Evaluation of the Effects of Electrical and Carbon Dioxide Stunning Methods on Quality Attributes of Pork Meat

Abstract

In our experiment, the effect of electrical and CO₂ stunning on pork meat quality attributes was studied. A total of thirty PIC337 female pigs were allocated to two equal groups which were stunned electrically (50 Hz, 210 V, 2.6 A, 15 s) or by CO₂ (85 V% CO₂, 15 V% O₂, 90 s) and slaughtered at a commercial slaughterhouse. For 24 h post mortem, the carcass m. gluteus medius (GM) was used to evaluate meat quality attributes such as pH, color, hardness, water holding capacity (WHC), and total pigment content, and meat classification was also carried out. The CO₂ stunning resulted in muscle with significantly lower pH (p < 0.01) and poorer water holding capacity (p < 0.05) compared to electrical stunning. The GM of pigs stunned electrically showed significantly increased lightness (L*) and redness (a*) (p < 0.05) compared to CO₂-stunned. The time course of development of rigor mortis was similar for both stunning methods. Pale, soft, exudative (PSE) or dark, firm, dry (DFD) meat defects were not observed. Based on total pigment content, stunning methods did not significantly affect the amount of removable blood.

1. Introduction

The Regulation (EC) No 1099/2009 emphasizes that at the time of slaughter or killing animals should be spared any avoidable pain or suffering. Therefore, stunning before slaughtering animals is required to render them unconscious and insensible to any pain. The stunning serves animal welfare and avoids irreversible meat quality defects [1]. The stunning is part of the slaughtering procedure and its method depends on the animal’s species [2,3]. During pig slaughtering, two stunning methods are used: electrical and carbon dioxide (CO₂).

In the case of CO₂ stunning, pigs are led into a vertically moving gondola then lowered into a deep pit containing at least 80% CO₂ and argon or nitrogen as inert gas [4]. After inhalation of CO₂, it dissolves in the blood and reaches the brain where it acidifies the brain cells resulting in a decrease in brain activity and causing a loss of consciousness [5,6] until the animal inhales air again and the blood pH returns to normal level [7]. The advantages of CO₂ stunning are that the modern systems allow the animals to stay in a group that is close to their natural behavior and minimize the incidence of hematomas and bone fractures [8,9,10]. However, the disadvantage is that its effect is not instant and during long stunning animals may become excited [11,12].

The principle of electrical stunning is to pass current through the brain that disrupts the electrical activity of neurons. Two types of electrical stunning are used: ‘head-only’ and ‘head-body’ stunning. In the case of ‘head-only’ stunning, electrodes are placed on both sides of the animal’s head and therefore current passes through the brain. With appropriate electrical parameters, it causes instantaneous but reversible loss of consciousness [13]. During ‘head-body’ stunning, current passes through the brain and heart. Similar to ‘head-only’ stunning, two electrodes are placed on the head and a third one on the chest. The electrical current disrupts the electrical activity of the brain causing membrane depolarization or hyperpolarization of the neurons inducing epileptic seizure and unconsciousness [14]. After the electric shock, neurological and metabolic states are normalized and consciousness returns.

Early studies pointed out that electrical stunning leads to physiological stress in pigs causing bone fractures and hemorrhages due to strong muscle contraction [15,16]; furthermore, rapid decrease in pH and WHC compared to CO₂ stunning was also observed [9,17,18]. In the case of CO₂ stunning, the occurrence of PSE meat defects and petechiae reduced and meat quality markedly improved [8,10,19]. Electrical stunning may increase the rate of glycogen metabolism causing accelerated acidification and leading to PSE meat [20]. The stunning method may affect meat quality such as color, texture, and WHC as main attributes of meats that determine their suitability for further processing and storage and furthermore consumer acceptance as well. These are also important from an economic point of view regarding losses due to undesirable color changes, early deterioration, or due to weight loss during cooling [21].

In our study, the meat quality attributes of pork from pigs stunned electrically were evaluated and compared with those of pigs stunned by CO₂. The experiment hypothesized that the incidence of meat defects resulting from electrical stunning could be reduced using CO₂. Furthermore, pH as an indicator of meat quality is modeled in relation to temperature allowing us to predict meat defects.

