DOI: https://doi.org/10.21203/rs.3.rs-1640587/v1
This research aimed to determine how the pre-slaughter restraint method affected broiler chickens' bleeding efficiency, carcass characteristics, and physicochemical qualities. Before slaughter, 30 male Ross broiler chickens were randomly allocated to either shackling or cone restraint. The individual blood loss of each bird was determined by comparing their body weight before and after 90 seconds of exsanguination. On the Pectoralis major muscle, meat quality measures including pH, water-holding capacity, color, tenderness, and total bacterial counts were determined. At the same time, the incidences of hemorrhage on the breast and thigh of each carcass were analysed morphologically. It was found that shackling produced less blood loss than cone restraint. Except for the final pH, shackling significantly affected the quality of the end product, as muscle lightness, shear force, drip loss, cooking loss, lipid-protein oxidation, and bacterial counts increased (p < 0.05). In addition, broiler birds held by the shackle exhibited larger (p < 0.05) hemorrhages than those restrained by the cone. The results indicated that the method of restraint might affect bleed-out and carcass and meat quality in broiler chickens; consequently, it should be examined in future research.
Since prehistoric times, poultry meat, namely broiler chicken, has been an essential part of the human diet (Sabow and Majeed, 2019). Its high biologically valuable nutritional content and processing utilisation positioned it among the most popular foods (Parpia et al., 2018). Additionally, Broiler chicken meat meets the needs of modern consumers due to its low fat, mostly saturated fatty acid, and cholesterol content compared to equivalent cuts of red meat such as mutton and beef (Wideman et al., 2016). Despite its low lipid content, broiler chicken flesh contains many unsaturated fatty acids and is a source of conjugated linoleic acid, which has anti-inflammatory, anti-thrombotic, and atherosclerosis-preventing properties (Mir et al., 2017; Moussa et al., 2019). Broiler meat also has high biological value protein constituting all essential amino acids that cover human requirements. In addition, the broiler is an essential source of vitamins, especially vitamin A, E, and D, and minerals such as potassium, sodium, calcium, magnesium, iron, copper, zinc, and manganese (Angelovičová et al., 2016).
Modern customers place a premium on meat quality and safety at the time of sale, influenced by genetic and environmental factors (Bostami et al., 2021). Among the environmental factors are occurrences that occur just before slaughter that is known to be stressful for broiler chickens and may substantially impact the meat quality of the birds (Saraiva et al., 2020). For instance, it has been observed that pre-slaughter restraining measures influence the pre-slaughter and post-slaughter physiological responses in broiler chickens, particularly energy metabolism inside the skeletal muscle, which influences postmortem muscle metabolism. While shackling is the most prevalent method of restraining broilers in commercial settings, it has been recognised that it negatively affects broilers' welfare and meat quality (Vinco et al., 2016; Fuseini et al., 2018). Struggling or wing flapping during pre-slaughter shackling has been reported to fasten the initial rate of pH drop, which is often associated with poor meat quality features such as undesirable color and poorer water-holding capacity (Huang et al., 2018). Likewise, pre-slaughter conditions may cause stress and physical damage, which significantly influences lipid-protein oxidation in meat and meat products during the early post-slaughter period (Bostami et al., 2021Limiting this behaviour in shackled broilers prior to slaughter may help to create meat with optimal final pH, juiciness, and lowers the occurrence of pale, soft, and exudative meat and protein degradation, thereby boosting production, profitability, and meat quality (Huang et al., 2018). In recent years, cone restraint has been discovered to limit the mobility of birds prior to slaughter, during slaughter, and shortly after slaughter. An earlier study found that confining birds in a cone constraint and then slaughtering them reduced fowl struggle and improved meat quality compared to shackling (Ismail et al., 2019). 2016 (Ismail et al.). However, there is a lack of detailed information on carcass defects, such as the incidence of hemorrhage, which is the primary concern in the poultry meat industry because it is one of the negative carcass characteristics that affect consumer acceptance of meat and meat quality in terms of physicochemical attributes and microbiological quality of broiler chickens during refrigerated storage.
