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. 2023 Aug 23;102(11):103033. doi: 10.1016/j.psj.2023.103033

Traditional halal meat production without stunning versus commercial slaughter with electrical stunning of slow-growing broiler chicken: impact on meat quality and proteome changes

Prasad M Govindaiah *, Naveena B Maheswarappa †,1, Rituparna Banerjee †, Bidyut Prava Mishra ‡, Balaji B Manohar †, Sowmya Dasoju †
PMCID: PMC10506100  PMID: 37708767

Abstract

Impact of traditional halal meat production without stunning (NST) and commercial slaughter with electrical stunning (ST) of 100 slow-growing broiler chicken on blood plasma and different biochemical, enzymatic, hormonal, meat quality, and proteomic changes was evaluated. The results revealed lower (P < 0.05) postmortem pH values and higher redness (a*) scores for ST samples relative to NST group. Myofibrillar fragmentation index and bleeding efficiency (%) were lower (P < 0.05) in ST compared to NST samples. The ST group had higher (P < 0.05) creatinine, total protein, alanine aminotransferase (ALT), and triiodothyronine (T3) than NST group, however, no difference (P > 0.05) in blood glucose, lactate dehydrogenase (LDH), creatine kinase (CK), thyroxine (T4), cortisol, and aspartate aminotransferase (AST) was observed relative to NST samples. The 2-dimensional gel electrophoresis (2-DE) coupled to MALDI-TOF MS of meat samples has identified 14 differentially abundant proteins between 2 groups. Proteins demonstrating positive correlation with stress namely adenylate kinase isoenzyme-1, Rho guanine nucleotide exchange factor (NST), and apolipoprotein A-I (ST) were overabundant. From the current study, it is concluded that electrical stunning of broilers prior to slaughter or traditional halal slaughter without stunning does not adversely affect the meat quality.

Key words: meat quality, poultry, electrical stunning, proteomics, MALDI-TOF MS

INTRODUCTION

Slaughtering is a critical step in the meat production process that affects meat quality, safety, and public health (Maqsood and Ayyub, 2023). Different methods of slaughtering may have an impact on postmortem muscle metabolism (Riggs et al., 2023). The slaughtering of animals without stunning is a practice that is popular in commercial halal meat production and is approved by several international organizations (Shahdan et al., 2016). Humane slaughter of animals combines ethical care with a technical indicator of how to reduce any distress connected to slaughtering. Most frequently, the phrase “humane slaughter” refers to successful stunning, which results in an instantaneous loss of consciousness and lasts until the animal dies by exsanguination or cardiac collapse. Due to technological advancements and increased application skills, methods for stunning and slaughtering animals have improved in industrialized nations (HSA, 2023). The Humane Slaughter Act, 1958, section 2, mandates that all animals should be made insensible to pain before being slaughtered to enforce humane slaughter of animals. However, the legislation allows for the killing of animals in line with religious rituals if they must be put to death by having the carotid artery severed with a sharp object. A controversial aspect in terms of blood biochemistry and meat quality is the practice of slaughtering animals without stunning. The OIE (World Organisation for Animal Health), most of the European Union (EU) nations, USA, UK, Canada, Brazil, France, and several other nations all accept the ritual slaughter of animals without stunning (DEFRA, 2015).

Head-only electrical stunning of poultry has been approved by many Islamic authorities if the method is reversible. However, the use of high-voltage electrical stunning has already been highlighted as being detrimental to meat quality because it causes muscle hemorrhage (Sirri et al., 2017). Further, death may result from heart failure and lack of oxygen to the brain during electrical shock (Turcsan et al., 2003). Preslaughter stunning of animals before exsanguination can affect both the efficacy of the bleeding process and the steps involved in meat processing that are significantly impacted by biochemical changes that take place during slaughter. Studies in broiler chicken have shown the effects of different stunning methods on meat quality parameters, and substantial changes in blood biochemical, enzymatic, and hormonal changes associated with stress (Huang et al., 2014a; Sirri et al., 2017; Li et al., 2022; Riggs et al., 2023).

In the last 2 decades, understanding the biochemical basis of meat quality with regard to preslaughter handling and postmortem muscle metabolism is facilitated by proteomic studies. High-throughput proteomic technologies, particularly in the field of animal stress, have been designated as a major platform in plasma/serum/muscle proteome research for the discovery of next-generation biomarkers. The changes in blood biochemical parameters, meat quality, and proteomic profile of Nellore-crossbred sheep have been investigated (Kiran et al., 2019). However, to the best of our knowledge, studies regarding traditional halal slaughter without any stunning and the effect of electrical stunning on meat proteome changes in broilers are nil. Hence, the present study aimed to identify the protein biomarkers and to evaluate the serum biochemical parameters, enzymatic, and hormonal changes, meat quality, and proteome profile in traditional halal slaughtered vs. electrically stunned and slaughtered slow-growing broiler chicken.

MATERIALS AND METHODS

Ethics Statement

All animal experiments were performed according to the protocols approved by the Institutional animal ethics committee of ICAR-National Meat Research Institute (IAEC No. 007/NRCM/IAEC-9). All animal procedures followed the regulations and guidelines established by this committee and minimized the suffering of animals.

