Table C.1.
Assessment of the reporting quality against the EFSA guidance criteria
| Paper/study | Information as provided in the dossier and related comments |
|---|---|
| Mackie et al. (2016) (Paper 5) |
Introduction The primary objective of the study was to carry out a detailed behavioural analysis of broiler chickens undergoing LAPS, both in groups and individually, with a focus on behaviour occurring during induction to unconsciousness. The secondary objectives were to investigate the effects of bird weight, and whether slightly adjusted decompression settings (automatically applied in relation to ambient temperature) had any effect on behavioural responses. Materials and methods Commercial broilers, Ross 708, aged 49–50 days. Weighed 3.4 ± 0.5 kg (range 2.6–4.3 kg). The birds were reared in a commercial flock. Before undergoing LAPS, the birds were feed restricted for 8 h and water restricted for 2 h to mimic commercial practice. Missing data resulted from birds being out of view during behavioural observations – see results for details. Experimental units were 50 individual birds and 50 focal birds in groups of 20. Each individual/group was exposed to LAPS in a different run, giving true replication. Sample sizes were determined using power calculations (Cohen J, 1992. A Power Primer. Psychological Bulletin, 112, 155–159) based on differences in behaviour durations and variances reported using similar measurement methodologies in related studies on controlled atmosphere stunning and emergency killing using anoxic gas mixtures (e.g. Abeyesinghe et al., 2007. Brit. Poult. Sci., 48, 406–423; Coenen et al., 2009. Poult. Sci., 88, 10–19; McKeegan et al., 2007a. Brit. Poult. Sci, 48, 430–442; McKeegan et al., 2007b. Anim. Welf. 16, 409–426). The birds all underwent an identical intervention, except that three temperature settings were applied in relation to ambient temperature (see methods). Outcomes recorded in this study were behaviour (postures and movements and general behaviour). These are described in detail in Table 1 of the paper. As appropriate, behavioural latencies, durations and counts were recorded. Birds were randomly selected from a commercial flock and treated in similar manner during transportation, care and husbandry and during handling prior to exposure to the intervention. Birds were randomly allocated to numbered groups and randomly allocated to individual or group killing. Application of temperature setting treatments was sequential and unbalanced, this was unavoidable based on ambient temperature change throughout the two trial days. A single observer conducted behavioural measurements using a specialised behavioural recording program (Noldus Observer). No blinding took place. Variables were created relating to the latencies, durations, bout numbers and bout durations (where appropriate) of the behaviours. Following testing for normality with the Anderson Darling test, using the nortest R package version 1.0‐2 (Grossand Ligges, 2012), and checking normality with a histogram of the data, a one‐way analysis of variance (ANOVA) or Kruskal–Wallis tests were carried out with temperature setting applied as a factor. In individuals, correlations between behavioural parameters and body weight were computed using Pearson's correlation and Spearman's rank correlation, using the pspearson test R package version 0.3‐0 (Savicky, 2014). Where temperature setting did not have an effect, data was pooled for further analysis, but if temperature setting was significant then weight correlations were carried out within each temperature setting. To compare results between individuals and groups, Mann–Whitney U tests and independent two sample t‐tests were used where appropriate. When comparing individuals and groups, if temperature setting had a significant effect, analysis was carried out within temperature setting.
