1. Introduction
Two main types of stunning systems are used commercially in broiler chickens: electrical waterbath stunning (WBS) and controlled atmosphere stunning (CAS). WBS is by far the most frequently applied method worldwide [
1,
2]. It consists in hanging birds by their legs in metal shackles on a moving line which takes them to a waterbath, where they are immersed up to the base of the wings in electrified water, where they are stunned. The contact of the head and neck with the water completes the electric circuit between the water (positive electrode) and the shackle line, which acts as the earth or negative bar electrode, so that the electric current passes through the bird’s head and body [
3]. If sufficient current passes through the brain, its normal function is disrupted, and the animal is immediately rendered unconscious. This unconsciousness is a result of temporary or permanent damage to normal brain function, and the individual is unable to perceive and respond to external stimuli, including pain [
4].
Although WBS is intended to induce unconsciousness until death occurs due to bleeding, it is not always effective [
5]. For this reason, in order to protect the welfare of birds, it is mandatory in the European Union that the state of consciousness is monitored at the exit of the waterbath (WB) stunner and that the animals do not regain consciousness before death. Thus, ineffectively stunned birds can be re-stunned using back-up methods to avoid causing them unnecessary pain, stress and suffering.
One of the main challenges in monitoring the state of consciousness in broiler chickens after WBS is the selection of animal-based indicators (ABIs) ensuring the consistency of assessments. Recording electroencephalogram (EEG) is the most objective available method to ascertain the induction and maintenance of unconsciousness following stunning in broiler chickens [
6]. In EEG analysis, the occurrence of a profoundly suppressed isoelectric EEG as well as epileptiform activity is normally associated with loss of consciousness [
7,
8,
9]. However, at present, EEG can only be used under laboratory conditions. For this reason, under commercial conditions, the alternative is to combine the two main categories of indicators: resource-based indicators (RBIs) and animal-based indicators (ABIs). Traditionally, welfare assessments have focused on RBI assessments, such as on the key electrical parameters used and the minimum time that the birds spent submerged in the waterbath; these have been linked to the efficiency of stunning and set down in legislation. It was considered that if the resources were appropriate, animal welfare was guaranteed. However, RBIs may not reflect high welfare standards since, although the key electrical parameters established by the legislation are used, not all birds are successfully rendered unconscious and some may be ineffectively stunned or recover consciousness before death. Thus, animal welfare assessments based on an integrated approach in which both RBI and ABI are taken into consideration are necessary [
10,
11]. ABIs can more directly reflect the state of consciousness. Indeed, the results of the RBI indicate the risk of ineffective stunning but not the actual state of consciousness of each animal [
12].
Hence, ABIs should meet three requirements, namely validity, feasibility and repeatability, in order to be relevant for assessing the state of consciousness. Validity tells us the extent to which an indicator is meaningful in terms of providing information about the presence of conscious birds, whereas feasibility refers to the applicability to different WBS equipment, slaughterhouse (SH) designs and different line speeds. Repeatability tells us the extent to which results are largely the same when the same observer repeats assessments, or the agreement between two or more observers after they have received reasonable training. If inter-observer repeatability is poor, then the indicator is probably inappropriate for welfare assessments.
The validity of the ABIs for the assessment of the state of consciousness in poultry was correlated with EEG outputs [
7,
13,
14]. However, it was not until 2013 that EFSA [
15] carried out the first step toward refining the perceived validity (i.e., sensitivity) and feasibility considered by experts in this field. In addition, they pointed out that birds should be monitored at two different stages of the slaughter line: before and during bleeding. Hence, a shorter list of ABIs was recommended according to the stage (with the highest validity and feasibility), with other parameters being seen as optional. However, inter-observer repeatability of any of these ABIs has not been assessed yet.