Slow-growing broilers (50-days old, male, multicolored, Plymouth Rock x Red Cornish breeds) weighing approximately 2.0 kg live weight reared under a deep litter system at Indbro Research and Breeding Farm, Hyderabad in accordance with normal husbandry practices were used in the current study. The birds were fed ad libitum with a single diet containing 20% CP and 2,900 kcal/kg ME for 50 d. The major feed ingredients include maize, soya deoiled, sunflower deoiled, vegetable oil, dicalcium phosphate salt, DL-methionine, L-lysine, vitamin premix, and trace minerals. Birds were transported to NMRI during the early hours in cages and rested for 4 h before slaughter at the Institute's experimental poultry processing facility.

Experimental Animals, Traits Measurement, and Calculation

The birds (n = 100) were divided into 2 groups: head-only electrically stunned (ST) and nonstunned (NST) birds. For head-only electrical stunning, live birds were shackled, and passed through the electrical water bath stunner, and allowed their heads to be dipped inside a water bath maintained at 100 mA current, 50 V, and 300 Hz frequency for 5 s followed by slaughtering through ventral neck cut within 5 s of the end of stunning (Girasole et al., 2016; HSA, 2023). In the second group, live birds were slaughtered according to the traditional halal method without prior stunning by manually severing jugular vein, carotid artery, trachea, and esophagus in a single stroke without lifting the knife by an adult Muslim. The spinal cord was kept intact, and birds were allowed to bleed for 3 to 4 min followed by scalding (52°C–54°C for 4 min), defeathering, evisceration, removal of skin, and chilling at 4°C ± 1°C. Prior to chilling, the samples (Pectoralis major and Biceps femoris) were collected and examined for pH, R value, and instrumental color (L*, a*, and b*), whereas the remaining samples underwent a 4-h chilling (4°C ± 1°C) and analyzed for meat quality and proteome analysis. Blood samples were collected at the time of exsanguination/bleeding from the ST and NST groups, and assessed for bleeding efficiency, biochemical, enzymatic, and hormonal assays.

Bleeding Efficiency, Biochemical, Enzymatic, and Hormonal Assay

Birds were sticked and shackled until the bleeding stops (3–4 min). The volume of the blood that was released was collected and instantly measured to determine the bleeding efficiency (Satyaningtijas et al., 2020). Blood samples were collected in ethylene-diamine-tetra acetic acid (EDTA) blood collection vials. Blood tubes were maintained at 4°C ± 1°C and immediately centrifuged for 15 min at 3,000 rpm. Aliquots of the obtained plasma fraction were made and kept at −80°C until further analysis. Plasma samples were analyzed for biochemical (glucose, total protein, and creatinine), enzymatic (lactate dehydrogenase, LDH; creatine kinase, CK; alanine aminotransferase, ALT; aspartate aminotransferase, AST), and hormonal analysis (cortisol, triiodothyronine, T3 and thyroxine, T4). All blood samples were analyzed according to the method demonstrated by International Federation of Clinical Chemistry (IFCC) in an automated chemistry analyzer (Model; TurboChemNeo, CPC Diagnostics, Chennai India) with commercial kits (Everlife CPC Diagnostics, Chennai, India).

Meat Quality Analysis

pH. The pH was determined by homogenizing 10 g of meat with 50 mL of distilled water in a tissue homogenizer at postslaughter intervals of 0, 4, 8, and 12 h. A standardized electrode attached to a digital pH meter was used to test the pH (HANNA instruments, HI 2216, Europe).

R Value. The R value was measured by homogenizing 10 g of meat sample with 50 mL of distilled water in a tissue homogenizer at 0, 4, 8, and 12 h postslaughter (Honikel and Fischer, 1977). Ten milliliters of 1.0 M perchloric acid was used to homogenize 2 g of meat sample. After filtering the homogenate, 0.1 mL of the filtrate was diluted with 4.9 mL of 0.1 M phosphate buffer (pH 7.0). The absorbance at 250 nm and 260 nm were measured with UV-VIS spectrophotometer (Model: UV-1700 Pharma-Spec, Shimadzu, Tokyo, Japan) using phosphate buffer as a standard, and the R value was determined as the ratio of 250:260.

Instrumental Color. A colorimeter (Model: CR-20, Konica Minolta, Inc., Osaka, Japan) was used to measure the lightness (L*), redness (a*), and yellowness (b*) of the breast meat samples at postslaughter intervals of 0, 4, 8, and 12 h. The precalibrated colorimeter surface aperture was placed on the surface of Pectoralis major muscles at 5 random locations to obtain the measurement in terms of lightness (L*), redness (a*), and yellowness (b*) as per Commission Internationale de l'Eclairage (CIE).

Water-Holding Capacity. The water-holding capacity (WHC) was determined according to the method described by Wardlaw et al. (1973). Ten grams of minced breast meat sample were mixed with 15 mL 0.6 M NaCl using glass rod in a centrifuge tube followed by centrifugation at 9,000 × g (Sorvall Biofuge Stratos, Thermo Electron LED GmbH, D-37520, Osterode, Germany) at 4°C ± 1°C for 15 min. After centrifugation, the volume of solution retained by the meat as a percentage was calculated from the supernatant's measurement.

Myofibrillar Fragmentation Index. The myofibrillar fragmentation index (MFI) was determined by adding 10 g of minced meat sample with 50 mL of cold 0.24 M sucrose and 0.02 M potassium chloride, allowed to stand for 5 min. The mixture was blended for 40 s at high-speed using tissue homogenizer (WiseMix homogenizer, LHG-15D, Daihan Scientific, Wonju, Kangwon-do, South Korea). The homogenate was collected and filtered through a 250 µm pore size preweighed muslin cloth into a beaker while being constantly stirred using a glass rod. The muslin cloth was gently and consistently squeezed and kept overnight for drying at room temperature. The MFI was calculated as the residue's weight in g and expressed as a percentage (Olson et al., 1976).