This study was conducted to determine the effects of pre-slaughter shackling and cone restraining methods on broiler chickens' bleeding efficiency, carcass, and meat quality.
All birds were treated humanely, following Sabow's established standards (2020). The experimental procedure was approved by the agriculture college's animal care and use committee, Salahaddin University-Erbil, Kurdistan Region, Iraq.
Thirty 42-day-old male Ross broiler chickens reared under similar management system with an average live body weight of 1.925 ± 0.008 kg were purchased from a commercial poultry farm. The broiler chickens were transported from the farm to a commercial poultry abattoir (Erbil's slaughterhouse for poultry- Kurdistan Region, Iraq). They were slaughtered 60 min after arrival (Ismail et al., 2019). Following transportation and release, the live bodyweight of each bird was measured and recorded. Using either a shackle or a cone, broiler birds were restrained for 30 seconds before slaughter and humanely slaughtered according to the standard halal slaughtering method. A licensed slaughterman carried out the slaughtering procedure. Each bird's head was pulled dorsally to stretch its neck and facilitate exsanguination. Using a sharp knife, a transverse section was severed. The neck cut severed skin, muscle, esophagus, trachea, carotid arteries, jugular veins, and major nerves to drain excess blood from the carcass without decapitating the head.
Individual blood loss during the 90 seconds of exsanguination was measured by changing body weight before and after slaughtering (Kranen et al., 1996). Calculate the percentages of blood loss using the formula provided below:
Blood loss(%) = [(W1-W2)/W1] ×100
Where,
W1 = liveweight
W2 = weight after neck cut.
Following evisceration and carcass dressing, 20 g of pectoralis major muscle from the left side was taken, labeled, vacuum-packed, and stored at 4 oC for drip loss assessment. The breast muscles of the dorsal side of the pectoralis major were removed from the chilled carcass and divided into two portions. The first portion (right pectoralis major muscle) was labeled correctly, vacuum packaged, and stored at -20 ºC for subsequent determination of pH, color, cooking loss, shear force, lipid-protein oxidation, and microbial enumeration at d 1, whereas the second portion (left pectoralis major muscle) were vacuum packed and directly stored at 4 ºC chiller for 5 days. Upon completion of the aging period, the left pectoralis major muscle was labeled, vacuum packaged, and stored at -20 ºC until subsequent analyses.
Using a modified version of the Bostami et al. (2021) method, we observed characteristics of the corpse, such as hemorrhages, 1 day after postmortem (2021). The breast and thigh muscle hemorrhages were quantified using a visual grading system. The classification was performed independently by three observers knowledgeable in the carcass and meat quality. A threshold model consisting of a discontinuous 5-point scale with 4 cutoff points was used for classification. Cutoff points were formed by photographs of breast and thigh muscles showing a particular severity of hemorrhages; class 1: hemorrhage free; class 5: numerous and severe hemorrhages (Lambooij et al., 1999). The severity of red wingtips on the carcass was also estimated visually using a number scale with 0 (no defects), 1 (moderate redness), and 2 (severe redness) (McNeal et al., 2003).
The pH of the pectoralis major muscle was evaluated carefully and indirectly with a portable pH meter (EZDO PP-203, Taiwan). 1 gram of beef was homogenised in 20 milliliters of ice-cold deionized water for 30 seconds. Using a pH meter pre-calibrated at pH 4.0 and 7.0, the pH of the homogenates was used.
The meat samples' water holding capacity (WHC) measured cooking and dripped loss. Individual fresh flesh samples from the pectoralis major muscle were weighed (about 20 g) and recorded as the initial weight for drip loss (W1). The samples were placed in polyethylene plastic bags, properly labeled, vacuum sealed, and stored at 4 degrees Celsius for five days. At the prescribed storage duration, samples were removed from the polyethylene plastic bags, blotted dry, weighed, and recorded asW2. The percentage of drip loss was computed using the following formula:
Drip loss (%) = [(W1-W2) ÷ W1] × 100).
