Exposure to Carbonic Gas Enriched Atmosphere or Electrical Water Bath to Stun or Kill Chickens

The objective of this study was to compare the effects of two methods (electrical water bath or carbonic gas atmosphere) for stunning or killing broiler chickens prior to bleeding on weight loss due to bleeding and meat traits. A completely randomized design with 2 x 2 factorial arrangement (electrical or gas system x stunning or killing) was applied. The time required for stunning and killing and the birds’ behavior were evaluated for the gas exposure method. The birds killed by the electrical method retained more blood than those killed by the exposure to gas and, therefore, presented redder meat. The exposure to 10 to 15% CO2 atmosphere caused discomfort reactions in 100% of birds, and the intensity of reaction presented a wide variation: 35% for weak reaction, 40% for intermediate reaction, and 25% for strong reaction. The time required to stun or kill the birds by CO2 exposure varied from 28 to 97 seconds and from 42 to 158 seconds, respectively. It was concluded that the time required to stun or to kill the birds by CO2 exposure widely varied and caused discomfort reactions; however, meat traits were not influenced by the methods used in this experiment.


INTRODUCTION
Reducing the suffering of animals at slaughter is becoming an important quality attribute for an increasing share of the meat consumer market.Brazilian legislation requires the stunning of animals before bleeding (Brasil, 1998;Brasil, 2000).
In Brazil, as well as in other countries where broiler slaughter is automated, birds are stunned in electrical water bath systems.In this method, the steps prior to electric stunning cause clear discomfort to the birds.During hanging, for instance, there must be maximum contact of the birds' legs with the metal shackle to allow effective application of the electrical current, which means that, depending on broiler size, the feet must be forced into the hooks.
Moreover, the upside down position can cause birds to flap their wings due to fear and discomfort, resulting in distress, suffering, and injuries (Ludtke et al., 2010).Other disadvantages are the difficulty to adjust the equipment to the size of the birds, the occurrence of preshocks, inadequate bird positioning, and ineffective stunning.
Some research studies suggest the exposure to controlled atmosphere to replace the electrical systems, either to stun or kill broiler chickens (Raj & Gregory, 1990;Kang & Sams, 1999;Gerritzen et. al., 2000).According to those studies, the exposure of birds to air containing high levels of carbon dioxide caused anesthesia and showed good results.However, carbon dioxide is acidic and irritates mucous membranes during inhalation, causing birds to immediately

MATERIAL AND METHODS
Forty 42-day-old male Cobb 500 broilers were divided into four groups of 10 birds each.Birds were individually identified, weighed and transported to the experimental pilot scale abattoir.Each group was submitted to one of the following treatments: 1) electrical stunning (electronarcosis); 2) electrical killing (electrocution); 3) CO 2 stunning, or 4) CO 2 killing .
The experiment followed a completely randomized design in a 2 x 2 factorial arrangement (electrical or gas system x stunning or killing) with 10 replicates per treatment.Parameters were evaluated per individual bird, and therefore, each bird was considered as an experimental unit for the analysis of variance (Perret, 2012).The slaughter procedures were performed according to the Brazilian legislation (Brasil, 1998).

Electrical method
A pilot scale electrical equipment, consisting of overhead rails with metal shackles to hang the birds and an electrified water bath, with capacity to apply electric current to one bird at a time, was used.The electrical current parameters were fixed at 240 V AC, 60 Hz, and 120 mA.Therefore, the only parameter that was changed in order to cause electronarcosis or electrocution was the exposure time of the bird to electric current.The electrical stunning was performed by immersing the bird's head for 3 seconds in electrified water to produce effective unconsciousness.Birds were considered stunned when both nictitating membrane reflex and rhythmic breathing were absent.Rhythmic breathing was indicated by contraction of the abdominal muscles near the cloaca (Ludtke et al., 2010).
In order to kill the broilers by the electrical method, the bird's head was immersed in electrified water for 8 to 10 seconds, until death, as confirmed by the observation of the complete respiratory arrest.

CO2 exposure method
The birds were exposed to CO 2 enriched atmosphere in a pilot scale stainless steel experimental chamber with acrylic ceiling, in order to allow the observation of birds' behavior, attached to a CO 2 gas cylinder.CO 2 concentration was monitored by a Scenty GDZ 203 HTK Hamburg GMBH gas analysis system, equipped with a 760 GMF 0 to 100% CO 2 sensor inside the chamber.The Brazilian legislation determines a minimum CO 2 concentration of 30% for the stunning of chickens (Brasil, 2000).However, according to Bitencourt (2011), birds suddenly submitted to this CO 2 level may show agitation and/or convulsive reactions.Therefore, this experiment used an initial 10% CO 2 concentration, which was gradually increased until the bird was completely stunned, at approximately 13% CO 2 .Birds were individually placed inside the chamber.Prior to each new stunning procedure, the gas concentration was set back to 10%.The broilers were considered stunned when they fell and did not present any rhythmic breathing movement or nictitating membrane reflex.
The same procedures were carried out to kill the birds.In this case, however, the bird was maintained in the chamber until it died, as confirmed by complete respiratory arrest, which happened at a CO 2 concentration of approximately 15%.