Results Means (and standard deviation) of continuous variables are presented in the results. Numbers are provided in results tables. Results are stated in absolute numbers when feasible. We report the results for each behaviour outcome for individuals and groups, in both table and graphical form. These include means, standard deviations and ranges. No adverse effects were observed. Discussion The discussion summarises the key results with reference to the study objectives and provides an interpretation of results considering objectives and limitations and previous studies. The sequence of behaviours seen at the different temperature settings was similar to those seen previously during LAPS (Thaxton et al., 2010) and also to those seen in experimental and field studies of controlled atmosphere stunning using anoxic gas mixtures. There were variations in the latencies and duration of behaviours with LAPS; in general, latencies were longer than those seen on some gas anoxic systems. The results are relevant to the LAPS procedure as applied to slaughter weight broiler chickens at these temperature settings. Although this study provided more detailed information using precise definitions of behavioural activities, the sequence of behaviours seen at the different temperatures was broadly similar to those seen previously during LAPS (Thaxton et al., 2010). The results also suggest that LAPS produces a sequence of behaviours which are equivalent to those seen with CAS. Other |
| Martin et al. (2016a) (Paper 6) |
Introduction There are concerns that birds undergoing LAPS could experience discomfort or pain. Here, it is investigated whether subjecting birds to LAPS with and without administration of an opioid analgesic (butorphanol) affected behavioural responses, with the rationale that abolition of suspected pain related behaviour with analgesic is circumstantial evidence of pain. The primary objective of this study was to investigate whether subjecting birds to LAPS with and without administration of an opioid analgesic would affect their behavioural responses, especially those that have been previously thought to relate to pain and discomfort. Materials and methods Commercial broilers, Cobb 500, aged 36–37 days (mean bodyweight 2.30 ± 0.12 kg). The birds were reared in a research facility at the University of Arkansas. Before undergoing LAPS, the birds were feed and water restricted for 2–6 h before LAPS, dependent on the pair kill order. Missing data resulted from birds being out of view during behavioural observations. Experimental units were 45 pairs of birds arranged in three types of blocks in relation to treatment (analgesic/analgesic, analgesic/sham, or sham/sham). Each individual/group was exposed to LAPS in a different run, giving true replication. There were 15 replications of each block (AA, AS, and SS), each containing a pair of birds. The birds underwent LAPS in 45 consecutive pairs over 2 days (day 1 = 23 pairs; day 2 = 22 pairs). Sample sizes were determined using power calculations (Cohen J, 1992. A Power Primer. Psychological Bulletin, 112, 155–159, Snedecor and Cochran Statistical Methods 1967 Iowa State University) based on differences in the range of behaviour durations and variances reported using similar measurement methodologies in related studies on controlled atmosphere stunning and emergency killing using anoxic gas mixtures (e.g. Abeyesinghe et al., 2007. Brit. Poult. Sci., 48, 406–423; Coenen et al., 2009. Poult. Sci., 88, 10–19; McKeegan et al., 2007a. Brit. Poult. Sci, 48, 430–442; McKeegan et al., 2007b. Anim. Welf., 16, 409–426; McKeegan et al., 2013. Poult. Sci., 92, 1145–1154). Outcomes recorded in this study were behaviour (postures and movements and general behaviour). These are described in detail in Table 1 of the paper. As appropriate, behavioural latencies, durations and counts were recorded and related to analgesic treatment. Birds were randomly selected from a larger flock reared for a series of research trials. All birds were treated in similar manner with regard to husbandry and during handling prior to exposure to the intervention. Birds were randomly allocated by individual wing tag number into three types of blocked pairs (analgesic/analgesic (AA), analgesic/sham (AS), and sham/sham (SS)) and pair kill order following a Graeco‐Latin square design (Martin and Bateson, 2007. Measuring Behaviour: An Introductory Guide First published 2007. Printed in the United Kingdom at the University Press, Cambridge. ISBN‐978‐0‐521‐82868‐0 hardback ISBN‐978‐0‐521‐53563‐2 paperback). A single observer conducted behavioural measurements using a specialized behavioural recording program (Noldus Observer). The observer was blinded to both individual bird treatment (analgesic/sham) and pair type (analgesic/analgesic, analgesic/sham, or sham/sham). All data were summarised in Microsoft Excel (2010) spreadsheets and analysed using Genstat (14th Edition). Statistical significance was based on F statistics and p < 0.05 threshold level. Summary graphs and statistics were produced at bird level. Statistical comparisons of behavioural variables were conducted via generalised linear mixed models (GLMM) (Poisson distribution) or linear mixed models (LLM) (normal distribution) dependent on the data distributions for each variable. Data transformations were attempted when necessary via logarithm function. All models included bird ID, companion bird ID and pair block type as random effects. All fixed effects were treated as factors and all interactions between factors were included in maximal models. All models included treatment, pair order, and marked bird as fixed effects and bird weight, ambient temperature, ambient humidity as covariates. Correlations between variables and fixed effects were performed as Pearson's Correlations for parametric data, and Spearman's rank correlations for non‐transformable non parametric data. For behaviours which were not exhibited by all birds, the effect of treatment on the proportions of birds showing the behaviour was compared with chi‐square tests using two by two contingency tables.
Results Means, standard deviations and ranges of continuous variables are presented in the results. Numbers analysed are provided in results tables. Results are stated in absolute numbers when feasible. The results are reported for each behaviour outcome by analgesic treatment, in table. These include means, standard deviations and ranges. For behaviours which were not exhibited by all birds, the effect of treatment on the proportions of birds showing the behaviour was compared with chi‐square tests using two by two contingency tables.
