## 2 Materials and Methods
### Meat Quality Measurements  
After slaughter, each carcass was weighted individually. Meat quality measurements were performed on the right carcass at the height of the first and second lumbar vertebrae on the m. gluteus medius (GM) muscle. Meat pH was measured using a Testo 206-pH2 digital pH meter with temperature compensation (Testo GmbH, Wien, Austria). The instrument was previously calibrated to pH 4.01 and pH 7.01 buffer solutions.  
Meat color was determined using a Minolta CR 400 (Konica Minolta Inc., Tokyo, Japan) chromateter with D65 illuminant, a 10\({}^{\circ}\) standard observer, and an aperture size of 5.0 mm with a closed cone at 90\({}^{\circ}\) to the muscle surface. Data were evaluated in the CIELab color space where L* represents lightness, a* redness, and b* yellowness. Color differences (\(\Delta\)E*) between CO2 and electrical stunning were calculated from average L*, a*, and b* values at given times by using the following standard equation.  
\[\Delta\text{E}^{*}=\sqrt{\Delta\text{L}^{*2}+\Delta\text{a}^{*2}+\Delta\text{b} ^{*2}} \tag{1}\]  
Color differences in human perception were evaluated based on [22] where the human perception was described by [23] (Table 1).  
The meat hardness was measured using a handheld penetrometer with an 8 mm diameter head that was pushed into meat tissue to an 8 mm depth with constant speed. The displayed kg values were expressed as penetration force (F\({}_{\text{p}}\)) (kg = 9.80665 N).  
The pH, color, and hardness were measured in three replications on each carcass at 45 min, 2, 3, 4, 5, 12, and 24 h post mortem.  
The water holding capacity (WHC) was determined using the Grau and Hamm method [24]. For this purpose, 0. g meat was placed on a 90 mm diameter filter paper (VWR 516-0814) and put between two glass plates under a weight of 0.5 kg for 5 min. The wet spot which appeared was used to calculate WHC as the proportion of spot area and meat weight and expressed in (cm\({}^{2}\)/g) dimension. Three parallel measurements were performed on each carcass 24 h post mortem.  
The cooking loss was determined from the 24 h post mortem GM sample based on [25] and expressed as the percentage of loss related to the initial sample weight.  
To determine the total pigment content, meat was minced; then, a 10 g sample was mixed with 40 mL acetone and 1 mL of 12 N hydrochloric acid. The mixture was agitated and then stored at 4 \({}^{\circ}\)C for 1 h. The mixture was filtrated and submitted to spectrophotometric readings (Hitachi U2400, Hitachi Corp., Tokyo, Japan) of optical density at wavelength 640 nm as described by [19]. The result was expressed as parts per million (ppm) of hematin and was obtained by multiplying the optical density found by 680, according to the methodology described by [26]. Three parallel measurements were performed on each carcass 24 h post mortem.  
The cooling loss was calculated as the difference in carcasses' weight before and after cooling and expressed as the percentage (%) of weight before cooling.  
\begin{table}
\begin{tabular}{c c} \hline \(\Delta\)E* & **Visual Perception** \\ \hline
0 \(<\Delta\)E* \(<\) 1 & observer does not notice the difference \\
1 \(<\Delta\)E* \(<\) 2 & only an experienced observer can notice the difference \\
2 \(<\Delta\)E* \(<\) 3.5 & an inexperienced observer also notices the difference \\
3.5 \(<\Delta\)E* \(<\) 5.0 & clear difference in color is noticed \\
5 \(<\Delta\)E* & observer notices two different colors \\ \hline \end{tabular}
\end{table}
Table 1: Perceived color difference (\(\Delta\)E*) by human perception [22; 23].  
Based on pH and color, meat was classified as described by [27].  
\begin{tabular}{c c c} PSE & L* \textgreater{} 50 & pHu \textless{} 6.0 \\ Normal & L* \textless{} 50 & pHu \textless{} 6.0 \\ DFD & L* \textless{} 42 & pHu \textgreater{} 6.0 \\ \end{tabular}