Pectoralis major muscle samples were weighed (W1), packaged in polyethylene bags, and vacuum-sealed to quantify the cooking loss. The samples were cooked in a preheated water bath (HAAKE C10, UK) set to 80 oC for 10 minutes after reaching an internal temperature of 78 oC, as measured with a piercing temperature probe. After removing the cooked samples from the water bath and allowing them to cool to room temperature, they were carefully dried and reweighed (W2). The proportion of cooking loss was computed using the following formula.:
Cooking loss (%) = [(W1- W2) ÷ W1] × 100
The meat samples used to determine cooking loss were prepared to evaluate the shear force values using the Volodkovitch bite jaw attached to a Brookfield Texture Analyzers (CT3™, USA). The equipment was calibrated at a 10 mm return distance for height, and the blade speed was set at 10 mm/s. Samples were prepared according to Sazili et al. (2005) method. Parallel to the direction of the muscle fibers, 1 cm (height) × 1 cm (width) × 2 cm (length) blocks were cut from each sample. Each block was sheared with the Volodkevitch bite jaw in the center and perpendicular to the fibers' longitudinal orientation. Measurements of shear force were recorded in kilogram (kg) units as the average peak positive force of all subsample values for each sample.
A Color Flex spectrophotometer was employed to assess the meat's color (Shenzhen 3nh Technology Co., Ltd, China). Before use, the colorimeter was calibrated against black and white tiles. Blooming was applied for 30 minutes to 12 mm thick samples of the pectoralis major muscle (AMSA, 2012). The flower sample was placed on the facing base of the colorimeter cup. Each sample's L* (lightness), a* (redness), b* (yellowness), c* (chroma), and h* (hue angle) values were measured and averaged in triplicate.
Determination of thiobarbituric acid reactive substances (TBARS )
According to Aminzade et al. (2012) and modified by Sabow et al., the TBARS of the pectoralis major muscle was determined (2020). 5 grams of pectoralis major muscle were homogenised in 48 milliliters of sterile water and 1.25 milliliters of 4N HCl for 2 minutes. The liquid was distilled to a volume of 25 mL. The distillate and the TBA reagent were boiled for 35 minutes (15 percent trichloroacetic acid, 0.375 percent thiobarbituric acid). After 10 minutes of chilling under running water, a spectrophotometer measured absorbance at 538 nm against a blank (Spectronic Instruments, USA). Increasing the optical density by 7.843 percent yielded the TBARS results. The oxidation products were measured in malondialdehyde equivalents (mg MDA per kilogram of beef).
Morzel et al. (2006) extracted myofibril proteins with slight modifications (Sabow et al., 2016). 2.5 g of pectoralis major muscle was homogenised for 30 seconds in 20 ml of extraction buffer consisting of 150 mM NaCl, 25 mM KCl, 3 mM MgCl2, and 4 mM EDTA, with protease inhibitor (Sigma-Aldrich, USA). The homogenate was filtered to remove any remaining collagen. The homogenate was incubated at 4oC for 15 minutes following filtration, shaking before centrifuging at 4 oC at 2000 g for 15 minutes. The pellet was washed twice with 25 ml of a 50 mM KCl solution at pH 6.4 and once with 25 ml of 20 mM phosphate buffer at pH 6. The pellet was finally re-suspended in the same phosphate buffer. The protein quantification for the final suspension was assessed (Bradford, 1976) using Bio-Rad Protein Assay Kit II 500-0002 following the colorimetric analytical procedure. Bovine serum albumin was used to generate the standard solutions for protein measurement. Each sample's protein concentration was evaluated using a standard curve relating protein concentration to absorbance at 595 nm wavelength.
Spectrophotometry was utilised to determine the thiol content by employing Ellman's method of using 2, 2-dithiobis (5-nitropyridine) DTNP and 2, 2-dithiobis (5-nitropyridine) (Morzel et al., 2006). Myofibrillar protein pellets containing 4 mg were dissolved in 3 ml of 100 mM phosphate buffer with 8 M urea at pH 8. After adding 30 µl of 10 mM DTNP (ethanol stock solution), the mixture was incubated at room temperature for one hour. A spectrophotometer (Spectronic Instruments, USA) was utilised to compare the absorbance at 386 nm to a protein-free solution. With an absorption value of 14 mM− 1 cm− 1, the thiol concentration was estimated after subtracting the absorbance of the blank. The outcome was reported in terms of nanomoles of free thiol per milligram of protein.