Cooking Loss and Cooked Meat pH. Sample weights were measured both before (raw weight) and following cooking at 100°C for 30 min. The percent change in weight following cooking was calculated as cooking loss, and further, the pH of cooked meat was determined.

Warner-Bratzler Shear Force. The breast meat samples were placed in low-density polyethylene pouches and cooked in a water bath (JEIO TECH BS-11, South Korea) at 100°C until the internal temperature of sample reached 72°C. Cooked samples were refrigerated at 4°C ± 1°C for 12 h, then allowed to acclimatize to room temperature. Ten cores of 1.25 cm diameter were collected from each sample using a tissue borer with the muscle fibers running parallel to the borer's direction. The Warner-Bratzler shear force (WBSF) was measured in Newtons (N) using a texturometer (Model H1KF; Tinius Olsen, Redhill, UK) with a V-shaped stainless-steel blade (60° angle). The crosshead speed was set at 200 mm/min, and a load range of 75 Newtons (N) was used to shear the cores perpendicular to the orientation of the muscle fibers.

Proteome Analysis

SDS-PAGE. The method described by Laemmli (1970) was used for SDS-PAGE, with a midi-electrophoresis apparatus (Model SE-600 Ruby, GE Healthcare, Uppsala, Sweden). The total protein extract, consisting of sarcoplasmic and myofibrillar proteins, was obtained by homogenizing 100 mg of muscle tissue with 1 mL of lysis buffer containing 8 M urea, 2 M thiourea, 1% dithiotheritol (DTT), 2% 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), and 2% protease inhibitor. To load the 12% acrylamide gel, approximately 50 μL of protein sample (containing 50 μg of protein) was denatured by boiling for approximately 5 min. Electrophoresis was performed at a constant voltage mode of 80 V/slab for 3 to 4 h. Coomassie blue was used to stain the gel for 4 to 5 h, followed by destaining.

Protein Purification and 2-Dimensional Gel Electrophoresis. The protocol for the 2-dimensional gel electrophoresis (2-DE) was adapted from Lametsch and Bendixen (2001), with some modifications. About 100 mg of muscle tissue was homogenized in 1 mL of lysis buffer containing 8 M urea, 2 M thiourea, 1% DTT, 2% CHAPS, 2% protease inhibitor, and 2% IPG buffer pH 3 to 10. An equal volume of the extracted protein sample and a solution containing 50% TCA and 20 mM DTT were mixed and incubated on ice for 1 h with intermittent vortexing. The mixture was then centrifuged at 16,000 × g for 10 min and the resulting supernatant was carefully removed. The protein pellet was washed with 500 µL of ice-cold 100% acetone containing 20 mM DTT, which was mixed with the pellet by vortexing and inversion for 15 to 30 min in an icebox. The mixture was then centrifuged at 16,000 × g for 10 min and the supernatant was decanted to remove unextracted cellular components and insoluble proteins. This acetone wash step was repeated twice. The protein content was quantified using the 2-D Quant Kit (GE Healthcare Bio-Sciences, Chicago, IL) and was found to be approximately 10 μg/μL. The protein pellet was dried for 5 min and then dissolved in Rehydration buffer (7 M urea, 2 M thiourea, and 2% w/v CHAPS) and used for the first dimension (isoelectric focusing, IEF). The IPG strips (13 cm, pH range 3–10) were passively rehydrated in 200 μL of protein solution containing 750 to 800 μg proteins for 12 h under drystrip covering fluid in an IPG Box. The rehydrated IPG strips were then subjected to IEF in an Ettan IPGPhor3 gel apparatus at 18°C. Focused IPG strips were equilibrated for 15 min in 6 M urea, 20% glycerol, 2% SDS, 0.375 M Tris, pH 8.8, 2% DTT, and then for an additional 15 min in the same solution except that DTT was replaced by 5% iodoacetamide. After equilibration, proteins were separated in the second dimension using the midi-electrophoresis apparatus at 100 V with 60 mA/gel until the tracking dye reached the lower end of the gel. The gels were stained with Coomassie brilliant blue staining for 2 to 3 h, and after destaining, they were scanned using a Melonie 9 classic/DIGE analyzer software (Melonie 9 classic/DIGE, version 9.2.5 [2022.6.1.0] - SIB [Swiss Institute of Bioinformatics], Geneva, Switzerland). The (2DE) spots were analyzed for significant changes in normalized volume, normalized intensity, and area and significant spots were chosen for further identification using mass spectrometry.

Mass Spectrometry Analysis. The process of in-gel digestion and protein identification using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF/TOF MS) was conducted in accordance with Shevchenko et al. (2006) with slight modifications. The proteins separated by 2-DE were subjected to in-gel digestion, and subsequently enriched and purified using Zip-Tip pipette tips. The peptide mixtures were dissolved in 0.5 μL of CHCA matrix solution (5 mg/mL CHCA in 50% ACN/0.1% TFA) and applied onto a freshly cleaned MALDI target plate. The spots were then air-dried for 30 min at room temperature before being analyzed using a 4800 MALDI-TOF/TOF mass spectrometer (AB Sciex, Framingham, MA) linked to 4000 series explorer software (version 3.5.3). The mass spectra were recorded in a reflector mode within a mass range of 800 to 4,000 Da, using a Nd:YAG 355 nm laser. The acceleration voltage and extraction voltage were maintained at 20 and 18 kV, respectively. All the MS spectra were obtained from an accumulation of 900 shots. For the 15 most abundant precursor ions, MS/MS spectra were acquired with a total accumulation of 1,500 laser shots and collision energy of 1 kV. The combined MS and MS/MS peak lists were then subjected to a search using the GPSTM Explorer software version 3.6 (AB Sciex) against the Swiss-Prot and NCBI database. Protein identification was performed by MS/MS ion search using MASCOT version 2.1 (http://www.matrixscience.com) search engine.

