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INTRODUCTION
Livestock slaughterhouses in Korea use electricity and carbon dioxide (CO2) stunning methods during slaughter. Chicken slaughterhouses have also introduced CO2 stunning methods due to improved animal welfare compared with the traditional electric method. Electrical stunning is the most common pre-slaughter stunning method used for animals in the meat industry. Among the different types of electrical stunning systems, electrical water bath stunning is commonly practiced in commercial poultry plants [1]. It is expedient, inexpensive, has few space users, and can be readily tailored to commercial processing plants [2]. To induce unconsciousness in a water bath system, the EU recommends a minimum current of 120 mA per chicken [3]. This recommended minimum current causes muscle contraction, broken bones, and breast muscle hemorrhage in broilers [4–6]. On the other hand, Islamic countries perform slaughtering without stunning, following religious requirements, using the halal method [7].
Gas stunning can be considered an alternative method to the pre-slaughter stunning system to maintain animal welfare and meat quality, and is widely used in traditional electric-based slaughter houses [8]. According to Raj et al. [9], gas use as a novel method for stunning animals has increased daily. Minimizing the stressful handling of awake broilers using gas stunning prior to electrical stunning in a commercial slaughterhouse increases animal welfare and reduces the need for workers [10]. CO2 is commonly used in pre-slaughter stunning of broiler chickens [11] and pigs [12]. CO2 has been demonstrated to be aversive to chickens and induces suffering and pain [13,14]. Mice, rats, and pigs also have also shown an aversion to CO2 stunning [15-20], including irritation of the nasal mucosal membranes [18], severe respiratory distress [20], and strong head shaking [21]. Additionally, meat and small intestine discoloration (dark-red colored) occurs following the CO2 stunning method [22], so the market value of CO2 stunned meat and by-products, especially the intestine, is low in Korea. According to KOSIS [23], ~77,000 cows, 1.5 million pigs, and 84 million chickens are slaughtered each month in Korea. The intestines of animals have been widely utilized globally for the food ingredients and production of casings [24,25]. Therefore, any deterioration in the quality of intestines due to gas stunning can render them unsuitable for sale, leading to potential economic losses. Consequently, it is necessary to study an alternative to the CO2 stunning method that does not affect the quality of meat or animal welfare.
On the other hand, inert gases, such as argon (Ar) or nitrogen (N2), have been used in different proportions in conjunction with CO2 in the stunning of animals to help reduce CO2 aversion [20,26]. Exposure to high concentrations of inert gases has been found to induce hypoxic conditions in animals [18,27]. The displacement of oxygen (O2) in the atmosphere by a high concentration of inert gases can create hypoxic conditions with less than 2% O2, leading to a decrease in blood oxygen levels of animals [28]. This, in turn, affects the central nervous system (CNS) and induces unconsciousness to animal [29]. N2 is the most abundant gas in the air (78%), and can be used widely for commercial purposes due to its low price. Different concentrations of N2 (70%–92%) can be mixed with CO2 to maintain hypercapnic-hypoxia conditions for pig stunning [30,31]. Poultry showed less aversion to stunning with low atmospheric pressure and N2 than CO2, which may offer a significant welfare refinement [32]. No adverse effects on the quality traits of meat or the small intestine were observed using the high-concentration N2 gas stunning of pigs [22]. According to the syncope method, in broilers, fewer stress hormone changes occurred following N2 stunning than with the CO2 and halal methods [33]. Therefore, it is expected that N2 gas could be effective in preventing excessive discoloration and softening compared to conventional stunning methods. However, the feasibility of using only high concentrations of N2 gas (98%) in the stunning of birds (broiler chickens) and its effects on the quality traits of meat and the small intestine have not yet been investigated.
The objective of this study is to evaluate the impact of high-concentration N2 (98%) stunning on the quality of meat and intestines in poultry, compared to conventional stunning methods such as electrical stunning, CO2 stunning, and halal stunning. Additionally, the study aims to validate the industrial applicability of high-concentration N2 stunning.