2. Materials and Methods

The experiment was performed in an approved, veterinarian-supervised plant (Royal-Hús Kft., 6075 Páhi, Izsáki Street III. 51., Hungary, approval number HU 234). The experiment was continuously supervised by the Animal Welfare Officer of the establishment and all animal welfare requirements, in particular Regulation (EC) No 1099/2009 on the protection of animals at the time of killing, were complied with during the handling and slaughtering of the animals.

2.1. Animals

In this experiment, a total of thirty PIC337 female pigs with a live weight of 104.8 ± 5.1 kg were slaughtered in a commercial slaughterhouse (Royal-Hús Kft., 6075 Páhi, Izsáki Street III. 51., Hungary). The pigs arrived at the slaughterhouse 12 h before slaughter and rested in pens (5 pigs/pen) with approx. 100 kg body weight/m². During the resting period, pigs were fasted, but water was continuously available. Before stunning, pigs were randomly chosen from the stock received and then allocated to two equal groups (n = 15 pigs/group) completely randomly which were stunned electrically or by CO₂.

2.2. Stunning and Slaughtering Procedure

Electrical stunning was carried out using a restrainer and application of a manual ‘head-only’ stunning fork (Hubert Haas, Neuler, Germany) placed behind the ears on the back of the neck (50 Hz, 210 V, 2.6 A) for 15 s. Sound agitation was used to lead pigs to the restrainer without any physical impact.

The CO₂ stunning system (Banns GmbH, Wels, Austria) was an elevator system with 90 s (45 s downward, 45 s upward) stunning duration and an 85 V% CO₂–15 V% O₂ gas mixture was used. The pigs were CO₂-stunned in 5 pigs/group. A straight corridor with a length of 5.5 m (height 0.9 m, width 0.6 m) and sound agitation were used to lead pigs to the gondola without any physical impact.

After stunning, pigs were shackled and the brachiocephalic vein and artery were cut. Bleeding started within 20 s after stunning and its duration was 330 s. After bleeding, scalding, hair removing, eviscerating, splitting, and inspection were performed. After the inspection, carcass cooling was performed with 3 °C air in the slaughterhouse cooling room.

2.3. Meat Quality Measurements

After slaughter, each carcass was weighted individually. Meat quality measurements were performed on the right carcasses 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) chromameter with D65 illuminant, a 10° standard observer, and an aperture size of 5.0 mm with a closed cone at 90° to the muscle surface. Data were evaluated in the CIELab color space where L* represents lightness, a* redness, and b* yellowness. Color differences (ΔE*) between CO₂ and electrical stunning were calculated from average L*, a*, and b* values at given times by using the following standard equation.

$$\mathsf{\Delta }{\mathrm{E}}^{\mathrm{*}}=\sqrt{\mathsf{\Delta }{\mathrm{L}}^{\mathrm{*}2}+\mathsf{\Delta }{\mathrm{a}}^{\mathrm{*}2}+\mathsf{\Delta }{\mathrm{b}}^{\mathrm{*}2}}$$

(1)

Color differences in human perception were evaluated based on [22] where the human perception was described by [23] (

Table 1. Perceived color difference (ΔE*) by human perception [22,23].

ΔE*	Visual Perception
0 < ΔE* < 1	observer does not notice the difference
1 < ΔE* < 2	only an experienced observer can notice the difference
2 < ΔE* < 3.5	an inexperienced observer also notices the difference
3.5 < ΔE* < 5.0	clear difference in color is noticed
5 < ΔE*	observer notices two different colors

).

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_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 (cm²/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 °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.

Based on pH and color, meat was classified as described by [27].

PSE	L* > 50	pHu < 6.0
Normal	L* < 50	pHu < 6.0
DFD	L* < 42	pHu > 6.0

2.4. Statistical Analysis

An independent t-test was performed to evaluate the significant differences between stunning methods post mortem. The test was carried out using SPSS 23.0 (Chicago, IL, USA). Differences were considered significant at p < 0.05 (2-tailed). The grouping variables were the CO₂ and electrical stunning methods. Levene’s test was used to check the equality of variances (p < 0.05) of measured values before the t-test.