Statistical Analysis

A total of 100 birds were divided into 2 experimental groups (ST and NST) in a completely randomized design replicated on 5 different occasions with 10 birds in each group. The statistical analysis was carried out using OriginPro software (OriginPro, Version 2023. OriginLab Corporation, Northampton, MA). A t test was employed to discern significant effects between the NST and electrically stunned (ST) groups in terms of bleeding efficiency, blood biochemical parameters, water holding capacity, myofibrillar fragmentation index, shear force, cooked pH, and cooking loss. Furthermore, a 2-way analysis of variance (ANOVA) was conducted to ascertain the combined impact of electrical stunning and storage period (0, 4, 8, 12, and 24 h) on pH, R value, and instrumental color measurements. The differences between means were then identified using the least significant difference (LSD) at a 95% confidence level (P < 0.05). The significance of the difference was assessed using Duncan's multiple range tests and Tukey test if the impact of electrical stunning was shown to be significant.

RESULTS AND DISCUSSION

Bleeding Efficiency, Biochemical, Enzymatic, and Hormonal Assay

The total blood content ranges from 6 to 10% of the body weight of live birds (Shaw et al., 2009) and during sticking, 30 to 50% of the total blood is drained out. Irrespective of stunning methods, effective bleeding is an essential component of the slaughtering process as poor bleeding results in reduced shelf life and eating qualities of meat. Studies on the effect of slaughter methods on blood loss have yielded contrasting results. In the current study significantly (P < 0.05) higher bleeding efficiency was observed in NST (3.72%) group compared to ST (2.74%) (Table 1), which might be due to the incidence of ventricular fibrillation and arrest of the heart at the time of electric shock (stunning). However, bleeding efficiency and meat quality are positively correlated (Nakyinsige et al., 2014). Slaughtering of animals without stunning ensures the severing of the main vein supplying blood to the brain, allowing the maximum amount of bleeding. The bleeding efficiency in electrically stunned (water bath, 0.146 A for 5 s) and NST broiler chickens showed 1.14 and 2.25%, respectively (Satyaningtijas et al., 2020). An optimal combination of electrical frequency and voltage during the stunning process can result in reduced stress levels for broiler chickens while ensuring their recovery and bleeding efficiency remain unaffected. To exemplify, it has been documented that the utilization of low-voltage electrical stunning can result in subpar bleeding outcomes and elevated carcass defects, as noted by Gregory (2005). Conversely, the application of high voltage carries the potential risk of inducing heart fibrillation, subsequently giving rise to inadequate bleeding, excessive hemorrhages, and even premature fatality prior to exsanguination, as highlighted by Hayat et al. (2023). The electrical stunning might result in vasoconstriction and was considered to reduce the blood flow and the amount of drained-out blood than typical halal (NST) slaughtered birds (Sabow et al., 2017). On the contrary, Velarde et al. (2003) found that lambs subjected to electrical stunning before sticking, recorded slightly more blood loss than lambs which are not subjected to electrical stunning. The different animal species subjected to stunning, differences in stunning voltage, durations, and methods employed to quantify blood loss could be contributing factors for these inconsistent findings.

Table 1.

Bleeding efficiency and blood biochemical, enzymatic, and hormonal changes between electrically stunned and halal slaughtered (nonstunned) slow-growing broiler chicken.

Parameters NST1 ST1 P value
Bleeding efficiency (%) 3.72 ± 0.32a 2.74 ± 0.37b 0.026
Glucose (mg/dL) 244.33 ± 24.00a 260.60 ± 26.61a 0.476
Creatinine (mg/dL) 0.37 ± 0.06a 0.50 ± 0.00b 0.016
Total protein (g/dL) 3.67 ± 0.25a 4.43 ± 0.35b 0.037
Lactate dehydrogenase (LDH) (U/L) 642.27 ± 23.17a 667.07 ± 32.71a 0.344
Creatine kinase (CK) (U/L) 4751.17 ± 145.22a 4547.03 ± 136.92a 0.151
Aspartate transferase (AST) (IU/L) 276.03 ± 37.61a 323.70 ± 42.71a 0.220
Alanine aminotransferase (ALT) (IU/L) 3.23 ± 1.56a 9.63 ± 2.87b 0.028
Cortisol (µg/dL) 0.12 ± 0.03a 0.17 ± 0.04a 0.163
Triiodothyronine (T3) (ng/dL) 1.53 ± 0.09a 1.77 ± 0.09b 0.029
Thyroxine (T4) (µg/dL) 3.54 ± 0.12a 3.42 ± 0.14a 0.297
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a−b

Mean ± standard deviation without a common superscript were determined to be significantly different between slaughter methods (P < 0.05).

1

NST, nonstunned; ST, electrically stunned.