Carbonyl content was measured by reactivity with 2, 4-dinitrophenylhydrazine (DNPH) to create protein hydrazones, using a technique modified from Morzel et al. (2006). Briefly, 200 l of myofibrillar suspension was treated with 1 ml of 0.2% (w/v) DNPH in 2 N HCl before being incubated at room temperature with shaking for 1 hour. After 10 minutes of centrifugation at 4000g to remove free DNPH, the precipitates were washed three times with 1 ml of ethanol: ethyl acetate (1:1) solution. The precipitates were subsequently dissolved in 2 ml of 6 M guanidine hydrochloride buffered with 20 mM sodium phosphate at a pH of 6.5. Using an absorption coefficient of 21.0 mM− 1 cm− 1 and the absorbance at 370 nm of the sample obtained from the DNPH-treated precipitate, the carbonyl content was calculated and expressed in nanomoles of carbonyl per milligram of protein.
On every sampling day (1 and 5), 1 g of meat samples from pectoralis major muscle were aseptically weighed, transferred to a plastic centrifuge tube containing 9 ml of deionised water, and homogenised for 120 s at room temperature. For microbial enumeration, 100 µl samples of 10-fold dilution in deionised water were spread on the surface of dry media. Tenfold dilution was spread on Petri dishes in duplicate for enumerations of total aerobic count (TAC) on Plate Count Agar (NEOGEN-NCM0033A, UK). The plates were incubated at 32 ºC for 24 h. After the incubation period, all bacteria colonies (cfu/g) on TCB plates were counted and then converted to log10 cfu/g prior to statistical analysis.
The experimental design was a completely randomized design (CRD). The General Linear Models (GLM) technique of the Statistical Analysis System (SAS) software programme, version 9.1, was utilised for statistical analysis. The results were analysed using the ANOVA procedure, with the restraining method (shackle and cone) and storage (day 1 and day 5) as the main effects and their interaction (restraining method storage) as the interaction. Duncan's multiple range test was employed to compare means when significant effects were identified. The statistical significance level was (p < 0.05).
The fundamental objective of the meat processing industry is to increase blood loss during slaughter, as better blood loss can improve meat quality during storage. Blood loss in broiler chicks subjected to various pre-slaughter restraint procedures is presented in Fig. 1. The restraint method significantly (p < 0.05) affected blood loss as restraining the birds using the cone restraint resulted in higher blood loss than restraining the birds using a shackle restraint. The average blood loss during shackle restrain was 3.025%, while the loss following cone restrain was 3.682%. The consumption of blood is forbidden. Therefore, religious and modern slaughter requires that animals slaughtered for food be properly bled before consumption (Nakyinsige et al., 2014).
Numerous research on restraining measures on blood loss has produced contradictory findings. Our findings are congruent with Ismail et al. (2019), who discovered that the pre-slaughter restraint method substantially affects broiler chicken blood loss. According to the author (Ismail et al., 2019), birds with cone restraints lost more blood (3.26 percent) than birds with shackles (2.97 percent ). In contrast, Lambooij et al. (1999) examined the effect of two post-slaughter restraining devices, the shackle, and the cone, on bleeding efficiency in broiler chickens. Blood loss relative to body weight was significantly higher in shackled birds than cone birds. They utilised light broiler chicken (about 1.5 kg live body weight) with a bleed-out rate of around 2.68 and 2.46 percent after stunning while chained or restrained, followed by neck cutting. Although hanging may promote rapid bleeding by prompting birds in a shackle line to straighten up and flap their wings, it is established that wing-flapping occurs only for a few seconds immediately following shackling. Despite this, many birds continue to flap their wings if they are unexpectedly exposed to sunlight, startled, or receive electric shocks in the bath (Ismail et al., 2019).