Discussion The discussion summarises the key results with reference to the study objectives and provides an interpretation of results considering objectives and limitations and previous studies. The sequence of behaviours seen at the different temperature settings was similar to those seen previously during LAPS (Mackie et al., 2016) and also to those during controlled atmosphere stunning using anoxic gas mixtures. Administration of butorphanol had no effect on the range and patterning of behavioural responses during LAPS. Latencies to ataxia, mandibulation and deep inhalation were slightly delayed by analgesic treatment but the duration of ataxia and other behaviours related to loss of consciousness were unaffected. These effects appear to be most readily explained by potential sedative, dysphoric and physiological side effects of butorphanol. The results are relevant to the LAPS procedure as applied to slaughter weight broiler chickens at this temperature setting. Although this study provided more detailed information using precise observation techniques and detailed behavioural definitions, the patterning and range of behaviours seen was very similar to those reported previously during LAPS (Mackie and McKeegan, 2015). Other N/A |
| Martin et al. (2016b) (Paper 7) |
Introduction To date, no studies on LAPS have been carried out in which EEG and behaviour have been recorded in the same individual and the timings for loss of posture have not been consistent between studies (ranging between 40 and 80 s, McKeegan et al., 2013; Mackie and McKeegan, 2015). There is a need for brain and behavioural measures in the same bird to allow a more robust assessment of the welfare impact of the process and corroborate indicators of loss of consciousness.
Scientific background and rationale for the investigation are reported adequately. The primary objective of the paper is “to comprehensively examine responses to LAPS by recording behaviour, EEG and ECG in individual broiler chickens, and interpret these with regard to the welfare of birds undergoing the process”. However, behavioural indicators are not used to assess aversion before loss of consciousness. Materials and methods Commercial broilers, Cobb 500, aged 34–35 days. The birds were reared in a research facility at the University of Arkansas. Before undergoing LAPS, the birds were feed restricted for 2–6 h before LAPS, dependent on the triplet kill order. Missing data resulted from birds being out of view during behavioural observations – see results for details. Missing data in EEG and ECG traces resulted from movement artefacts and these were excluded from analysis. During the experiments, 31 birds underwent EEG implantation surgery – one dislodged its implant soon after surgery and was humanely euthanised. An additional bird underwent surgery to replace this loss (bird ID 217 replaced bird ID 385). Ninety birds (30 sets of three) were exposed to LAPS over 2 days (day 1 = 15 triplets; day 2 = 15 triplets. Each group was exposed to LAPS in a different run, giving true replication. The aim was to apply each temperature setting to 15 replicates, but changes in ambient temperature resulted in 16 replicates for setting 3 and 14 replicates for setting 4. Sample sizes were determined using power calculations (Cohen J, 1992. A Power Primer. Psychological Bulletin, 112, 155–159; Snedecor and Cochrane (1967) Statistical methods Iowa State University Press) based on differences in the variables reported using similar measurement methodologies in related studies on controlled atmosphere stunning and emergency killing using anoxic gas mixtures (e.g. Abeyesinghe et al., 2007. Brit. Poult. Sci., 48, 406–423; Coenen et al., 2009. Poult. Sci., 88, 10–19; McKeegan et al., 2007a. Brit. Poult. Sci, 48, 430–442; McKeegan et al., 2007b. Anim. Welf., 16, 409–426; McKeegan et al., 2013. Poult. Sci., 92, 1145–1154). According to ambient temperature, two of six possible temperature settings were applied in this study (settings 3 – applied between 13 and 18°C – and 4 – applied between 5 and 12°C). Outcomes recorded in this study were behaviour, electroencephalogram and electrocardiogram (see Annex table A7/4). For the behaviour observations, postures and movements and general behaviour were recorded, described in detail in Table 1 of the paper. The EEG was analysed in non‐overlapping 2 s epochs to produce latencies indicating unconsciousness, including F50 < 12.7 Hz (non‐responsive state) and < 6.8 Hz (general anaesthetic plane) (Martin, 2015; Sandercock et al., 2014), latency to total power equal to 10% of baseline and the onset of isoelectric EEG signal by visual interpretation and by identification of validated spectral characteristics. ECG signal was used to determine heart rate (bpm derived from the number of QRS complexes in a 5s epoch) at six baseline time points before LAPS (three outside chamber, three inside chamber with door open) and every 5 s during the LAPS cycle. Birds were randomly selected from a larger flock reared for a series of research trials. All birds were treated in similar manner with regard to husbandry and during handling prior to exposure to the intervention. The experimental birds were randomly selected from a larger flock by a random number generator (Microsoft Excel 2010) based on wing tag number. The birds underwent LAPS in triplets where one bird was implanted and instrumented to record EEG and ECG; behavioural observations were carried out on all birds. The triplet treatment order was generated by a Graeco‐Latin square design (Martin and Bateson, 2007) to balance day, temperature treatment and source pen for EEG implanted birds. A single observer conducted behavioural measurements