MATERIALS AND METHODS
The experiment was performed at the research of the National Institute of Animal Science of Korea (NIAS). The animal care and use committee of NIAS reviewed and approved the protocol for this study (Approval Number: NIAS20191536).
An experimental trial was conducted at the NIAS, RDA, Korea to assess the quality of hypoxic chicken breast meat and small intestine following N2 gas-stunning and to compare it with meat stunned using standard electrical- and high-CO2-based stunning and non-stunning (halal) methods. Four treatments were used: (i) electrical stunning (Elec-stun), (ii) 80% CO2 gas stunning (CO2-gas), (iii) 98% N2 gas stunning (N2-gas), and (iv) the non-stunning method (halal). An electrical- and high-CO2-based stunning (80%) trial was conducted at the nearest contact commercial slaughter house; a high-N2 gas stunning (98%) and a non-stunning (halal) method trial was conducted at the NIAS pilot plant abattoir. Forty Cobb-500 commercial broiler chickens were assigned to each treatment (n = 40). The chickens were collected from the nearest commercial farm. Just before the day of the experiment, broilers were transported to the institutional abattoir and stored in a commercial slaughterhouse. The chickens rested for 12 h in pens with ad-libitum water and finisher diet. Fasting conditions were maintained overnight before slaughter. Body weight was measured during the evening of slaughter. The body weight range was 2.51–2.68 kg at 42 days of age.
In the electrical stunning and non-stunning (halal) methods, single broilers were used, whereas gas stunning (both CO2 and N2) was performed on a crate containing eight broilers. Electrical stunning was performed within the contact commercial slaughterhouse, using a water bath stunner delivering a constant current of 120 mA (50 Hz in the form of sinusoidal waveform) for 4–5 s. In the case of the halal method, according to the rules of Islam, broilers were slaughtered without stunning [34]. The chickens in the CO2 and N2 treatment groups were stunned using specially designed gas chamber. A modern digital gas chamber (length 220 cm × width 100 cm × height 135 cm, Supreme Thermal Instrument [STI], Daegu, Korea) was used for gas stunning. The CO2 or N2 gases flow was continued into the pit of the chamber until the desired concentration (80% or 98%) was reached. After reaching the desired concentration level, the gas flow was stopped, and the time (s) counting was started until the stunned state was reached. It was found that within 70 s, broilers were completely stunned by 80% CO2, and in the case of N2 (98%), it required approximately 110 s for complete stunning.
After gas stunning, each crate of broilers was removed from the gas chamber as quickly as possible and heart activity was measured using an electrocardiogram (ECG) with two adhesive patches (Unilect, 5 cm in diameter; Unomedical, Stonehouse, UK) placed on the left and right pectoralis muscles under the wing base, and then slaughtered within 30 s. In electrical stunning, each stunned chicken was kept on a movable table, heart activity was measured using ECG, and then the chicken was slaughtered within 15–20 s. After slaughter, for proper bleeding, one leg (hock joint) of each broiler was tied with an iron chain and hung for 2–3 min. This process was observed in all treatments, including those treated with the halal method. Subsequently, chickens from the different treatments were inserted separately in a pluckier machine for feather plucking. After 7–8 min, the chickens were removed from the pluckier and eviscerated manually. Individual carcasses were cleaned, washed with tap water, and stored in polythene bags. The small intestine (100 cm) was collected from each broiler in every treatment (n = 40). After cleaning and washing, each small intestine was placed in a polythene bag. All carcasses and small intestines were stored in a cold room overnight at 2°C. After chilling, all carcasses and small intestine samples were transported (after slaughter at 18 h) to the laboratory, and then the breast muscle of all carcasses were obtained for further study.
To determine the pH of the raw meat, 5 g of the sample was mixed with 50 ml of distilled water using a hand blender. For the small intestine, 3 g of sample was mixed with 30 ml of distilled water. The resulting sample solutions were then measured using a digital pH meter (FP 20, Mettler Toledo, Greifensee, Switzerland). The pH readings were taken four times for every sample of chicken breast meat and small intestine.