Several problems with carcass quality can be directly attributed to pre-slaughter bird handling, including the restraining method. In Fig. 2, details of the comparison of the carcass quality scored in terms of breast hemorrhage, thigh hemorrhage, and red wing tips between different restraining methods. Cone restrain birds had a lower (p < 0.05) incidence of hemorrhage in the breast, thigh, and red wingtips than those from the shackle. No incidence of broken bones was observed in both slaughter restrain groups. Hemorrhages found in broiler carcasses could be due to struggling or wing-flapping (an escape or discomfort behavior) that occurred during shackling restraint which may result in the occurrence of blood circulation disturbances as well as capillary rupture (hemorrhage). This explanation is supported by Cockram et al. (2020). They stated that hemorrhages occur due to blood leakage from ruptured capillary vessels caused by flooding of capillaries due to vasodilation of arterioles following vasoconstriction stimulated by sudden muscular contraction accompanying shackling prior to slaughtering. In poultry, Lambooij et al. (1999) compared cone restraining and shackling methods on carcass quality of broiler chickens. The authors found that thigh muscle hemorrhaging was higher in shackled birds than those restrained in cones. Similarly, Kittelsen et al. (2015) found a significant increase in wing fractures after shackling. Ismail et al. (2019) also observed that the shackled form of pre-slaughter restraining could lead to hemorrhages in the muscles and skin covering the muscle.
The amount and rate of glycogen breakdown and lactate release in the muscle pre- and post-slaughter of fowl chicken are influenced by events that occur before slaughter handling. Table 1 presents the average pH of the pectoralis major muscle of hens subjected to various restraint methods. Although restraining methods did not affect ultimate pH (p > 0.05), the shackle group exhibited lower muscular pH readings than the cone group. The lower value of final pH in shackled birds could be due to struggle or free wing flapping during shackling, which raises lactate concentration in breast muscle, reducing muscle pH. Ismail et al. (2019) and Lambooij et al. (1999) discovered comparable muscle pH in shackle- and cone-restrained broiler chicks. Regardless of treatment, the pH levels steadily rose over time. The accumulation and proteolytic cleavage of metabolites due to bacterial action on meat may account for the increase in pH during preservation (Kim et al., 2019). Similar findings were made while chilling chicken breast meat (Kim et al., 2020).
| Parameter | Storage (day) | Pre-slaughter restraint method | P value | ||
| Shackle | Cone | RM | RM×S | ||
| pH (unit) | 1 | 5.514 ± 0.04y | 5.657 ± 0.05y | 0.8894 | 0.5495 |
| 5 | 5.808 ± 0.08x | 5.821 ± 0.02x | 0.5508 | ||
| P value | < .0001 | < .0001 | |||
| Drip loss (%) | 1 | 2.958 ± 0.15ay | 2.267 ± 0.13by | 0.0039 | 0.3713 |
| 5 | 3.713 ± 0.22x | 3.419 ± 0.36x | 0.0891 | ||
| P value | < .0001 | < .0001 | |||
| Cooking loss (%) | 1 | 29.879 ± 0.53ay | 27.46 ± 0.43bx | 0.0025 | 0.1996 |
| 5 | 26.449 ± 0.71y | 24.33 ± 0.77y | 0.0599 | ||
| P value | < .0001 | < .0001 | |||
| Shear force (kg) | 1 | 1.133 ± 0.02ax | 1.084 ± 0.01bx | 0.0492 | 0.1121 |
| 5 | 1.078 ± 0.01ay | 1.034 ± 0.01ay | 0.0055 | ||
| P value | 0.0012 | 0.0024 | |||
| a,b Means within the same row with different letters are significantly different at p < 0.05. | |||||
| x,y Means within the same column with different superscripts are significantly different at p < 0.05. | |||||
| Values are mean ± standard error. | |||||
Table 1 depicts the water holding capacity in drip and cooking losses of chickens subjected to different pre-slaughter restraint methods. On the first postmortem day, there was a significant (p < 0.05) increase in drip and cooking loss among birds subjected to shackle restraint compared to cone restraint. Increased water retention in broiler chicks may be attributable to shackling-induced lactate production in the muscles. Additionally, a reduction in meat pH results in protein denaturation and a decrease in the meat's water-holding capacity (Ismail et al., 2016; Huang et al., 2014). These results were compared with Ismail et al. (2019) and (2016), who observed that pre-slaughter shackling caused significantly greater drip and cooking loss than birds held in a cone.