using a specialised behavioural recording program (Noldus Observer). The observer was blinded to temperature setting treatment. It was not possible to fully blind the observer, as the physiological recording equipment was visible on birds wearing it. All data were summarised in Microsoft Excel (2010) spread sheets and analysed using Genstat (14th Edition). Statistical significance was based on F statistics and 5% threshold level (i.e. p value < 0.05). Summary graphs and statistics were produced at bird level. Statistical comparisons of behavioural variables were conducted via generalised linear mixed models (GLMM) (Poisson distribution) or linear mixed models (LLM) (normal distribution) dependent on the data distributions for each variable. Data transformations were attempted when necessary via logarithm function. All models included bird ID and triplet number as random effects. All fixed effects were treated as factors and all interactions between factors were included in maximal models. All models included treatment, triplet order, implanted and marked bird as fixed effects and bird weight, ambient temperature, ambient humidity as covariates. Correlations between variables and fixed effects were performed as Pearson's Correlations for parametric data, and Spearman's Rank Correlations for non‐transformable non‐parametric data. Summary statistics and graphs were produced at bird level, while statistical comparisons focussed on estimated means and differences between means. GLMMs (Poisson distribution) or LLMs (normal distribution) dependent on the data distributions for latency variables to unconsciousness (F50 b 12.7 Hz (non‐responsive state); and b6.8 Hz (general anaesthetic plane); latencies to visual inspection characteristics (presence of slow‐wave and three consecutive isoelectric 2 s epochs); latencies for the signal to have a total power equal to 10% of baseline; and finally, latencies to isoelectric (Ptot b170 mv and F50 N22 Hz). All models included bird ID and triplet number as random effects. All fixed effects were treated as factors and all interactions between factors were included in maximal models. All models included treatment and triplet order as fixed effects and bird weight, ambient temperature, ambient humidity as covariates. GLMMs (Poisson distribution) or LLMs (normal distribution) were carried out, dependent on the data distributions for each heart rate interval, including the six baseline intervals and latencies to bradycardia. All models included bird ID and triplet number as random effects. All fixed effects were treated as factors and all interactions between factors were included in maximal models. All models included treatment and triplet order as fixed effects and bird weight, ambient temperature, ambient humidity as covariates. A Pearson's correlation matrix was produced to examine associations between latencies to key behavioural responses (latency to ataxia, loss of posture, loss of jaw tone and motionless), EEG (latency to slow wave based on visual inspection, latency to isoelectric EEG based on visual inspection and spectral characteristics, latency to Ptot b10% of baseline, latency to F50 b 7 Hz and F50 b 12 Hz) and ECG (latency to bradycardia) events during LAPS.
The rate of change in partial pressure of oxygen in relation to time is provided in a Figure in the dossier. It is not clear why two different temperature settings are used when the ambient temperature falls only in Tset_3. There are discrepancies between the annex and the paper about temperature and humidity as well as in terms of replicates No description of the meaning of each behaviour. E.g. L260 ‘On completion of the LAPS cycle, the birds were removed from the chamber and reflexes were immediately assessed (e.g. presence of rhythmic breathing, nictitating membrane) to confirm death’. Some definitions are difficult to replicate and seem subjective (e.g. apparently conscious). The rationale behind the behaviours assessed is lacking. Some EEG variables are not scientifically supported. The onset of isoelectric EEG signal was determined in two ways, by visual interpretation, and by identification of validated spectral characteristics (Ptot less than 170 mv and F50 > 22 Hz). Latency variables to unconsciousness were defined as time for F50 < 12.7 Hz (non‐responsive state) and < 6.8 Hz (general anaesthetic (GA) plane). However, the reference of the validated spectral characteristics of Ptot less than 170 mv and F50 > 22 Hz is not scientifically supported in any the three references provided in the paper. Furthermore, the statement that the latency variables to unconsciousness of F < 12.7 Hz (non‐responsive state) and < 6.8 Hz (GA plane) is supported mainly by the thesis of the first author, which have not scientifically validated. The main scientific support to this statement is based on the paper of Sandercock et al. (2014) that reported that ‘A conservative threshold for F50 in hens and turkeys when unconscious (equivalent to a surgical plane of anaesthesia) was estimated at 7 Hz’. In the same studies the authors reported a F50 < 14 for the semi‐conscious (sedated) state. The relationship between sedation and non‐responsive state is not described. Results Means, standard deviation and ranges of continuous variables are presented in the results. Numbers analysed are provided in results tables. Results are stated in absolute numbers when feasible. The results are reported for each outcome by temperature treatment, in tables and figures. Individual bird data is provided in the form of scatter plots for latency to ataxia, loss of posture, jaw tone and wing flapping. The data presented include means, standard deviations and ranges.