In the present study, the main goal is to evaluate the inter-observer repeatability of the four most valid and feasible ABIs according to EFSA [
15], both before (i.e., tonic seizure, breathing, spontaneous blinking and vocalization) and during bleeding (i.e., wing flapping, breathing, spontaneous swallowing and head shaking) in different commercial slaughterhouses and with different batches of broilers. In addition, the prevalence of the outcomes of the ABIs are calculated to determine which are more prone to occur in cases of ineffective stunning and to identify correlations among them in terms of their reliability and relationship with the level of consciousness. From those observations, a reduced list of the most relevant ABIs is proposed for use in assessments of the consciousness of broiler chickens in commercial slaughterhouses, ensuring consistency between observers.
4. Discussion
One of the aims of this study was to gain insights into the inter-observer repeatability of valid and feasible ABIs for the state of consciousness after WBS in broiler chickens. In addition, the prevalence of the outcomes of consciousness of the ABIs and correlations among them were computed in order to give insights into their reliability and relationship with the state of consciousness. This study compared the assessments of three observers of 5241 broilers from 19 batches in six different slaughterhouses and 11 different key electrical parameters applied in waterbaths from two main broiler producer countries in the EU. Even though the SHs were not randomly sampled, they represented a diverse range of equipment designs, key electrical parameter combinations and line speeds. In addition, it should be highlighted that not were the only observers well trained, but they also agreed on the definition of the indicators before assessing the birds. The number and position of the observers was intended to result in minimal interference to the operators. Although there was a restriction in terms of the available space for assessments, the observers stood next to each other, assessing the same animals at the same time.
4.1. Inter-Observer Repeatability of Animal-Based Indicators
Data were analyzed at individual broiler level and the combination of κ and PoA was used to assess the inter-observer repeatability of the outcomes of some ABIs regarding the state of consciousness. This repeatability among observers could be interpreted as poor to excellent, based on to the calculated κ value [
18]. Our results showed that for most of the indicators, the κ interpretation varied according to the SH assessed. This was mainly because κ values are strongly influenced by prevalence, and this differed among SHs; the lower the prevalence, the higher the agreement among observers (as all observers agreed on the absence of ABIs) and the poorer the interpretation of κ. The higher the prevalence, the higher the likelihood of disagreement. The only exceptions to this were in the assessment of spontaneous swallowing and spontaneous blinking, where κ was interpreted as showing poor agreement among observers in all cases. In contrast, regarding vocalizations, the κ could not be computed due to a lack of outcomes of consciousness of this indicator. These results suggest that when prevalence is low, the calculation of κ does not give much information. A similar outcome occurs when paying attention to the PoA. A high PoA may suggest that there is a high level of agreement among observers. However, it may happen that the agreement is high because the outcome of consciousness of the indicator is very clearly detectable for all when present (e.g., head shaking), or because the outcome of consciousness is rarely (e.g., spontaneous blinking and swallowing) or hardly ever observed (e.g., vocalizations). It should be noted that the level of noise in the SHs did not impair the assessments of vocalizations. This was so because vocalizations are high in pitch and were clearly detectable before the broilers entered the waterbath and were still conscious. However, vocalizations after waterbath stunning were only detected in one out of 2608 broilers, and in this instance, all the three observers were in agreement.
Inter-observer repeatability in broilers is high for some ABIs regarding state of consciousness after WBS. The most repeatable indicator before bleeding is vocalization and spontaneous blinking, followed by tonic seizure and breathing. However, spontaneous blinking and vocalization were artificially highly repeatable because they were seldom observed. When considering these results, we recommend keeping tonic seizure and breathing at this stage, despite their being less repeatable among observers.
