**4. Discussion**

This study demonstrated that the penetrating spring-loaded CBE device can be a useful technique in the humane euthanasia of experimental animals by successfully producing instant and irreversible unconsciousness in 100% of the guinea pigs. This assessment of loss of consciousness is based on the absence of reflexes and the significant palpable physical damage to the skull, indicating sufficient damage to the vital regions of the brain [5,16,53]. The small but consistent result in this study exceeds the accepted animal welfare standard proposed by Temple Grandin for livestock in abattoirs, whereby 95% of animals should be immediately stunned with a single shot [54]. It also further supports the results from the study conducted by Limon et al. on South American commercial guinea pigs and is comparable to findings for other vertebrate species. In this study, some guinea pigs displayed involuntary muscle fasciculations and slow hindlimb pedaling motions after the initial shot. Both pedaling and head/neck movements are known autonomic movements controlled by reflex circuits in the spinal cord; thus, they commonly occur even after spinal severance due to the residual activity of these circuits [7,53,55].

The CB device used in this study was purchased from a commercial supplier which advertised it as a penetrating CB device. From the above results, all the skulls had extensive fractures, which is characteristic of a penetrating CB, although the skin remained intact after the procedure. This appearance is more characteristic of a non-penetrating CB which has a concussive effect without the bolt entering the cranium or penetrating the skin. Hence, while the skin remained intact, this paper refers to the device as a penetrating CB based on the photographs post-euthanasia as the bolt has clearly entered the cranium. An unexpected outcome of the experiment was that the overlying cranial skin of all guinea pigs was not compromised and remained intact. This observation was different to what was anticipated, especially given the extent of the fractures observed, and is not a typical characteristic of a penetrating CB. The intact skin may potentially be the result of the increased elasticity of the skin around the head in a guinea pig compared to the skin of other animals, such as cattle and rabbits, where the skin is more tightly adhered to their skulls. The cranial fractures associated with the position of the CB on the skull was expected given the nature of the device. The combination of the absence of measured parameters and post-mortem examination (hemorrhages and fractures) demonstrated that CBE resulted in effective stunning and insensibility in all the guinea pigs studied, even though the overlying skin was not compromised.

### *4.1. E*ff*ectiveness of Captive Bolt in Animals*

Currently, there is only one published review of CBE in guinea pigs. This study was performed on South American commercial guinea pigs and four different slaughter methods were compared (cervical dislocation, electrical head-only stunning, carbon dioxide stunning and penetrating CB) [13]. Similarly to this paper, a small sample size of guinea pigs was used (10) with one guinea pig requiring a second shot. This could be argued as a 10% fail rate which is above the Temple Grandin recommendations for humane euthanasia with CB. Possible reasons for this guinea pig requiring a second shot may have been related to handling and restraint, incorrect placement of the CB, differences in devices or device failure. These problems have been described in other studies investigating CB in other species, in which insufficient pressure and/or penetration of the device, inaccurate placement of the device, inappropriate CB chosen for the intended species or prior head injury in animals were associated with failure of CBE or the need for multiple attempts [10,20,23,26,28]. These are all aspects to be managed and explored appropriately when using CBE. It is noteworthy that the use of CBE in our study for experimental tissue collection did not result in failure for any animal involved, despite the relative inexperience of the researchers with this technique.

The findings from these papers, along with this present study, support the conclusion that CBE can be an effective and humane method of euthanasia across many vertebrate species. In contrast, BFT has been shown to have a comparatively high failure rate. BFT also has the potential to more frequently fail the international standard of having at least 95% of animals being stunned after an initial blow/strike to preserve animal welfare. Therefore, when compared to CBE, BFT has a higher potential to negatively impact the welfare of guinea pigs and other vertebrate animals. The use of CBE appears to support the 3R framework principle of refinement by utilizing a more humane and preferable method to BFT in animals used for research.

### *4.2. Uncontaminated Tissues*

At times, high quality chemically uncontaminated non-brain tissues are required for medical or biomedical research purposes. According to the operators in this study, the tracheal tissues collected were of a higher quality compared with tissues obtained from previous experimental work utilizing the BFT method. In these types of experiments, the majority of other euthanasia options available for guinea pigs (see Table 1) are not ideal as they have the potential to contaminate tissues, decrease tissue quality and affect metabolic serum biomarkers [45,46]. These issues can be potential confounders that can affect or compromise experimental outcomes, which could mean the information obtained may be incorrect or inconclusive. As such, the use of CBE over BFT supports the 3R principle of reduction as fewer animals may be required when higher quality tissues are used with better results. By utilizing high quality tissues, fewer animals may be used as research outcomes which may be more robust with a lower rate of rejected tissues. In regards to guinea pigs raised for human consumption, there is a legal and ethical requirement to ensure that meat products do not have any chemical residues unsuitable for human consumption and that animals are humanely killed. The use of CBE can be seen as a preferred method to support refinement due to potential improvements in animal welfare and research outcomes whenever intact brain or nasal tissues are not required [5].