Discussion The discussion summarises the key results with reference to the study objectives and provides an interpretation of results considering objectives and limitations and previous studies. Birds showed a consistent sequence of behaviours during LAPS (ataxia, loss of posture, clonic convulsions and motionless), which were observed in all birds. Leg paddling, tonic convulsions, slow wing flapping, mandibulation, head shaking, open bill breathing, deep inhalation, jumping and vocalisation were observed in a proportion of birds. Spectral analysis of EEG responses at 2 s intervals throughout LAPS revealed progressive decreases in median frequency at the same time as corresponding progressive increases in total power, followed later by decreases in total power as all birds exhibited isoelectric EEG and died. There was a very pronounced increase in total power at 50–60 s into the LAPS cycle, which corresponded to dominance of the signal by high amplitude slow waves, indicating loss of consciousness. ECG recordings showed a pronounced bradycardia during LAPS. There was a good correlation between behavioural, EEG and cardiac measures in relation to loss of consciousness. There were some effects of temperature adjusted pressure curves on behavioural latencies and ECG responses, but in general, responses were consistent and very similar to those reported in previous research on controlled atmosphere stunning with inert gases. The results are relevant to the LAPS procedure as applied to slaughter weight broiler chickens at these temperature settings. This study provides detailed information on behavioural, EEG and ECG responses to LAPS. There is an apparent discrepancy or at least a lack of clarity regarding the effect of the temperature settings on the latencies of the different behaviours. Considering, in particular, the time to onset of LOP, it appears that under temperature setting 4 latency is shorter. The authors make some hypothesis to explain this phenomenon, spotting, in addition, that it is a contradictory result when compared to previous studies where longer latencies were recorded with colder temperatures. However, no mention is made on the fact that in the Material and Methods section it is reported that the ambient temperature has a mean value of 16 ± 0.3°C, which does not justify the use of two different temperature settings. In theory, only temperature setting 3 should have been used and surprisingly enough, the shortest latencies were found in temperature setting 4. It is not clear if this difference in latency in the LOP of almost 3 s is due to the apparent forcing of the use of temperature setting 4, normally used when the ambient temperature ranges between 5 and 12 degrees, while the actual ambient temperature was reported to be 16 ± 0.3°C. It is curious, though, that the shortest latency to LOP was recorded for the temperature setting that should be used when the actual ambient temperature is lower (setting 4). A possible explanation for this phenomenon is that when the LAPS system is set to temperature setting 4, it assumes that the atmosphere is denser (i.e. higher oxygen concentration), compared to situations when the ambient temperature is higher, and as a consequence the pump runs more intensively, reducing the actual available oxygen concentration quicker than specified in the protocols. No discussion about the behavioural indicators of aversion is provided. Other N/A |
| Martin et al. (2016c) (Paper 8) |
Introduction The primary aim of this study was to determine how behavioural, electroencephalogram and electrocardiogram responses to LAPS are influenced by illumination of the decompression chamber. The standard LAPS procedure is done in darkness. It has been noted that slow‐wave EEG patterns are seen early in the LAPS process, before behavioural evidence of loss of consciousness such as ataxia and loss of posture (McKeegan et al., 2013; Martin et al., 2016b). It is well known that birds being placed in darkness fall asleep rapidly and demonstrate similar slow‐wave brain activity (Ookawa and Gotoh, 1965; Gentle and Richardson, 1972). Thus, darkness in LAPS might introduce a confounding factor changing animal behaviour. To disentangle the influence of decompression from the effect of darkness, the LAPS process has been performed in light conditions. A secondary aim was to determine the influence of the decompression chamber itself on birds without submitting them to decompression. Materials and methods The birds were reared in a research facility at the University of Arkansas. Before undergoing LAPS, the birds were feed restricted for 2–8 h before LAPS, dependent on the pair kill order. Missing data resulted from birds being out of view during behavioural observations – see results for details. Missing data in EEG and ECG traces resulted from movement artefacts and these were excluded from analysis. Eighty birds (40 pairs) were exposed to LAPS over 2 days (day 1 = 20 pairs; day 2 = 20 pairs). Each pair was exposed to LAPS/SHAM in a different run, giving true replication. A two by two‐factorial design was employed, with LAPS/light, LAPS/dark, sham/light and sham/dark treatments (10 pairs in each). Sample sizes were determined using power calculations (Cohen J, 1992. A Power Primer. Psychological Bulletin, 112, 155–159; Snedecor