The pH trend versus temperature within stunning methods was modeled as a natural cubic spline regression method by the SRS1 Cubic Spline add-on for Microsoft Excel 365 using all measured data within the stunning. The fitting of model prediction to measured values was characterized by the following R-square equation.

$${\mathrm{R}}^2={\displaystyle \frac{\sum {({\mathrm{Y}}_{\mathrm{p}}-{\overline{\mathrm{Y}}}_{\mathrm{p}})}^2}{\sum {({\mathrm{Y}}_{\mathrm{p}}-\mathrm{Y})}^2+\sum {({\mathrm{Y}}_{\mathrm{p}}-{\overline{\mathrm{Y}}}_{\mathrm{p}})}^2}}$$

(2)

where ${\mathrm{Y}}_{\mathrm{p}}$ is the predicted value of $\mathrm{Y}$, ${\overline{\mathrm{Y}}}_{\mathrm{p}}$ is the average of predicted Y.

3. Results and Discussion

3.1. pH

Post mortem pH is usually characterized by two measured values: 45 min as the initial and 24 h after slaughter as the ultimate pH [28]. Figure 1 shows the change in pH post mortem. The CO₂ stunning resulted in a slightly lower initial pH compared to electrical stunning (p > 0.05). This might be caused by inhaled CO₂ during stunning which led to slight acidification in the blood and muscle. Studies found that inhalation of 80% or 90% of CO₂ for 15, 45, or 58 s increases the pressure of CO₂ (mmHg) in the blood which results in pH decreasing [5,6,29]; furthermore, CO₂ gas stunning accelerated early post mortem pH decline compared to electrical stunning [30]. The CO₂ stunning resulted in muscle with significantly (p < 0.01) lower pH compared to electrical stunning post mortem. The pH decreased to pH_CO2 5.46 ± 0.07 and pH_electric 5.75 ± 0.20 in the case of electrical stunning as the ultimate pH. These results are consistent with earlier data from the literature which found that CO₂-stunned m. quadriceps femoris showed slightly lower ultimate pH than electrical stunning [31,32]. The more rapid pH decrease in the case of CO₂ stunning is attributed to increased anoxic convulsion which leads to an increased ATP depletion and more rapid lactic acid production [33]. Furthermore, inhaled CO₂ causes acidification of the blood and reduces the muscle cells’ capacity to maintain intracellular pH [13]. The suggestion in [34] of a quick or moderate pH decrease was not observed for PSE or DFD; the GM pH decreased normally over time.

The pH 6.0 and temperature (T_pH6.0) combination is important for the meat quality. Furthermore, certain regions in the pH/T combinations have to be avoided, which are called pH/T windows. One of them is T_pH6.0 < 12 °C and the other one is when T_pH6.0 > 35 °C. The first one leads to cold shortening and the latter to heat shortening of meat [35]. The post mortem pH values were plotted in a pH/Temperature diagram with non-linear estimation of pH as shown in Figure 2. The ‘ideal pH/Temperature window’ and ‘cold shortening window’ based on [35] are also shown as solid lines. The CO₂ prediction model showed R² = 0.7584–0.9547 fitting and the electrical R² = 0.5221–0.9407. The pH decreasing indicates the difference between the CO₂ and electrical stunning methods. The predicted pH data show the CO₂-stunned meats reached the ‘ideal window’ at 35 °C < pH_6.0 < 12 °C suggested by [36]. However, in eight cases of electrical stunning, the predicted pH crossed the boundary between ‘ideal’ and ‘cold shortening’ and three of them showed pH_u > 6.0. In the latter cases (pH_6.0 < 12 °C), cold shortening may have occurred. Besides these, the predicted pH_u values were in the range of 5.4–5.8 which is midway between the onset of rigor pH 6.0 and the isoelectric point (pI) which offers a good compromise between the water holding capacity and toughness of the meat. When pH reaches the pI of main proteins (e.g., myosin, pI = 5.4), the negative and positive charges are equal to each other and the protein has a minimal net charge. As a result, the protein’s negative and positive groups attract one another which results in minimal swelling of myofibers and results in a poor water holding capacity of meat [37].