The effect of slaughter methods on biochemical changes namely blood glucose, creatinine, and total proteins were tabulated in Table 1. No difference (P > 0.05) in blood glucose level was observed between the 2 groups, however, the creatinine and total protein contents were higher (P < 0.05) in ST compared to the NST broilers. Significantly higher blood creatinine levels were reported in ostriches subjected to transport stress probably due to the actions of catecholamines and glucocorticoids as a result of stress conditions (Vazquez-Galindo et al., 2013). Increased plasma total proteins might be due to vasoconstriction and decreased plasma volume during stress (Ribeiro et al., 2018). The results of the present study were in accordance with the previous findings (Huang et al., 2014a), where no significant change in blood glucose level was observed between ST and NST groups. On the contrary, Sabow et al. (2016) found significantly higher blood glucose level in NST goats than those animals subjected to stunning. Glucose levels are less variable and not a reliable measure of stress since it may decrease due to feed withdrawal and increases due to stress, which causes a release of glucagon and cortisol stimulating glycogenolysis and glyconeogenesis.

The plasma enzymes that have been widely used as indicators of stress in food animals/poultry include creatine kinase (CK), aspartate aminotransaminase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH). Elevation of plasma concentration of these intracellular enzymes reflects alterations in tissue function or is indicative of cell damage (Klein et al., 2020). The plasma levels of AST, CK, and LDH correlate with meat quality traits in cattle and pork, indicating that these markers have the potential to predict meat quality in live animals. The concentration of ALT, AST, CK, and LDH in electrically stunned and halal slaughtered broilers was tabulated in Table 1. No difference (P > 0.05) was observed for AST, CK, and LDH concentration between the 2 groups, however for ALT, significantly (P < 0.05) higher concentration was found in ST broilers compared to NST birds. Menon et al. (2014) observed that the serum ALT and AST did not increase significantly after 6 h of transportation, but increased significantly after lairage, and noticed a significant increase in creatine kinase activity after 6 h of transportation, with a further significant increase after lairage. In contrast, the activities of the enzymes AST, ALT, and CK in plasma increased significantly following loading, transporting, and handling the animal (Menon et al., 2014) due to increased permeability of muscle cell membranes and their concentrations were significantly elevated at slaughter indicating physical stress and muscle damage. Huang et al. (2017) assessed the impact of heat stress on the serum biochemical parameters of broilers and reported that ALT levels significantly increased with the duration of heat stress and a significant difference in CK level was observed between control and boilers subjected to heat stress.

The metabolic regulators are important in elucidating the modulation in physiological mechanisms during stress conditions and are best assessed by determining the hormones governing various metabolic reactions in plasma or serum (Gupta et al., 2013). Physiological responses to stress occur in the form of differences in body temperature, T3, and T4 hormone levels (Altın et al., 2018). The findings of hormonal analysis namely cortisol, T3, and T4 in both stunned (ST) and halal (NST) groups were tabulated (Table 1). A significantly lower (P < 0.05) level of T3 in NST broilers was observed compared to stunned birds, however, no significant (P > 0.05) changes were found in cortisol and T4 between ST and NST groups. The plasma corticosterone concentrations of the low-frequency stunned broilers were higher than in the groups subjected to higher-frequency electrical stunning, demonstrating that stunning with too low a frequency was more stressful to the broilers (Huang et al., 2017). Wing flapping of shackled broilers during stunning and bleeding also results in increased plasma corticosterone concentrations (Huang et al., 2014a). On the contrary, the concentration of plasma corticosterone was not found to be affected by 35, 50, or 65 V or 160, 400, or 1,000 Hz with 18 s of stunning (Xu et al., 2011). Li et al. (2022) reported considerably lower cortisol levels in electrically stunned group (50 V, 48–52 mA, 50 Hz for 10 s) compared to the NST broilers. The difference in the findings might be due to differences in frequency, voltage, and duration of electrical stunning in animals. Thyroid hormones T3 and T4, which play a major role in controlling thermoregulation, metabolic rate, and heat production in vertebrates, are influenced by several internal and external factors including stressors (Oladokun and Adewole, 2022). Significantly reduced blood T3 concentrations have been reported in broilers exposed to thermal stress at various conditions (Zaglool et al., 2019). Jiang et al. (2020) observed that while heat stress increased the T4 concentration, the T3 concentration was significantly decreased. Taken together, the current and previous results suggested that thyroid hormones in broilers are affected by multiple factors including stress level and duration, bird age, and also nutritional status.

Meat Quality Analysis

There was a consistent and significant (P < 0.05) decrease in meat pH as postmortem storage time increased from 0 to 24 h between both groups, and a significantly lower pH in breast and thigh meat of stunned birds was observed in comparison to NST breast and thigh meat throughout the postmortem storage period (Figure 1A). However, there is no difference in pH at 24 h of postmortem between ST and NST samples. During the complete postmortem storage period, there was a significantly greater (P < 0.05) pH in thigh samples than in breast samples. All the samples had an ultimate pH ranging from 5.5 to 5.8 at 24 h postmortem. Any acute stress during slaughter accelerates the rate of muscle glycogenolysis and elevates the concentration of lactic acid by anaerobic glycolysis, which lowers the muscle pH (Terlouw and Bourguet, 2022). In view of this, the pH can be used as a benchmark for stress assessment, with lower pH values suggesting greater levels of stress during slaughter (Li et al., 2022) but present results revealed, electrical stunning had no effect on pH at 24 h postmortem and similar results were found by various researchers in broilers (Lambooij et al., 2010; Huang et al., 2014b, 2017; Riggs et al., 2023) and in other animals (Velarde et al., 2003; Önenç and Kaya, 2004; Sabow et al., 2017; Al-Amri et al., 2023) where slaughtering method had no significant effect on ultimate muscle pH value at 24-h postmortem.