and Cochrane (1967) Statistical methods Iowa State University Press) based on differences in the variables reported using similar measurement methodologies in related studies on controlled atmosphere stunning and emergency killing using anoxic gas mixtures (e.g. Abeyesinghe et al., 2007. Brit. Poult. Sci. 48, 406–423; Coenen et al., 2009. Poult. Sci., 88, 10–19; McKeegan et al., 2007a. Brit. Poult. Sci, 48, 430–442; McKeegan et al., 2007b. Anim. Welf. 16, 409–426; McKeegan et al., 2013. Poult. Sci., 92, 1145–1154). The four treatments applied were LAPS/light, LAPS/dark, sham/light and sham/dark treatments. See details in intervention table above. One of six possible temperature settings was applied in this study 4, applied between 5 and 12°C. According to treatment, illumination was applied at 500 lux and in sham treatments birds were identically handled but remained undisturbed in the LAPS chamber without decompression for 280 s. Outcomes recorded in this study were behaviour, electroencephalogram and electrocardiogram (see Annex table A8/4). For the behaviour observations, postures and movements and general behaviour were recorded, described in detail in Table 1 of the paper. The EEG was analysed in non‐overlapping 2s epochs to produce latencies indicating unconsciousness, including F50 < 12.7 Hz (non‐responsive state) and < 6.8 Hz (general anaesthetic plane) (Martin, 2015; Sandercock et al., 2014), latency to total power equal to 10% of baseline and the onset of isoelectric EEG signal by visual interpretation and by identification of validated spectral characteristics. ECG signal was used to determine heart rate (bpm derived from the number of QRS complexes in a 5s epoch) at six baseline time points before LAPS (three outside chamber, three inside chamber with door open) and every 5 s during the LAPS cycle. Birds were randomly selected from a larger flock reared for a series of research trials. All birds were treated in similar manner with regard to husbandry and during handling prior to exposure to the intervention. The experimental birds were randomly selected from a larger flock by a random number generator (Microsoft Excel, 2010) based on wing tag number. The birds underwent LAPS in pairs where one bird was implanted and instrumented to record EEG and ECG; behavioural observations were carried out on both birds. The pair treatment order was generated by a Graeco‐Latin square design (Martin and Bateson, 2007) to balance day, temperature treatment and source pen for EEG implanted birds. A single observer conducted behavioural measurements using a specialized behavioural recording program (Noldus Observer). It was not possible to blind the observer, as the physiological recording equipment was visible on birds wearing it and it could be seen on the video recording whether the lights were on or not. All data were summarised in Microsoft Excel (2010) spread sheets and analysed using Genstat (14th Edition). Statistical significance was based on F statistics and p < 0.05 significance level. Summary graphs and statistics were produced at bird and treatment level. Statistical comparisons were conducted via generalised linear mixed models (GLMM) (Poisson distribution) or linear mixed models (LLM) (normal distribution) dependent on the data distributions for each variable. Data transformations were attempted when necessary via logarithm function. All models included bird identification number (ID) and pair number as random effects. All fixed effects were treated as factors and all interactions between factors were included in maximal models. All models included LAPS/sham treatment, light/dark treatment and whether the bird was implanted as fixed effects and bird weight, ambient temperature, ambient humidity and feed withdrawal time as covariates. It was necessary to group behavioural data for analysis dependent on treatment (LAPS/sham) due to the majority of behaviours not being exhibited when birds did not undergo LAPS. The complete data set was analysed for some behaviours shown in all treatments (notice, standing, sitting, headshake, mandibulation, vigilance and vocalisations). Spearman correlations were used to determine directional associations between temperature and humidity (ambient and within chamber) and behavioural measures. EEG summary statistics and graphs were produced at bird level, while statistical comparisons focussed on estimated means and differences between means. GLMMs (Poisson distribution) or LLMs (normal distribution) were performed dependent on the data distributions for latency variables to unconsciousness (F50 < 12.7 Hz (non‐responsive state); and < 6.8 Hz (general anaesthetic plane); latencies to visual inspection characteristics (presence of slow‐wave and three consecutive isoelectric 2 s epochs); latencies for the signal to have a total power equal to 10% of baseline; and finally latencies to isoelectric (Ptot less than 170 mv and F50 greater than 22 Hz). These spectral variable thresholds were never reached in sham treatment groups, therefore as with behavioural observations data were split into subsets for modelling of other effects. The ECG data were analysed by carrying out GLMMs (Poisson distribution) or LLMs (normal distribution), dependent on the data distributions for each heart rate interval, including the 