The measurements of the surface color was carried out samples of the raw chicken breast meat and the raw and cooked small intestine were bloomed in air at room temperature for ~40 min. The surface color were measured using a chroma meter (CR-400, Minolta Camera, Osaka, Japan). First, the chroma meter calibrated using a white plate (Y = 86.32, X = 0.3165, and y = 0.3242). In the chicken breast meat, for each rectangular sample (size 12 × 5 cm), color values were measured from 8 different sections on the surface and bone-side. For the small intestine, 100 cm of a freshly cut clean small intestine was measured from 8 different sections, and there were intervals of 10 cm minimum between different locations. The color values were reported as CIE L*, CIE a*, and CIE b*.
The Proximate components were measured by using a Food Scan Lab 78810 (Foss Tecator Hillerød, Denmark). Approximately 100 g of raw chicken breast meat was blended using a hand mixer and used as a sample. The sample was measured in triplicates.
In measurement the WHC of a raw chicken breast meat, the sample was ground, and subsequently, any fat, fibers, and coarse particles were removed. The fine meat (0.5 g) was inserted into an ultra-centrifugal tube. These tubes were heated at 80°C for 20 min using water-bath.The next step in determining the WHC of the meat sample involved using a centrifugal machine (Avanti(R) J-E, Beckman coulter, Brea, CA, USA). The ground samples were centrifuged for 10 min at a speed of 470×g at 4°C. After centrifugation, the tubes were allowed to stand for 10 min at room temperature. The samples were weighed, and the WHC of the chicken breast meat was calculated using a formula. The above method was performed twice for each sample, and the average value was used for the study [35].
To measure the CL of the chicken breast meat, they were weighed and placed in heat-durable plastic bags. The mouths of the bags were folded and blocked using steel clips. Then, these sample was heated using a water bath at 71.3°C. The inner temperature of each sample was evaluated using a thermorecorder. When the core temperature reached 70°C, the samples were transferred an ice water for cooling for 30 min. Next step, the surface of cooked samples remove moisture with absorbant tissue paper. The cooked samples were weighed, and the CL was determined using a formula.
The WBSF were measured using an Instron Universal Testing Machine (5543, Instron Corp., USA). The WBSF for chicken breast meat was prepared using a sample with completed CL measurements. From cooked chicken breast meat, five samples (length 2.0–2.5 cm, diameter 1.25 cm) were made using a metal corer according to the muscle fiber direction. In the small intestine, WBSF was determined in both fresh and cooked samples. For samples of fresh small intestine, each 5–7 cm of samples from the small intestine was taken and directly cut using an Instron machine. To measure the WBSF of the cooked small intestine, a clean sample of 50 cm in length was taken from each small intestine. Also, in case of the cooked sample, the fresh small intestine were cooked under the same heating conditions as meat (cooking time, ~40 min) and were then used to measure the WBSF. Machine with a speed of 200 mm/min and a load cell of 40 N; a 1 cm distance was maintained from one cut to another.
The thickness was measured using a digital caliper scale of both fresh and cooked small intestines. The sample was longitudinally spread on a white plastic board. Readings were obtained from six different locations of the small intestine. The minimum interval distance from one location to another was 5–7 cm. The cooking conditions used to prepare the cooked samples were equivalent to the CL of chicken breast meat, however, the cooking time was ~40 min.
RESULTS AND DISCUSSION
Table 1 shows the pH24h for chicken breast meat and small intestine with stunned and non-stunned. The results revealed significant differences in the pH24h of the chicken breast meat and small intestine among different stunning methods. Among the stunning treatments, the pH24h was comparatively high in the Elec-stun and low in the CO2-gas compared to other treatments (p < 0.05). The pH24h of halal was also significantly higher than that of all other stunning treatments in both the chicken breast meat and the small intestine.