Figure 1.

Figure 1

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A. Effect of halal slaughter without stunning (NST) and slaughter with electrical stunning (ST) on pH of slow-growing broiler chicken (breast, Pectoralis major and thigh, Biceps femoris) during postmortem storage. (B) Effect of halal slaughter without stunning (NST) and slaughter with electrical stunning (ST) on R value of slow-growing broiler chicken (breast, Pectoralis major and thigh, Biceps femoris) during postmortem storage.

The R value, which is an indirect measurement of ATP depletion in the muscle during rigor mortis development, increases as it represents the ratio of inosine: adenosine-containing compounds in the muscle (Khan and Frey, 1971). No significant (P > 0.05) difference was observed in R values between ST and NST groups throughout the postmortem storage (Figure 1B). However progressive (P < 0.05) increase in R value was recorded from 1.3 to 1.42 in all the groups throughout the storage period. Similar results were also observed by Huang et al. (2014b), who reported that although a significant difference in ATP level was observed between free struggle and electrically stunned broilers in 0.3 h postmortem, no significant changes in ATP, ADP, AMP, and glycogen level between the treatment groups was recorded. Similarly, Kiran et al. (2019) noticed no significant change in R value between stunned and not stunned meat samples in sheep.

In the present study, no difference (P > 0.05) in WHC was observed between ST and NST groups (Table 2), however significant differences (P < 0.05) were observed between thigh and breast samples among stunned and NST group, which might be due to the net-charge effect (Vadehra et al., 1973). In the present study, the WHC was not affected by the electrical stunning which was in accordance with the pH values obtained between ST and NST groups throughout the storage period. Similar results were observed in goat meat (Sabow et al., 2017; Al-Amri et al., 2023) and in beef (Önenç and Kaya, 2004).

Table 2.

Meat quality changes between electrically stunned and halal slaughtered (nonstunned) slow-growing broiler chicken.

Parameters NST1 ST1 P value
2WHC (%)
Breast* 15.60 ± 4.34a 19.80 ± 1.10a 0.069
Thigh# 29 ± 4.24a 35 ± 9.95a 0.249
3MFI (%)* 12.78 ± 0.44b 8.95 ± 0.23a <0.001
4WBSF (N)* 11.78 ± 1.06a 11.06 ± 1.02a 0.302
Cooked pH* 5.74 ± 0.05a 5.73 ± 0.11a 0.887
Cooking loss (%)* 30.40 ± 1.95a 30.55 ± 0.64a 0.872
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a−b

Means ± standard deviation without a common superscript were determined to be significantly different between slaughter methods.

⁎

Pectoralis major.

#

Biceps femoris.

1

NST, nonstunned; ST, stunned.

2

WHC, water-holding capacity values were expressed in percentage and determined at 4-h postmortem.

3

MFI, myofibrillar fragmentation index values were expressed in percentage and determined at 4-h postmortem.

4

WBSF, Warner-Bratzler shear force values were expressed in N, Newton and determined after 4-h postmortem.

No difference (P > 0.05) was noticed in cooking loss, cooked pH, and shear force values between the ST and NST groups (Table 2). These results were in accordance with the findings of Sabow et al. (2017). Contradictory results were reported for cooking loss in electrically stunned animals compared to NST (Önenç and Kaya, 2004; Nakyinsige et al., 2014), whereas Li et al. (2022) reported significantly (P < 0.05) higher cooking loss in NST birds compared to stunned birds. Shear force values were not significantly (P < 0.05) affected by the electrical stunning and similar results were reported by Sirri et al. (2017). An inconsistent result was reported by Huang et al. (2014a) depicting significantly higher shear force using high current flow stunning (48–52 mA/bird vs. 106–110 mA/bird, both at 50 Hz for 10 and 5 s, respectively). On the other hand, a significantly lower shear force was reported by Huang et al. (2017) in low- and high-frequency electrically stunned groups compared to medium-frequency stunning, which was supported by fast pH decline in muscles, and increasing cytoskeleton degradation due to earlier activation of calpains.

The disintegration of structural proteins within the sarcomere's I-band, as well as intermyofibrillar linkages, has been shown to be reflected by the myofibrillar fragmentation index (MFI), making it a helpful indicator of the degree of proteolysis (Kim et al., 2013). The present study recorded a higher (P < 0.05) MFI in NST meat samples compared to ST meat samples (Table 2). Our findings were in accordance with results of Nakyinsige et al. (2014), in which meat from NST rabbits had significantly higher MFI than meat from gas-stunned rabbits. The MFI and shear force were discovered to have a strong inverse association (Hou et al., 2014). Al-Amri et al. (2023) found higher MFI in NST samples compared to stunned samples from goat meat. Contradictory results were reported in goats, that the electrical stunning did not affect the myofibrillar fragmentation index (Sabow et al., 2015) and observed significantly higher MFI in stunned goat meat samples compared to NST samples (Sierra et al., 2012; Sabow et al., 2017).