### *4.3. Physical, Emotional and Compassion Fatigue*

Operators anecdotally reported increased satisfaction with using a CBE compared to BFT due to the decreased variability of the effectiveness and easier application of the technique. This could potentially reduce the likelihood of physical, emotional and compassion fatigue being experienced by the operators. This is similar to statements made by other operators in different experiments which also rated CBE as the most aesthetically pleasing method of euthanasia when compared to BFT [8,17,22]. Aspects of operator fatigue include physical, emotional and compassion fatigue. Physical fatigue can be reduced when the CB is correctly placed on the cranium as the kinetic energy is delivered by the device [56,57]. This is different to the use of BFT which relies on the force applied by operators.

Emotional and compassion fatigue can also be linked to the human–animal bond. This bond is thought to be a contributing factor in the compassion fatigue experienced by people working in the animal profession [29]. Thus, by reducing the strain on the human–animal bond, both emotional and compassion fatigue can possibly be reduced. In this study, the operators using the CB reported this method to be more aesthetically appealing, as the bolt did not appear to significantly compromise the skin (Figures 3 and 4). They also felt the method of euthanasia was more humane. As a result, this method appeared to be less confronting and overall more appealing to operators, and potentially less detrimental to their wellbeing.

While the human element is an important aspect of humane euthanasia, the psychological aspect of this euthanasia technique was not objectively assessed in this paper. However, the initial anecdotal evidence appears to be positive. In future studies, a survey could be used to assess the emotional effect of CBE use to further investigate the human welfare aspect of various techniques of humane euthanasia. Overall, there was a verbalized positive impact on the physical and psychological well-being of operators when using this method in this case study. However, for CBE to be successful in future experimental studies or settings, adequate training and correct restraint of the animals must be ensured, and an appropriately sized CB device must be used and maintained. Other causes for CBE failure can arise if personnel are not adequately trained, which can lead to incorrect restraint of animals and placement of the device. If any of the above are performed incorrectly, higher levels of physical and emotional fatigue can occur due to failure of the device or technique [5,7–9,20]. Therefore, correct training and device usage is essential to decrease the chances for physical, emotional and compassion fatigue, as well as reducing risks to personnel and animals [3,36,58].

### *4.4. Limitations and the Future*

Due to the small sample size of guinea pigs, aspects such as device fatigue and physical fatigue experienced by operators from loading the spring with each guinea pig were not evaluated. Other potential parameters that determine insensibility and success of stunning include measuring auditory evoked potential (AEP) and electroencephalogram (EEG) activity with electrodes. Both correlate to the cessation of convulsions, loss of reflexes and brain death [8,59,60]. These were not used in this study as the five parameters for humane livestock slaughter were deemed adequate to indicate insensibility. The use of EEG in CBE also risks damaging the electrodes and was deemed inappropriate for this study [8].

Additional parameters to measure could include the absence or presence of a heartbeat. However, cardiac contractions can continue in unconscious or brain-dead animals and thus this parameter was not used [8,18,61]. Whilst histological changes or scoring of the damage to the cranium and brain could have been assessed (skull fractures were easily observed and palpated), the degree of brain and cranial damage was not considered essential in this study [8,13,17,18]. An area that would benefit from additional investigation would be to explore alternate types of restraint and equipment. The current technique described requires a handler and a CB operator. It would be beneficial to develop or modify a device to enable the task to be performed by only one CB operator.

Finally, a di fferent type of CB device could be tested. The use of an industrial CB (gunpowder-powered, compressed air or CO2 blank cartridges) may be more suitable than the spring-powered CB in larger cohorts or in an abattoir setting for animals bred for human consumption. This is because the spring-loaded CB is designed to be used for <1000 animals per year and can experience device fatigue more quickly compared to an industrial CB [51]. The downside of an industrial CB is that they are more expensive and require more comprehensive training to use. However, the use of an industrial CB can result in less physical fatigue compared to a spring-loaded CB as there is no requirement to physically pull the spring with each use. Thus, the use of industrial CBs could be considered if a greater number of animals are to be used in a study due to larger cohorts or in other settings (i.e., an abattoir). It should also be noted that as of 2020, within Australia, CB is an accessible humane euthanasia technique as there are no legal requirements to hold a firearms license when using a CB in six out of the seven Australian states and territories. The authors are aware of similar legislation in other countries, which makes CBE a potentially accessible and humane euthanasia technique internationally in research and other settings.

From this study and other studies that have been undertaken, it can be tentatively proposed that CBE can be used as a suitable alternative for the collection of non-brain uncontaminated tissues. A drawback of this study is the small sample size available at the time. Further research should be undertaken to review the potential failure rate on a larger population to determine potential complications. Future studies in refinement could explore di fferent handling/restraint techniques or devices, and the e ffects of the use of di fferent CBs. Additional work can be performed to ensure consistency of the results from this study. As compassion and emotional fatigue are so prevalent, any future studies should incorporate formal surveys of operator preferences regarding their levels of physical, emotional and compassion fatigue and how this changes when performing euthanasia in di fferent conditions.