The pH level of the chicken breast meat and small intestine is one of the indicators that change according to stunning methods and reflects the biochemical state of the muscle following the development of rigor mortis [1,5]. Several reports imply that during stunning and exsanguination, muscular activity is elevated and glycolysis is increased, resulting in the accretion of lactic acid in at the muscle decreased the pH level [9,36,37]. The accumulation of lactic acid in the cells of chicken muscle is closely associated with pre-slaughter stress [38]. The detection of stunning stress in animals has been attempted by some researchers through the use of blood samples [2,33]. Exposing hogs to an 80% concentration of CO2 for 70 s results in the production of large amounts of lactic acid in the blood [39]. In pigs exposed to 80% CO2 for 60 s, lactic acidosis, hyperglycemia, hypercapnia, and increased hematocrit levels were observed [40]. According to Channon et al. [41], the pH decline of pork loin was faster in electrically-stunned pig meat than after CO2 stunning. However, the pH of pork meat with N2 stunning was higher than that with CO2 stunning, which was slightly lower than that with electrical stunning [22]. Similar to a previous study, the pH in chicken breast and small intestine was higher in N2-stunned chickens than in CO2-stunned chickens, and lower than in electrically-stunned chickens.
The surface color of chicken breast meat and small intestines is presented in Table 1. In chicken breast meat, the L* and b* was significantly highest in the halal treatment, while it was the lowest in the CO2-gas treatment (p < 0.05). On the contrary, CO2-gas treatment had the highest a*, while halal had the lowest a* value (p < 0.05). N2-gas treatment showed higher L* and b* values and lower a* values compared to CO2-gas treatment (p < 0.05). Compared to N2-gas treatment, Eelc-stun treatment showed similar L* valuse (p>0.05), lower a* value (p < 0.05), and higher b* value (p < 0.05). In both raw and cooked intestines, halal treatment had the highest L* and b* values, while CO2-gas treatment exhibited the lowest L* and b* values (p < 0.05). For a* values, CO2-gas showed the highest, while halal had the lowest values. Elec-stun and N2-gas treatments fell between CO2-gas and halal treatments in terms of color. Elec-stun had higher L* and b* values compared to N2-gas treatment (p < 0.05), while its a* value was lower (p < 0.05). The surface color of the chicken breast meat and intestines is a factor that can influence the purchasing decision of consumers. When purchasing meat and meat products, consumers prioritize physical appearance, especially color [42,43]. Discolored or dark-red meat and intestines are usually disliked by consumers, and they tend to prefer bright-red fresh meat and intestines [42,43]. The a* and b* are related to the pigment concentration, oxidation, and redox conditions [44,45]. The stunning method influences the color characteristic of chicken breast meat and the small intestines. It has been reported that bright-red colored meat and small intestine were observed in highly concentrated (98%) N2-stunned pigs and rabbits, and dark-red colored meat was found in highly concentrated (80%) CO2-stunned pigs and rabbits [22,46]. Electrical stunning showed a higher L* value in the Longissimus thoracis muscle of pigs than CO2 stunning [22,41]. Also, CO2-stunned pigs were observed to have high a* values and low b* values compared to electrically stunned pigs [22]. In the case of N2-stunned pigs, the color characteristics was intermediate between electrically- and CO2-stunned pigs [22]. Additionally, in rabbits, the N2 stunning method resulted in higher L* and b* values and lower a* values compared to CO2 and Ar stunning method [46]. In this study, the Elec-stun showed high L* and b*, and low a* values of chicken breast meat and small intestine, implying a pale color, and CO2-gas showed lower L* and b* values and higher a*, indicating a dark-red color. The color characteristics of N2-gas treatment was intermediate between that of Elec-stun and CO2-gas treatments, which implies bright-red chicken breast meat and small intestine.
The proximate components of the stunned and non-stunned chicken breast meat are presented in Table 2. The moisture content showed a significant difference among all treatments (p < 0.05). In proteins content, among the stunning treatments, a noteworthy dissimilarity was found (p < 0.05). The Elec-stun and N2-gas treatments were not significantly affected by fat content (p>0.05). In terms of ash and collagen content, Elec-stun treatment showed significant higher than the other stunning treatments (p < 0.05). In this study, although there was a significant difference, each component showed a small difference of ~1% among all treatments. Several factors can influence the nutrient composition of meat, including feeding, genetics, age, and gender [47]. In a previous study, it was shown that the stunning method did not have an effect on the proximate composition of the meat, except for moisture [22].