A higher (P < 0.05) a* value in ST group was recorded relative to NST during 4, 8, and 24 h of postmortem storage period and noticed no significant change in L* and b* values throughout the storage period (Table 3). The ST samples displayed a pinkish hue, which was probably caused by improper bleeding. Similar findings were also reported in chicken (Huang et al., 2014a) and this outcome is consistent with the findings of earlier researchers (Velarde et al., 2003; Önenç and Kaya, 2004), between electrically stunned and NST chicken breast samples.

Table 3.

Instrumental color of breast (Pectoralis major) and thigh (Biceps femoris) muscle between electrically stunned and halal slaughtered (nonstunned) slow-growing broiler chicken.

Postmortem storage period
 SEM
Instrumerntal color Treatments 4-h 8-h 12-h 24-h
L STB1 51.24 ± 2.32a,A 51.65 ± 2.02ab,A 53.34 ± 1.59ab,A 53.05 ± 1.21a,A 0.526
NSTB1 53.16 ± 1.77a,A 55.57 ± 0.51c,A 55.78 ± 1.78b,A 55.73 ± 2.39a,A 0.543
STT1 53.28 ± 2.40a,B 49.79 ± 0.04a,A 51.41 ± 1.20a,AB 53.90 ± 1.87a,B 0.631
NSTT1 53.16 ± 0.98a,A 53.72 ± 1.71bc,A 52.46 ± 0.91a,A 54.20 ± 1.79a,A 0.397
a* STB 1.24 ± 0.42ab,A 1.80 ± 0.64ab,A 1.03 ± 1.01a,A 1.00 ± 0.54ab,A 0.195
NSTB 0.86 ± 0.62a,A 0.83 ± 0.79a,A 0.84 ± 0.22a,A 0.81 ± 0.37b,A 0.134
STT 3.07 ± 0.55c,A 3.44 ± 0.25c,A 3.38 ± 0.29b,A 3.68 ± 1.30c,A 0.192
NSTT 2.08 ± 0.50bc,A 2.90 ± 0.75bc,A 2.87 ± 1.31b,A 2.39 ± 0.64bc,A 0.235
b* STB 7.36 ± 1.51a,A 6.55 ± 1.45a,A 6.63 ± 1.00a,A 9.83 ± 0.05b,B 0.492
NSTB 7.75 ± 2.16a,A 8.73 ± 0.76b,AB 7.96 ± 1.51a,AB 10.53 ± 0.14b,B 0.473
STT 6.53 ± 1.50a,A 6.89 ± 0.29a,A 7.19 ± 0.28a,A 7.32 ± 0.92a,A 0.241
NSTT 6.73 ± 0.43a,A 8.97 ± 0.25b,A 8.47 ± 1.98a,A 7.58 ± 1.96a,A 0.434
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a−c

Means ± standard deviation without a common superscript were determined to be significantly different between slaughter methods (P < 0.05).

A−B

Means ± standard deviation without a common superscript were determined to be significantly different between storage period (P < 0.05).

1

STB, stunned breast; NSTB, nonstunned breast; STT, stunned thigh; NSTT, nonstunned thigh; SEM, standard error of means.

Proteome Analysis

No apparent difference in band pattern or intensity was observed in the SDS-PAGE analysis (Supplementary Figure 1) of total and sarcoplasmic proteins between electrically stunned (ST) and halal slaughtered (NST) meat samples. Therefore, 2-DE and mass spectrometry (MS) were utilized to identify the protein biomarkers that vary between ST and NST meat samples. Analysis of 2-DE gels revealed the presence of 136 protein spots. Statistical analyses indicated that 14, 10, and 42 spots exhibited significant differences (P < 0.05) in normalized volume, normalized intensity, and area (P < 0.01), respectively (Supplementary Table 1) between ST and NST samples. Five protein spots with a spot intensity difference of 1.5-fold or more between ST and NST are indicated in Figure 2. The differentially abundant protein spots were excised from the gel, subjected to in-gel tryptic digestion, and analyzed with tandem MS. Proteins were identified using SwissProt and NCBI database. The proteins identified by tandem MS are presented in Table 4 with their accession number, species, molecular weight (MW), protein score, matched peptides, and sequence coverage. Overall, NST samples exhibited overabundance of 3 proteins, whereas 2 proteins were overabundant in ST.

Figure 2.

Figure 2

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Representative 2-DE gel of total muscle proteins extracted from Pectoralis major muscles of halal slaughtered birds without stunning (NST) and slaughtered with electrical stunning (ST). Gels were produced in 3 technical replicates. Protein spots identified through MALDI-TOF/TOF are highlighted.

Table 4.

MALDI-TOF MS identification of proteins extracted from chicken (Pectoralis major) subjected to electrical stunning or halal slaughtering without stunning.