After 7 days of aging, the restraining method did not influence drip and cooking loss (p > 0.05). Water holding capacity is the ability of meat to keep both inherent and added water when subjected to external forces such as cutting, heating, grinding, and pressing (WHC). It is a vital quality parameter in the chicken meat market since it affects palatability and economic attributes (Kaboshio et al., 2020). Regardless of the treatment group, water holding capacity increased (p < 0.05) over time. This conclusion may be attributable to the aging-related degradation of myofibrillar proteins and collagens, which reduces the ability of myofibrillar proteins to hold water (Soglia et al., 2018).
From a sensory perspective, tenderness is the most important meat quality characteristic determining customer acceptance of meat ( Li et al., 2021). The principal effects of restraint method and age on the shear force of broiler chicken meat (Table 1). Shear force values of pre-slaughter shackle restraints were significantly greater than those of cone restraints (1.133 kg vs. 1.133 kg at d 1 and 1.078 kg vs. 1.034 kg at d 7). This could be attributable to the higher water loss of meat from the shackle group during cooking. Ismail et al. (2019) gave a similar explanation, who attributed the higher force of meat from the shackled chickens to the coned chickens to significantly higher cooking loss.
Moreover, wing restraint treatment stretches the breast muscles and prevents contraction, resulting in longer sarcomeres, thus improving the tenderness of the meat. The values of shear force reduced significantly with aging. The reduction in shear force values could be due to rigor resolution caused by the enzymatic breakdown of collagen holding muscle fibers under refrigerated storage (Oliveira et al., 2021). It has been reported that the final meat tenderness depends on postmortem changes during aging, which affect the contractile system of the muscle or myofibrils (Shi et al., 2021; Bhat et al., 2018).
The meat's color is a useful indicator of its freshness at the time of purchase and is one of the most influential factors in determining customer acceptability (Wang et al., 2021; Hughes et al., 2020). Table 2 displays the color parameters of chicken pectoralis major muscle treated with various pre-slaughter restraint techniques. Restraining methods affected meat lightness, redness, and yellowness.
| Parameter | Storage (day) | Pre-slaughter restraint method | P value | ||
| Shackle | Cone | RM | RM×S | ||
| Lightness (L*) | 1 | 62.55 ± 0.74ay | 60.09 ± 1.26by | 0.0117 | 0.5283 |
| 5 | 63.71 ± 1.06x | 60.97 ± 0.91x | 0.1662 | ||
| P value | 0.0039 | 0. 0443 | |||
| Redness (a*) | 1 | 6.102 ± 0.15ax | 5.51 ± 0.18bx | 0.0261 | 0.8915 |
| 5 | 5.855 ± 0.20y | 5.47 ± 5.47y | 0.2295 | ||
| P value | 0.0211 | 0. 0224 | |||
| Yellowness (b*) | 1 | 8.698 ± 0.28ax | 7.074 ± 0.22bx | 0.0003 | 0. 7431 |
| 5 | 8.151 ± 0.24y | 7.821 ± 0.29y | 0.0882 | ||
| P value | 0.0307 | 0.0111 | |||
| Chroma (C*) | 1 | 10.36 ± 0.12x | 9.910 ± 0.28x | 0.0670 | 0.6586 |
| 5 | 9.986 ± 0.35y | 9.088 ± 0.20y | 0.3943 | ||
| 0.0022 | 0.0273 | ||||
| Hue (h*) | 1 | 48.83 ± 0.62y | 47.68 ± 0.80y | 0.2698 | 0.9661 |
| 5 | 49.94 ± 1.08x | 48.89 ± 1.05x | 0.4990 | ||
| P value | < .0001 | < .0001 | |||
| a,b Means within the same row with different letters are significantly different at p < 0.05. | |||||
| x,y Means within the same column with different superscripts are significantly different at p < 0.05. | |||||
| Values are mean ± standard error. | |||||
At 1 day postmortem, meat from birds confined using the shackle method had significantly (p < 0.05) higher lightness (L*) and lower redness (a*) and yellowness (b*) values than meat from birds confined using the cone method. This can be attributable to differences in pH values across various restraint methods. Salwani et al. (2016) found that the color of breast meat is frequently related to postmortem muscle pH change. According to the author (Salwani et al., 2016), breast muscles with a lower pH appear less red than those with a higher pH in broiler chickens. Saláková et al. (2009) observed a significant positive correlation between redness and yellowness. Mir et al. (2017) posited that the redness and yellowness of meat are linked, where meat with a higher a* tends to have higher levels of b*.