The most repeatable indicators during bleeding were found to be wing flapping, head shaking and spontaneous swallowing followed by breathing. Nevertheless, spontaneous swallowing and wing flapping were artificially highly repeatable because they were observed on few occasions. While the span of observation during bleeding was set from 10 s to 16 s post-neck cutting, some birds started to flap their wings at the end of this time. This generated doubts in terms of scoring and affected the consensus among the observers when wing flapping was present. This reflects the importance of setting an optimal observation duration where more accurate outcomes of consciousness may be observed within a slaughterhouse during bleeding. Additionally, but at lower scale, sometimes there were difficulties in differentiating wing flapping from movements of the wings caused by line shaking. Despite this, we recommend keeping breathing, wing flapping and head shaking as key ABIs during bleeding based on their high prevalence and repeatability.
Repeatability among the three observers could be influenced by impaired visibility of the animals because of the slaughterhouse design or because, when paying attention to a specific ABI, the evaluator is more prone to miss a positive outcome of another. However, it is likely that higher levels of inter-observer reliability could be achieved by standardizing descriptions and through training and wider testing. Hence, adequate training appears to be one of the key points to improve in animal welfare assessments of SHs. This can be achieved through theoretical lectures and video clips designed to train assessors, as well as practical field exercises in SHs until trainees harmonize their scoring with those done by experts [
20].
4.2. Correlation among Animal-Based Indicators
Based on our observations, pre-stun shocks or runt (small) animals could have been the cause of non-stunned birds at the exit of the waterbath due to a lack of contact of the head with the electrified water. This explains the presence of broilers that remained conscious and showed combinations of indicators of consciousness before bleeding. On the other hand, some birds did not exhibit tonic seizure, and this indicator was not correlated to other outcomes of consciousness before bleeding. This may have been because the tonic seizure occurred while the bird was submerged in the waterbath, as might happen in long baths or with slow line speeds. On the other hand, it is known that when the electrical parameters are set to stun-to-kill the birds, the induction of cardiac arrest leads to reduced or an absence of tonic seizure at the exit of the waterbath [
14], and does not mean that birds are conscious. In this sense, tonic seizure might not be as reliable as the other indicators of consciousness, since it depends on SH configuration and the current delivered.
Data on the order of the re-appearance of indicators during recovery in poultry are not described in literature. Despite the importance of these indicators, in the context of a slaughterhouse, their precise relationships with the brain state or with other indicators of consciousness are insufficiently known. Study of the relationships between different ABIs of state of consciousness may benefit from analyses by correlation [
21]. In the present study, 14% of birds with at least one outcome of consciousness during bleeding showed simultaneously more than one outcome of consciousness. The most observed indicator of consciousness during bleeding was the presence of breathing, indicating a return of consciousness. It seems that when a broiler starts breathing, it is more prone to show movements such as head shaking and/or wing flapping later in the line.
Taking all this into consideration, assessing if the broilers are breathing is the most recommended ABI among those assessed in the present study after WBS and before bleeding. However, wing flapping, although not included, should be also considered before bleeding, as some birds were suspected of missing the waterbath (and therefore were not stunned at all) flapped their wings. During bleeding, breathing is the most observed indicator of consciousness; when observed, it was sometimes accompanied by wing flapping and/or head shaking.
5. Conclusions and Recommendations
Before bleeding, breathing and wing flapping are the most relevant and commonly observed indicators of consciousness, whereas spontaneous blinking and vocalizations may be considered secondary parameters. Any broilers that showed at least one of these ABIs should be re-stunned with backup stunning equipment before performing the neck cut, while the shackling of runts should be avoided, since they are more prone to skipping the waterbath.
During bleeding, breathing, wing flapping and head shaking are the most relevant and commonly observed ABIs of consciousness. Like before bleeding, any broiler that shows at least one outcome of consciousness at this stage should be re-stunned with backup stunning equipment as soon as it is detected. If some broilers of the same batch regain consciousness, the electrical parameters should be readjusted to ensure that all birds remain unconscious until death.
The repeatability in terms of evaluating the consciousness of broilers is likely to be improved by standardizing descriptions, robust training and wider testing in slaughterhouses.
This work will proposed a refined and validated list of indicators for use in assessments of the consciousness of broiler chickens in commercial slaughterhouses.