation and primarily described the effect of high cortisol levels on color parameters b* and h* values, physiological parameter PCV, quality parameter drip loss, number of bruises (animals with two to four bruises), and grades 1 and 2 bruises in region 3 and grade 1 bruises in region 5. These types of bruises, however, had a negative association with color parameters L*, a*, and C*, with values indicative of DFD meat. As these numbers and bruise types were highly associated with cortisol, it can be inferred that these bruises were probably caused by stress-inducing situations during preslaughter and slaughter. Accordingly, if the stress response is strong enough, meat quality will be affected. In contrast, factor F2 represented 3.42% of the remaining variance related to the increase in pH, glucose, texture, and changes in color parameters L*, a*, and C* values. It is important to point out that F2 comprised grade 1 bruises in regions 1, 2, 4, and 6 and the number of bruises (animals with zero to two bruises), indicating that although these types of bruises were low in number, they produced stress as well. The current Federal Norm (NOM-033-SAG/ZOO-2014) and the World Organization for Animal Health (Office International des Epizooties) [38] state that cattle must be handled according to animal welfare guidelines before slaughter and animals should be slaughtered as soon as possible after arrival to the slaughterhouse. In the present study, a 30- to 90-min lairage period, palpebral reflex and vocalizations after stunning, and excessive use of the electric prods were observed, indicating that the personnel responsible for the cattle during the preslaughter period is not trained and certified on the humane handling of cattle.

In conclusion, under the conditions of this study, carcasses with pH≥5.8, low L* values, high shear resistance and drip loss values, were indicative of DFD meat. High plasma cortisol levels at exsanguination during slaughter were highly associated with the numbers and bruise types, color parameters and drip loss values. These results suggest the exposure of cattle to stressful situations before and during the slaughtering process, a poor infrastructure design, and not well trained staff. Therefore, handling routines and/or training of the operators in commercial operating conditions should be achieved to minimize the biological cost to animals during the slaughter process and to improve the meat quality.