The restraint method did not affect (p > 0.05) on the parameter of color, which consists of lightness, redness, and yellowness on 5 d postmortem. In agreement with the present findings, Ismail et al. (2016) also found a significant effect of the restraint method (shackle and cone) on the color coordinates (L*, a*, and b*) of broiler chickens. Although C* (Chroma) and h* (hue) values up to day 5 did not differ significantly between restraint methods, meat from birds restrained in the cone had lower chroma and hue color tone than meat from the birds restrained using the shackle method. Irrespective of the restraint method, lightness values increased (p < 0.05), while the redness and yellowness values decreased (P < 0.05) with increasing postmortem aging. A vital decrease in color characteristics could be due to myoglobin oxidation during aging, as it is the major hem protein responsible for the color of meat (AMSA, 2012).
Lipid oxidation is the major non-microbial cause of spoilage in meat and meat products, primarily under pro-oxidative conditions such as storage (Sabow and Majeed, 2019). Malondialdehyde (MDA) is one of the essential aldehydes produced during the secondary lipid oxidation of polyunsaturated fatty acids. It is considered the major marker of lipid oxidation. Thus, the thiobarbituric acid reactive substances (TBARS) test for malondialdehyde determination is the most commonly used method for assessing lipid oxidation in muscle because of its sensitivity and relatively simple procedure (Domínguez et al., 2019).
Table 3 shows the TBARS value of muscle from broiler chickens subjected to different pre-slaughter restraining methods. At 1 and 5 d postmortems, pre-slaughter shackle restraint resulted in significantly higher lipid oxidation value than cone restrain. These values were consistent with the results for residual blood, which increases the concentration of home proteins (mainly hemoglobin) in meat. Hemoglobin is an influential promoter of lipid oxidation (Sabow et al., 2016).
| Parameter | Storage (day) | Pre-slaughter restraint method | P value | ||
| Shackle | Cone | RM | RM×S | ||
| Lipid oxidation (mg MDA/kg meat) | 1 | 0.669 ± 0.01ay | 0.424 ± 0.01by | 0.0214 | 0.330 |
| 5 | 2.475 ± 0.01ax | 1.931 ± 0.01bx | < .0001 | ||
| P value | < .0001 | < .0001 | |||
| Free thiol (nmole/mg protein) | 1 | 48.24 ± 0.19x | 49.74 ± 0.29x | 0.198 | 0.302 |
| 5 | 41.37 ± 0.38by | 46.01 ± 0.73ay | < .0001 | ||
| P value | < .0001 | < .0001 | |||
| Carbonyl ( nmole/mg protein) | 1 | 2.320 ± 0.01y | 2.210 ± 0.007y | 0.622 | 0.347 |
| 5 | 2.976 ± 0.01ax | 2.523 ± 0.015bx | 0.011 | ||
| P value | < .0001 | < .0001 | |||
| a,b Means within the same row with different letters are significantly different at p < 0.05. | |||||
| x,y Means within the same column with different superscripts are significantly different at p < 0.05. | |||||
| Values are mean ± standard error. | |||||
Additionally, pre-slaughter conditions may cause stress and physical damage, greatly influencing lipid oxidation in meat and meat products during the early post-slaughter period (Bostami et al., 2021). In general, lipid oxidation increased significantly over storage in both groups. However, no group (shackle or cone) had an MDA value that reached detectable concentration for humans, as established by Abdulla et al. (2016). Lipid oxidation changes lead to off-odors, off-taste, discoloration, protein degradation, toxic compound accumulation, and shelf life decline, affecting consumers' health (Sabow et al., 2016).