Fig. 1 shows the WHC for chicken breast meat and small intestine with stunned and non-stunned. There were no significant differences observed in WHC among all treatments. The WHC value of halal (60.14 ± 1.51) was higher than that of the stunning treatments (p>0.05). Among the stunning treatments, Elec-stun (59.21 ± 2.06) had the highest WHC value and the lowest WHC value was observed in the CO2-gas (56.22 ± 0.99; p > 0.05). The WHC value of N2-gas (57.78 ± 1.42) was intermediate between Elec-stun and CO2-gas (p > 0.05).
Meat industries are concerned with the improved WHC of meat and meat products. Meats with a lower WHC percentage may not fulfill export quality regulations, resulting in huge economic losses [48,49]. The decrease in pH causes the meat to become more acidic, which in shift causes the protein filaments in the meat to tighten and reduces the WHC of the meat [50]. Previous studies have shown that because of pre-slaughter stunning, muscle glycogen quickly breaks down and produces large amounts of lactate in the blood, resulting in a low pH and decreased WHC [51,52]. Our present study is consistent with this, where the halal stunning observed a higher WHC value than any other treatment. According to Bond et al. [53] increased amounts of adrenaline were observed in the blood due to pre-slaughter stress, which can potentially lead to water loss from meat. During exposure to CO2, lambs inhaled a large amount of gas that severely influenced the breakdown of cells, resulting in decreased WHC of the lamb meat [54]. In previous studies, a lower WHC value was found in pigs and lambs stunned with 80% CO2 than in those stunned with electrical stunning [22,55]. It has been reported that N2-stunned (98%) pigs provide intermediate WHC values of meat, between those of electrical and CO2 stunning [22]. Also in this study, the CO2-gas treatment showed a lower WHC value and the Elec-stun demonstrated a higher value. The N2-gas treatment provided values between Elec-stun and CO2-gas treatments.
The CL values of the stunned and non-stunned chicken breast meat are shown in Fig. 2. There were no significant differences observed in CL among all treatments. The figure indicates that the halal treatment provided a higher value (19.45 ± 1.17) than the all stunning treatment (p > 0.05). Among the stunning treatments, Elec-stun showed the highest value (17.17 ± 1.59) and CO2-gas the lowest (13.81 ± 1.06). The value of N2-gas (15.84 ± 0.32) was intermediate in Elec-stun and CO2-gas (p > 0.05).
The method of pre-slaughter stunning and slaughtering may have an impact on the CL of meat. According to Önenç and Kaya [56], stunned meat shows lower CL than non-stunned meat. Our research supports this statement, where the halal provided a higher CL than that of the stunning treatments. The quicker pH decline in electrically-stunned animals resulted in higher CL [57]. Higher CL was observed in more pale-colored broilers and turkey meat [58,59]. Electrically-stunned lambs showed significantly increased CL at 72 h postmortem than CO2-stunned lambs [60]. Elevated CL was also found in electrically-stunned cattle [56] and lambs [60] at the 1st and 2nd weeks postmortem. The CL value of N2-stunned pigs showed an intermediate value between that of electrical and CO2 stunning [22]. The present study also supports these statements; Elec-stun provided a higher value of CL than CO2-gas, and the value of N2-gas was intermediate.
The WBSF values of the stunned and non-stunned broiler meat are shown in Fig. 3. There were no significant differences observed in WBSF among all treatments. The WBSF value of the halal treatment (1.96 ± 0.07 kg/cm2) was comparatively higher than the stunning treatments (p > 0.05). The highest value was found in the Elec-stun (1.80 ± 0.16 kg/cm2) and lowest value was found in the CO2-gas (1.35 ± 0.41 kg/cm2) amongst the different stunning treatments (p > 0.05). The WBSF value of N2-gas (1.67 ± 0.08 kg/cm2) was higher than CO2-gas but lower than Elec-stun.