3.2. Color

The most important factor in judging meat before purchase is the color. The preferred meat color is usually reddish-pink which shows the meat is fresh and wholesome [38]. There were no significant differences in initial L*, a*, and b* values (p > 0.05) between using CO₂ and electrical stunning.

The initial L*_CO2 46.96 ± 0.82 and L*_electric 46.92 ± 0.81 were in the normal range (L* < 50) recommended by [27] in both stunning methods (Figure 3A). A slight increase in L* was seen two hours post mortem, and then a decrease occurred until the fourth hour. After death, physicochemical, biochemical, and structural changes begin in the muscles. The major physicochemical change is the development of rigor mortis in which muscle contractions occur and its structure becomes compact. This increasingly compact structure results in less light dispersion on muscle surfaces and a less light appearance. Complete development of rigor mortis occurs at approx. 197 min in pigs [39] which can be traced in the changing of the measured L* values. Post rigor, muscle increasingly relaxes and its lightness increases again. The GM of pigs stunned electrically showed significantly higher L* values compare to those submitted to CO₂ stunning (p < 0.05). A similar significant relation was found in pig m. longissimus lumborum [19], m. longissimus thoracis [10], and lamb m. longissimus dorsi [40].

Using electrical stunning resulted in significantly higher a* values compared to using CO₂ stunning (p < 0.05) (Figure 3B). Increasing redness post mortem is likely related to the oxygen consumption of muscle cells. After slaughter, mitochondria in the cells remain capable of consuming oxygen if substrates are available for their operation. However, post mortem, mitochondrial activity decreases due to the decreasing amount of substrates and this allows the formation of oxymyoglobin resulting in higher red color [41]. In the yellowness of GM, no significant difference was shown between electrical and CO₂ stunning methods (Figure 3C).

Changes in color difference over time can be seen in Figure 4. The ΔE* values showed an increasing tendency post mortem. Two hours post mortem, the color difference was <1 which means a standard observer does not notice the difference between CO₂- and electrically stunned GM. After three hours post mortem, the color difference increased to the range of 1.5–2.0. From three hours post mortem, the color difference is already noticeable to an experienced observer. The decrease in ΔE* values can be seen at four hours post mortem which can be explained by a slight reduction in the differences between L* and b* values. This is associated with the development of rigor mortis. The contraction of muscle fibers in the meat results in a more compact structure. The contracted muscle fibers absorb more light and less light is reflected from them, resulting in a reduction in L* and b* differences.

3.3. Hardness

The hardness of muscles was characterized by penetration force and can be seen in Figure 5. In the first three hours post mortem, the penetration force of GM showed a decreasing tendency. Immediately after death and before the development of rigor mortis, muscles are pliable. Then, a slight increase in penetration force was observed for both stunning methods. This relates to the development of rigor mortis which is the major post mortem change in the muscle. There is no difference in the time course of the development of rigor mortis between stunning modes. In rigor mortis, muscle fibers are contracted due to ATP depletion which results in an increased hardness of muscles [42]. The penetration force of muscles reached the peak at F_p,electrical 45.13 N ± 1.97, and F_p,CO2 48.56 N ± 1.46 at four hours post mortem; then, at 24 h post mortem, decreased to F_p,CO2 40.01 N ± 0.63 and F_p,electrical 39.78 N ± 0.69. A previous study [43] found that the Warner–Bratzler shear force of lamb m. longissimus dorsi increased at three hours post mortem and then decreased up to 72 h post mortem. Post mortem, a significant difference was not observed between electrically and CO₂-stunned GM penetration force. During electrical and CO₂ stunning of pigs, significant differences were also not found in Warner–Bratzler shear force on m. longissimus thoracis [18].