Behavior Evaluation of the Birds Exposed to CO 2
During the exposure to the CO 2 atmosphere, the behavior of the birds before they fell was classified as "no reaction", when the bird presented only tachypnea; "weak reaction", when the bird gasped and/or shook its head, "intermediate reaction", when the bird presented weak and intermittent wing flapping; and "strong reaction", when the bird presented continuous and strong wing flapping.

Weight Loss after Bleeding
All birds were individually weighed, bled immediately after stunning or killing by either electrical or gas treatment, and then weighed again to determine weight loss due to bleeding, expressed as a percentage of live weight.

Meat pH
Breast and thigh meat pH was determined 24 hours after slaughter using a pHmeter (Mettler Toledo Model 1120-X) with a penetrating probe.

Meat Color
Breast and thigh meat color was evaluated after the skin was removed and in triplicate, using Hunterlab Miniscan XE Plus with D65 illuminant and 10º observation angle.Color was expressed according to the CIELab color system, which attributes L* (lightness), ranging from zero (absolute black) to 100 (absolute white); a*, ranging from green (negative values) to red (positive values ); and b*, ranging from blue (negative values) to yellow (positive value) (Minolta, 2007).
The equipment was positioned on the dorsal surface of the breast fillet and on the lateral surface of the thighs, avoiding hemorrhagic petechiae, discolored spots, and any other color interference.

Statistical Analysis
The results were submitted to analysis of variance and means were compared by Duncan's test at 5%significance level.Pearson's linear correlation coefficient among variables was determined (Zar, 2009).Statistical analyses were performed using the Statistical Analysis System (SAS, 2008).

RESULTS AND DISCUSSION
Broilers submitted to gas method presented lower and higher meat redness and lightness values, respectively, compared with those of broilers submitted to the electrical method (p <0.05), probably due to the smaller amount of residual blood in the muscles, as shown by the significantly lower weight loss due to bleeding of the birds submitted to the electrical method (Table 1).
The birds killed by the electrical method presented higher thigh meat pH compared with those in the other treatments (Table 2).This may be explained by the longer time of exposure of the birds to electricity, causing muscle contraction and depletion of muscle energy reserves (Judge, 1989).As muscle glycogen stores are depleted, less lactic acid is produced, and consequently lower muscle pH decrease after slaughter.
Weight loss due to bleeding was not different between broilers stunned and killed by either of the methods applied (electrical and gas).However, when both methods are compared, electrically killed broilers lost significantly less weight after bleeding than those killed by gas exposure.This may be explained by the application of 120 mA electrical current, which can cause the rupture of intramuscular blood vessels and blood retention in muscle tissue (Gregory & Austin, 1992;Lambooij et. al., 1999).The influence of the amount of blood drained from the carcasses on meat redness is confirmed by Pearson's correlation results, shown in Tables 3 and  4.There was a negative correlation between these parameters, indicating that the greater the weight loss caused due to bleeding, the lower was the a value.A higher and more significant correlation was found for thigh meat, which redness is more intense due to a higher concentration of myoglobin and to a more developed vascular system compared with breast muscles (Varnam & Sutherland, 1995).A significant correlation between breast and thigh meat pH and b values was also observed.However, considering that the breast meat pH values were not different between treatments, it is not possible to associate these results with the method of stunning or killing.According to Grandin (2010), there has been much controversy about the effect of different gases on bird discomfort during the induction of insensibility.This is why, in the present study, the initial CO 2 concentration used was 10%, and then was gradually increased after the birds were placed into the chamber.All birds reacted when exposed to CO 2 , both during the killing and the stunning procedures: 35% presented weak reaction, 40% intermediate reaction, and 25% strong reaction (Figure 1).The time required to stun and to kill the birds by the gas exposure method presented a wide variation, ranging from 28 to 97 seconds for stunning and from 42 to 158 seconds for killing.Under experimental conditions, birds' reactions to the gas may be different when compared with those observed in processing plants.Specific gas concentrations that can be easily maintained at laboratory scale may be difficult to maintain in commercial facilities (Grandin, 2010).Therefore, in practice, the adoption of gas stunning method presents some difficulties.

Figure 1 -
Figure 1 -Birds' reaction, in percentage, on the exposure to CO 2 .

Table 1 -
Weight loss due to bleeding and breast and thigh meat traits of broilers submitted to electrical or gas stunning or killing.

Table 2 -
Deployment of the interactions among treatments for weight loss due to bleeding, thigh meat pH, and breast meat redness.Means followed by different letters in the same column are significantly different by Duncan's test (p<0.05).

Table 4 -
Pearson's correlation coefficient for thigh meat parameters