The oxidation of protein is one of the most novel challenges in meat quality evaluation throughout processing and storage because muscle tissue has a large concentration of proteins, which have an important role in meat quality in terms of sensory, nutritional, and physicochemical aspects (Falowo et al., 2014). The principal oxidative changes of protein occur on the amino acid side chains and include the production of carbonyl groups, thiol oxidation, and aromatic hydroxylation (Morzel et al., 2006).
As a result, the quantity of thiols and carbonyl groups in meat is commonly used to assess protein oxidation (Guyon et al., 2016). Table 3 shows the results of the influence of pre-slaughter restraint measures on protein oxidation in terms of thiol and carbonyl levels in chicken. The pre-slaughter restricting approach did not influence protein oxidation values in broiler chicken flesh on day 1 postmortem.
Nonetheless, at 5 d postmortem, meat from birds restrained using the shackle method was significantly (p < 0.05) higher in the thiol and carbonyl content than meat from birds restrained using the cone. This observation coincides with the results obtained for lipid oxidation which showed that birds restrained using a shackle had greater TBARS value than those restrained with a cone. According to Falowo et al. (2014), protein oxidation occurs due to the interaction between proteins, especially the nitrogen or sulfur centers of reactive amino acid residues of protein and lipid hydro-peroxide or secondary lipid oxidation products such as aldehydes.
It was also observed that the onset of lipid oxidation in meat and meat products seems to occur faster than the oxidative degradation of myofibrillar proteins. Thus it is more likely that lipid-derived radicals and hydro-peroxides promote protein oxidation (Thanatsang et al., 2020; Domínguez et al., 2019). The free thiol content decreased, and carbonyl content increased (p < 0.05) over storage regardless of the pre-slaughter restraining method. These observations are consistent with those of Ferreira et al. (2018) and Smet et al. (2008), who showed that free thiol and carbonyl groups significantly decreased and increased as meat ages.
Figure 3 shows the effect of the pre-slaughter restraining method on microbial levels of breast meat from broilers during the first five days postmortem. At d 1, microbial counts were not significantly different for the two pre-slaughter restraining methods. However, at d 5 postmortem, more significant growth of total bacterial counts was indicated by meat samples obtained from the birds restrained using the shackle method than the meat samples obtained from birds restrained in the cone (p < 0.05). The higher bacterial growth exhibited by broiler chickens subjected to shackle restraint can be attributable to the low blood loss.
According to Bourbab and Idaomar (2012), residual blood in the carcass after bleeding is one of the most significant factors affecting contamination. It is a perfect medium for microorganisms' growth due to its high nutritive value. Some authors have also demonstrated that meat from broiler chickens that have suffered stress during the process of slaughtering is likely to have a shorter shelf-life due to spoilage since the glycogen levels in the muscles are not enough to develop the maximum level of lactic acid and an ideal pH (Iannetti et al., 2020; Petracci et al., 2010).
In general, increased growth of microorganisms with storage time was detected in meat samples from both pre-slaughter restraining groups and meat samples from the shackled chickens with the highest counts of total bacteria. However, the levels of total bacteria counts in both pre-slaughter restraining groups were within the acceptable limits. Rouger et al. (2017) reported that spoilage of poultry meat occurs when total bacterial counts reach 6–7 log cfu/g.
The present study results indicate that, except for ultimate pH, the pre-slaughter restraint method significantly affected meat quality characteristics such as color, water holding capacity, and tenderness. Due to the limited bleed-out, the shackle method had a detrimental effect on lipid-protein oxidation and overall bacterial counts in broiler chicken meat. There was a higher incidence of breast and thigh hemorrhage in the shackle group than in the cone group. Consequently, the cone can be substituted for conventional restraint methods to produce broiler chicken meat with increased meat quality and shelf life following postmortem aging.
FUNDING
The authors received no financial support for the research and publication of this article.
CONFLICT OF INTEREST
The authors declare that they have no conflicts of interest.
AUTHORS' CONTRIBUTION
ABS contributed to the original concept and design of the study. ABS and FAN conducted the tests and compiled the results. ABS and FAN assessed the carcass and the quality of the meat. ABS was responsible for statistical analysis. Both writers contributed equally to the writing of the final manuscript.