3.4. Water Holding Capacity, Cooling Loss, and Cooking Loss

The WHC expresses the ability of the muscle to retain its water content. In the case of the used method, a greater value indicates greater water loss, i.e., poorer WHC. If WHC is poor then weight loss may occur leading to financial loss; moreover, it influences the sensory quality of the meat, e.g., juiciness and toughness [44]. Meat with poor WHC called PSE is found more frequently in the case of pigs stunned electrically compared to CO₂-stunned [10]. Some authors have shown that pigs exposed to electrical stunning generally had higher drip loss, i.e., decreased WHC, than pigs stunned by CO₂ [8,9,18,19]. Results showed a noticeable effect of the stunning method on WHC. The CO₂ stunning resulted in significantly poorer WHC compared to electrical stunning (

Table 2. Effect of CO₂ and electrical stunning on water holding capacity, total pigment content, cooling loss, and cooking loss in pork m. gluteus medius 24 h after slaughter.

Quality Attribute	Stunning Method
	CO2	SEM	Electrical	SEM	p-Value
WHC 1, cm2/g meat	2.26	0.17	1.72	0.15	0.042
Total pigments, ppm	14.96	1.70	14.63	1.65	0.892
Cooling loss, %	3.01	0.12	3.06	0.15	0.740
Cooking loss, %	9.53	0.22	6.41	0.21	0.000

¹ WHC: Water holding capacity.

). To the best of our knowledge, the influence of stunning methods on muscle WHC is less known but may be related to post mortem metabolism and pH changes. Due to muscle fiber contraction during rigor mortis, the water placed between myofibrils could enter the space formed between the fibers and cell membranes due to the calpain decomposition effect. From these channels, water could release to the exterior [45]. Most of the water in muscle is held by capillary forces between actin and myosin filaments. The WHC of myofibrils is determined by interfilament spacing which is mainly affected by electrostatic forces through pH [37]. Close to the meat isoelectric point that can be found in the range pH 5.0–5.4, the WHC is at a minimum due to the equalization of myofibrils’ negative and positive charges which results in increasing interfilament distance [46]. Furthermore, decreasing pH reduces the ability to bind water to myofibrils’ hydrophilic side chains as well. As can be seen in Figure 1, post mortem pH values of CO₂-stunned pigs were significantly lower (p < 0.05) compared to electrically stunned pigs which resulted in decreased WHC. However, in the context of this, the cooling loss during post mortem cooling was 3.01% (electrical) and 3.06% (CO₂) of the initial weight which did not prove to be a significant difference between stunning methods (Table 2). The CO₂-stunned pig meats showed a significantly higher cooking loss compared to electrically stunned, which is linked to poorer WHC in CO₂-stunned meats.

3.5. Total Pigment Content

The residual blood is an important factor in meat quality. Blood remaining in the muscle decreases shelf life and causes undesirable discoloration and microbiological deterioration. The amount of residual blood is in positive correlation to residual levels of myoglobin and hemoglobin as the two main pigments of meat [47]. Therefore, residual blood in the muscle was characterized by total pigment content. As Table 2 shows, the bleeding efficiency was similar for both stunning methods, thus there was no effect of the stunning method on the amount of removable blood. Also, no significant difference was found in residual blood with the use of electrical or CO₂ stunning in the case of pork m. longissimus lumborum [19].

4. Conclusions

In some cases, electrical stunning results in meat defects, but the incidence of defects could be reduced by CO₂ stunning. The CO₂ stunning accelerates muscular acidification of the m. gluteus medius that may be caused by inhaling CO₂ during stunning and results in acidification in blood and muscle. As a consequence, pH approaches the isoelectric point which leads to poorer water holding capacity and higher cooking loss compared to electrical stunning. This has effects on quality attributes and thus on the technological and economic value of meat. The time course of the development of rigor mortis and the amount of removable blood was similar for both stunning methods. Although CO₂ stunning had a slightly greater effect on the m. gluteus medius muscle primary quality properties compared to electrical stunning, the differences are minor but the incidence of meat defects was avoided by the CO₂ stunning method.