Spot ID Species (accession no.) Identified protein Matched peptides Seq. coverage (%) Protein mass (kDa) Protein score Peptide position Peptides (m/z)
NST 32 Gallus gallus (TPM2_CHICK) Tropomyosin beta chain 6 20 32.871 46 92–101 R.IQLVEEELDR.A (1243.61)
168–178 R.KLVVLEGELER.S (1284.71)
NST 30 Gallus gallus (KAD1_CHICK) NP_990440.1 Adenylate kinase isoenzyme 1 6 33 21.783 116 11–22 K.IIFVVGGPGSGK.G (1130.63)
33–45 K.YGYTHLSTGDLLR.A (1495.72)
90–98 K.GFLIDGYPR.E (1037.51)
150–156 K.RLETYYK.A (972.5)
NST 31 Gallus gallus (CCD25_XENTR) NP_996856.1 Coiled-coil domain-containing protein 25 8 52 24.378 62 2–22 M.VFYFTSNVISPPYTMYMGKDK.Y (2488.18)
44–52 K.LSSAHVYLR.L (1045.58)
NST 31 Gallus gallus (ARHG6_CHICK) Rho guanine nucleotide exchange factor 6 11 26 86.6652 78 308–320 R .VGGCFMNLMAQFR.S (1562.87)
399–404 K.AITSFK.S (666.39)
405–415 K.SLVSQCQELRK.R (1347.69)
711–717 K.DEVKELK.Q (860.47)
ST 13 Gallus gallus (RFC2_CHICK) Replication factor C subunit 2 9 21 40.138 85 1–14 MEEEEVLEVVEDEK.A (1706.74)
24–46 R.GPTDTLGSAPAASGHYELPWVEK.Y (2383.16)
127–132 K.MFAQQK.V (768.56)
ST 29 Gallus gallus (APOA1_CHICK) Apolipoprotein A-I 15 44 30.661 199 120–130 K.IRPFLDQFSAK.W (1321.76)
164–172 K.LTPVAEEAR.D (985.57)
185–194 K.NLAPYSDELR.Q (1177.62)
230–238 K.MTPLVQEFR.E (1120.61)
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1NST, nonstunned; ST, stunned.

Adenylate kinase (AK), a monomeric enzyme, regulates adenine nucleotide metabolism and plays an important role in maintaining cell energy balance. Adenylate kinase isoenzyme 1 (AK1) is considered the main AK subtype in the cytoplasm of skeletal muscle cells (Tanabe et al., 1993) and has been demonstrated to play a role in regulating adenine nucleotide metabolism (Pucar et al., 2002). The enzyme catalyzes the production of ATP, which is a key molecule involved in cellular energy metabolism and its activity has recently been found to be closely associated with apoptosis. It has been suggested that the expression of this protein may be induced by stress, as previous research has shown that under conditions such as hypoxia or nutrient deprivation, cells may increase their reliance on AK1 to produce ATP and maintain energy balance (Panayiotou et al., 2014). Xing et al. (2020) also detected AK1 enzyme related to energy metabolism where the phosphorylation levels in muscle were affected by stress or postmortem time. The representative MS and MS/MS spectra of K.YGYTHLSTGDLLR.A (m/z 1495.72) peptide derived from AK1 is depicted in Supplementary Figure 2A. Rho guanine nucleotide exchange factor 6 (RGNEF) is a stress response protein that plays a crucial role in providing enhanced survival after axonal injury, oxidative or osmotic stress in cultured cells. The RGNEF has been identified as a prosurvival factor under stress conditions, and studies have shown that it can modulate mRNA stability and activate RhoA protein (Droppelmann et al., 2014). It has been demonstrated that RGNEF plays a role in protection against oxidative and osmotic stress in vitro (Cheung et al., 2017). The representative MS and MS/MS spectra of R.VGGCFMNLMAQFR.S (m/z 1560.83) peptide derived from RGNEF is depicted in Supplementary Figure 2B. Expression of AK1 and RGNEF in halal (NST) birds might be due to their role in protecting the cells from the damaging effects of stressors. Studies have shown that electrical stunning can cause an increase in reactive oxygen species (ROS) production, leading to cellular damage and apoptosis (Redza-Dutordoir and Averill-Bates, 2016). Apolipoprotein A-I (ApoA-I) the primary protein component of HDL particles influences cell function to protect endothelial cells by inhibiting apoptosis and increasing cell proliferation (Castaing-Berthou et al., 2017). The anti-inflammatory and antiapoptotic properties of ApoA-I may be beneficial in mitigating the effects of stress or slaughter stress on animals. Additionally, the protein's role in promoting cell proliferation and protecting cells from oxidative damage may be valuable in protecting animals' cells during and after the stunning process. The representative MS and MS/MS spectra of K.IRPFLDQFSAK.W (m/z 1321.76) peptide derived from ApoA-I are depicted in Supplementary Figure 2C.

CONCLUSIONS

Electrical stunning prior to slaughter of slow-growing broilers results in substantially lower bleeding efficiency relative to halal slaughtered birds without adversely affecting the meat quality. Blood plasma analysis revealed a significant increase of biomarkers such as creatinine, total protein, ALT, and T3 level in stunned (ST) birds. The 2-DE and MALDI-TOF MS has identified few key proteins that are abundant in electrically stunned (APOA-1) or halal slaughtered (AK1 and RGNEF) broilers. From the current study, it is concluded that electrical stunning of broilers prior to slaughter or traditional halal slaughter without any stunning does not adversely affect the meat quality even though, stunning induces the expression of biomarkers such as creatinine. The peptides identified in the current study may be used to authenticate the halal slaughtered meat, however further research is needed to determine the expression and specific impact of the proteins identified in the current study in different species.

ACKNOWLEDGMENTS

This study was supported financially by the Education Division, Indian Council of Agricultural Research (ICAR), New Delhi, India (Grant No. Ag. Edn./27/04/NP-NF(VP)2019-HRD). Special thanks to the M/s. Indro Poultry Farm, Hyderabad for providing slow-growing broilers.

DISCLOSURES

The authors declare no conflicts of interest.

Footnotes

Supplementary material associated with this article can be found in the online version at doi:10.1016/j.psj.2023.103033.

Appendix. Supplementary materials

mmc1.docx (6.6MB, docx)

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