6 baseline intervals and latencies to bradycardia. Latencies to bradycardia and bpm < 100 were never reached in sham treatment groups, therefore as before subsets of data were analysed. Paired t‐tests were used to do comparisons within treatment groups at individual bird level to compare heart rate at specific time points. The rate of decompression is described in detail but the use of the ‘tilde’ (i.e. ‘approximatively’) provides uncertainty to the range of variation of the chamber pressure by the end of each phase. A two by two factorial design was employed, with LAPS/dark, LAPS/light, sham/dark and sham/light treatments. The design of the experiment is reasonable and the procedures are adequately described. However, instead of visual inspection and elimination of movement artefacts from EEG used in the current study, one could apply automated tools for artefacts detection and rejection. These tools provide somewhat better purification of the data. Also, in case of large amount of artefacts, it might be helpful to use short epochs (1 s) instead of 2 s in the current study. This would increase the duration of non‐rejected episodes at a cost of slightly decreased spectral resolution that is not truly important in this case. It is a pity that heart rate variability (HRV) as indicated in EFSA guidelines was not analysed in the current study. One can note that it was not analysed in alternative stunning methods by other researchers as well. This precludes comparative analysis of HRV in the current and previously described stunning methods. However, inclusion of such analysis might be useful for future applicant and for science in general. Results Means, standard deviation and ranges of continuous variables are presented in the results. Numbers analysed are provided in results tables. Results are stated in absolute numbers when feasible. The results are reported for each outcome by LAPS/SHAM and light/dark treatment, in tables and figures. The data presented include means, standard deviations and ranges. Within the sham treatments, illumination increased active behaviour and darkness induced sleep. The time to loss of consciousness in the two groups of birds was similar (54.7 ± 1.3 s vs 55.9 ± 1.19 s, mean ± SEM, P = 0.25). Electrophysiological measures such as F50 and Ptot were similar under light and dark conditions in LAPS birds as well, although large amount of non‐rejected artefacts especially seen after 60 s after LAPS onset make a comparison difficult. The cardiac response in LAPS was unaffected by illumination. Discussion The discussion summarises the key results with reference to the study objectives and provides an interpretation of results considering objectives and limitations and previous studies. Birds which underwent the sham treatment exhibited standing, slow wing flapping, vigilance, mandibulation, headshakes, vocalisations, sitting, pecking and panting behaviours, while those exposed to LAPS exhibited these plus ataxia, open bill breathing, deep inhalation, jumping, loss of posture, convulsions, leg paddling and motionless. Behavioural latencies and durations were generally increased in the sham treatments, since the whole 280 s cycle time was available (during LAPS birds were motionless by 145 s on average). Within the sham treatments, illumination increased active behaviour and darkness induced sleep but slow wave EEG was seen in both light and dark sham treatments. Exposure to LAPS was associated with increased headshaking, probably relating to increased noise levels in the chamber and the hypoxic environment. The pattern of EEG response to LAPS (steep reduction in median frequency in the first 60 s and increased total power) was similar with and without illumination, though birds in darkness had shorter latencies to reach a non‐responsive state (F50 < 12.7 Hz), GA plane (F50 < 6.8 Hz) and isoelectric EEG. Cardiac responses to LAPS, such as pronounced bradycardia, closely matched those reported previously and were not affected by light treatment. Collectively, these results add to a growing body of evidence that behavioural, ECG and EEG responses to LAPS are consistent and indicative of a process that is largely equivalent to controlled atmosphere stunning with anoxic gases. The LAPS/dark results are relevant to the LAPS procedure as applied to slaughter weight broiler chickens at this temperature setting. This study provides detailed information on behavioural, EEG and ECG responses to LAPS dependent on lighting conditions and on sham exposure to the LAPS chamber, also dependent on lighting conditions.
In general, the given set of data provides a clear impression about the absence of significant influences of light on the LAPS process. Other |
| Holloway et al. (2017) (Paper 9) |
Introduction The primary objective of this study was to define the characteristics of the vacuum used in LAPS in terms of pressure time and to define the dynamics of chamber parameters such as pressure, temperature and relative humidity and their interrelationship with oxygen level. A second objective was to characterise the role of water vapour, which at low pressures can have an increased impact in reducing on oxygen levels. A third objective was to characterise the measured chamber parameters for pressure curves used for several temperature ranges.