The WBSF of chicken breast meat depends on several factors such as stress, genes, chilling system, and cooking situation [61,62]. The method of stunning is a factor that affects the WBSF value of meat. ÖnenC and Kaya [56] and Vergara et al. [63] found that stunned meat provides a lower WBSF value (more tendered) than non-stunned meat. We also found a higher WBSF value in non-stunned meat compared with any stunning treatment animals in our study. An elevated WBSF value was found in electrically-stunned meat compared with CO2-stunned suckling lamb meat [64]. Electrically-stunned animals demonstrated higher WBSF values owing to less calpain activity [65]. The muscle pH and number of enzymes present controlled the activity of calpain [66]. When animals are exposed to high levels of CO2, a large amount of gas is absorbed in the animal body, which is greatly soluble in muscle tissue and remains at prominent levels in the muscle tissues as residue, resulting in a lower WBSF value [67]. CO2 stunned pigs and lambs showed lower WBSF values than electrically-stunned meat [22,68]. One possible reason for these findings is that the gas stunning method may result in higher calpain activity compared to electrical stunning. This is a speculative hypothesis, and further analysis on the muscle calpain activity based on the stunning methods should be conducted in future studies to clarify this relationship. N2-stunned pigs provided slightly higher WBSF values than CO2-stunned pigs, but no statistically significant difference was found between them [22]. The findings of this study concur with these statements. We found lower WBSF values in the CO2-gas treatment and higher values in the Elec-stun, and N2-gas was intermediate. Therefore, it is believed that the softening phenomenon of CO2 can be improved by using N2 gas.
Table 3 shows the thickness and WBSF value for chicken small intestines with stunned and non-stunned. The thickness of both fresh and cooked small intestines was significantly higher in the halal treatment compared to the stunning treatments (p < 0.05). Among the stunning treatments, Elec-stun had the highest thickness value and the lowest value was found in CO2-gas (p < 0.05). The thickness of N2-gas treatment was higher than CO2-gas but lower than Elec-stun.
The thickness value of small intestine is influenced by various factors including genetics, sex, feeding habits, disease, nutrition, age, and bacterial load etc. The stunning method and pre-slaughter stress may affect the thickness value of the small intestine in chickens post-mortem. The thickness value of N2-stunned pigs’ small intestine was higher than that of CO2-stunned pigs, and lower than that of electrically-stunned pigs under both fresh and cooked conditions [22]. The present study demonstrates similar findings. The electrically-stunned (Elec-stun) broiler small intestine showed higher thickness values than the other treatments. The thickness value of N2-stunning chicken small intestine (N2-gas) was higher than those of CO2-stunning chickens (CO2-gas).
In both fresh and cooked conditions, the WBSF in small intestine of thickness differed significantly between all treatments. The halal treatment provided higher values of WBSF than all the stunning treatments. Among the stunning treatments in both fresh and cooked conditions, the Elec-stun treatment showed the highest values of WBSF and the lowest values of WBSF were found in the CO2-gas. The WBSF value of the N2-gas treatment was between those of the Elec-stun and CO2-gas.
The WBSF value of the small intestine can be influenced by pre-slaughter stunning. During exposure to high concentrations of CO2 during stunning, an enormous amount of CO2 is absorbed in the body. This gas was highly dissolved in the chicken breast meat and small intestine of the animals and remained elevated as a residue [67,69]. It was reported that the WBSF value of N2-stunned pork meat intestines was intermediate between those of electrically- and CO2-stunned pigs [22]. Similar findings were observed in the present study; here CO2-stunned broiler small intestine (CO2-gas) showed a lower WBSF value than electrically-stunned (Elec-stun) small intestine, and N2 stunning (N2-gas) provided intermediate results.
CONCLUSION
The purpose of this study was to assess the quality attributes of N2 gas (98%)-stunned broiler breast meat and small intestine compared with chicken breast meat stunned using conventional electrical, CO2-gas and halal stunning. Our research findings indicate that N2 gas stunning results in less discoloration in meat and small intestines compared to CO2 stunning and halal stunning. Additionally, N2 stunning exhibits color properties similar to electrical stunning and inhibits the phenomenon of softening caused by CO2 stunning method. Therefore, high-concentration N2 gas stunning method can be considered for new industrial applications in poultry slaughter. However, for the adoption of N2 stunning in the industry, further research is needed on factors such as changes in stress hormones and measures regarding the toxicity/safety of meat.