Materials and methods The trial was a grab sample conducted during commercial operations and to reduce bias from temperature serial effects. Runs with the chamber empty were recorded both before and after the runs with the birds. Due to operational constraints the runs with and without birds were not balanced. Sample size was estimated using Snedecor and Cochrane (1967) Statistical methods Iowa State University Press) based on variances in the pressure recorded from previous plant records of LAPS runs. A full description of the apparatus is given in detail. These observational studies were summarized using mean values and standard error and compared using Student t test and ANOVA using p < 0.05 (XLSTATBASE, Addinsoft Limited, Paris, France). The description of the system and its component is clear and well detailed. The information on the sample size reported in the annex to the paper are not clear to which animals it refers to. Results The annex to Paper 9 in the dossier provides all the data used for the parallel chamber fore pipe study, for and presence of bird's study and the chamber parameters. Means, standard errors deviation and ranges of continuous variables are presented in the results. Numbers analysed are provided in charts. Results are stated in absolute numbers when feasible.
The effect of altitude, hence environmental barometric pressure, was tested at 141 m, 434 m or 1048 m above the sea level and it is reported that the local barometric pressure was used to automatically compensate for changes in the starting pressure due to weather. The physics of gas flow regime and calculated pump‐down curve are presented, and these have been submitted for evaluation to a physicist. The performance of multiple parallel systems, e.g. four chambers operated simultaneously in this study, were reported to be equal. Effects of environmental temperature on LAPS was also studied. It is reported that temperature inside the chamber decreases approximately 4°C during the first 67 s of the LAPS cycle, when the pressure is reduced from 760 Torr (101.3 kPa) to 250 Torr (33.3 kPa), and then remains almost constant. Fogging inside the chamber is also reported, in this sense, fog is first observed during Region I, when the chamber pressure is reduced to approximately 660 Torr, and then clears when the chamber pressure decreases below 450 Torr. However, data presented in one of the figures indicate that the relative humidity in the chamber falls from about 85% at the start to about 40% at 67 s, and subsequently increases to 50% which is attributed to “outgassing from the birds”. The welfare consequences of this sudden decrease in relative humidity have not been elucidated or discussed. However, latency to some behavioural events (e.g. head shaking, gasping) observed during the period of reduced relative humidity would be of interest. It is also reported that the atmospheric equivalent oxygen concentration decreased from 20.68% to 3.77% at a final vacuum pressure of 150 Torr. Discussion The results/discussion summarise the key results with reference to the study objectives and provides an interpretation of results considering relevant atmospheric physics. The performance of the chamber matches that specified by the temperature based set curves, The layout of the four chambers with different designs and lengths of fore pipe did not affect the pump down curves. Normal loading with tow‐palletized crates of birds did not affect the pump down curve. Chamber temperature was found to fall about 4.5°C due to adiabatic cooling and RH first dropped and then rose which may relate to preferential pumping of water vapour/droplets during the short period of fog formation which occurs in some combinations of temperature and humidity. Measurements of oxygen levels showed a small reduction in fractional oxygen, which may relate to interactions with water vapour. Oxygen levels need to be adjusted to atmosphere equivalent to reflect the physiological impact. The oxygen levels measured were similar to those calculated and reflect those used by Purswell et al., 2007 which found that less than 5% oxygen for 2 minutes was effective to irreversibly stunning poultry from 160 to 280s of pump down. Observations presented in Papers 6, 7 and 8 show that the mean time to motionless is around 145 s and maximum times vary up to 191 s. Thus, most of the birds have fully succumbed to the effects of Hypoxia are motionless before the 5% oxygen level is reached at around 160 s depending on the pressure curve used. As has been shown in mountain medicine and aviation medicine the impact of hypobaric hypoxia is the cumulative effect of both the hypoxic deficit experienced and oxygen consumption. In man, alcohol and smoking have major effects on sensitivity to hypoxia. In broilers oxygen consumption varies with the energy costs of maintaining homeostasis, Blood glucose, pH, oxygen levels and body temperature as well as response to feeding, physical activity and levels of stress (See Bias J 2015 Stress in Sturkie's Avian Physiology Elsevier. The two‐minute period after almost all the birds are motionless is used to ensure that no birds exit the chamber, which are not irreversibly stunned. The results are relevant to the LAPS procedure as applied to poultry. This study provides detailed information on chamber parameters, temperature relative humidity and oxygen levels. The reduction in oxygen in LAPS is controlled by the pressure reduction, which accurately predicts oxygen concentration. There is no operational need to have oxygen meters on LAPS installations for killing poultry. However, there may be a case for using oxygen meters for monitoring purposes. EFSA 2014 suggested that oxygen meters should be used for LAPS and if they could be provided. If oxygen meters were used, the evidence presented by Paper 9 and this annex strongly suggests that the fractional meter reading needs to be adjusted to equivalent atmosphere before displayed to the operative on the HMI.
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