**1. Introduction**

For over 200 years, guinea pigs have played an important role as an animal model in biomedical research due to their greater physiological parallels with humans in comparison to other species of rodents and rabbits [1,2]. There is an increasing level of awareness and importance of animal welfare at each stage of research with animals which includes ensuring a humane death. A humane death or euthanasia is defined as achieving immediate stress-free insensibility and death [3–5] The very definition of humane euthanasia supports the 3R's framework of replacement, reduction, refinement as proposed by Russell and Burch in 1959 [6]. The full replacement of animals in some areas of research can be difficult, and at times animal use may still be required. Whenever the use of animals cannot be replaced, it is incumbent on the research community to apply the principle of refinement at all stages of research to improve animal welfare and care [6].

Captive bolt (CB) is a technique that can be applied in the humane euthanasia of animals. Despite its benefits during humane slaughter, it is ye<sup>t</sup> to be applied in the research setting for the collection of uncontaminated non-brain tissue. The CB devices used can be hand-held with a retractable bolt powered by either a spring, compressed air, gun powder or a blank cartridge, and placed on a specific area on the cranium of the animal [7]. Once triggered, it will lead to either stunning (reversible loss of consciousness) or death [7]. CB placements and their effectiveness differs between various animals. [4,7–10] There are two types of CB: non-penetrating and penetrating [7]. Non-penetrating CB leads to stunning via the transfer of kinetic energy into the cranium and brain and is usually followed by a secondary euthanasia method [4,5,7,11]. In contrast, penetrating CB results in death due to the bolt fracturing the skull and entering the brain, subsequently causing irreversible physical damage to the cerebral cortex, brain stem and the thalamus [10,12–14]. The five indicators of successful stunning are lack of corneal blink reflex, rhythmic breathing, righting reflexes, vocalization and head/neck tension [5,15,16]. These indicators are reliable to assess the effectiveness of the shot as CB damages the region of the brain responsible for these responses [12,15,16]. The technique has not been tested in a research setting using a non-commercial captive bolt device and a methodology for laboratory use is ye<sup>t</sup> to be developed. The application of this technique is thought to be transferable and potentially valuable in animal research models requiring chemically uncontaminated and intact non-brain tissues.

The use of a captive bolt to perform humane euthanasia (CBE) has been demonstrated through numerous experiments to be efficacious and humane across a wide range of species, including: guinea pigs [13], rabbits [17], piglets [8,18–22], joeys [11,23], ruminants [24–27], and avian species [28]. Some of these results showed CBE produced immediate death in 100% of animals [28]. The effective use of this method has also been documented in guinea pigs for humane slaughter in the meat industry, which demonstrates its feasibility in this species [22].

While blunt force trauma (BFT) is a common technique to euthanize guinea pigs in research requiring chemically uncontaminated tissues, there are no known published studies assessing the use of BFT in this species. In other vertebrates, this technique has been compared to CB. In a study based on 170 rabbits by Walsh et al., CBE was shown to be more effective than BFT, with BFT failure rates of up to 23% due to insufficient force [17]. Likewise, studies on piglets, sheep/lambs, and kids have also shown that CBE will produce extensive brain damage before pain can be perceived and caused instantaneous death. In these studies, the failure rate was between 0–6% in animals [8,18,19,22,25–27]. This is in contrast to a study on piglets which determined that BFT had a similar failure rate of up to 24%, hence the need for repeated blows [22].

Compassion fatigue is a form of post-traumatic stress disorder. It is defined as exhaustion from the stress of feeling empathetic towards animals and feeling unable to help them in any way [29,30]. This important concept is now readily recognizable in staff working with animals. Compassion fatigue is believed to be exacerbated by performing euthanasia on a regular basis and is partly dependent on the type of method used [29]. A study performed by Rohlf et al. on 148 animal workers in animal shelters, veterinary clinics and research laboratories reported that they experienced mild and moderate stress symptoms (39% and 11%, respectively) from performing euthanasia [31]. Similarly, a systematic review conducted by Scotney et al. demonstrated staff involved in performing euthanasia (animal shelters, veterinary clinics or research laboratories) was correlated with increased work stress and was hence a contributing factor to developing compassion fatigue [32]. In particular, laboratory staff may experience guilt when euthanizing research animals [33]. Therefore, if the euthanasia method has high animal welfare and acceptable aesthetics, then the staff member or "operator" may be less likely to experience compassion or emotional fatigue.

As part of a study requiring the collection of chemically uncontaminated tracheal tissues from guinea pigs (University of Melbourne Animal Ethics ID: 1814500.3), researchers would often use cranial blunt force trauma (BFT) followed by exsanguination or decapitation [34,35]. However, facility staff, animal welfare veterinarians and researchers all expressed concerns with BFT. It was found to be a

method operators wished to avoid and has been shown in other studies to potentially have a risk of low repeatability due to inappropriate force or technique, and it is very dependent on the individual operator [5,35,36]. Furthermore, there are known concerns that this method of euthanasia can lead to physical, emotional and compassion fatigue in personnel due to the human–animal bond [5,18,37–41]. Prior to identifying CBE as an alternative technique to BFT, other methods of humane euthanasia were considered. Table 1 outlines the currently available experimental methods of guinea pig euthanasia with their known respective advantages and disadvantages.

After a literature review of possible known options, it was extrapolated that CBE could be suitable for non-brain tissue collection with no chemical residues and possible improvements in experimental, animal and human welfare outcomes when compared to the BFT method [5]. The aim of this case study was to develop and trial a protocol to refine the method of humane euthanasia of guinea pigs for research requiring the collection of uncontaminated non-brain tissues. This paper introduces an alternative application of an existing method of humane euthanasia in a novel setting using CB and the criteria for humane slaughter to ascertain both the practicality and humaneness (animal and human) of using CBE on guinea pigs [15] for research.



### **2. Materials and Methods**

Equipment: A penetrating spring-loaded CB called 'The Ballista' (Bunny Rancher, Shapleigh, ME, USA), was used in this research (see Figures 1 and 2 below). This hand-held device was placed mid-forehead on the guinea pigs (see Figure 2 below). The device delivered 6.7 joules with each shot [51].

**Figure 1.** 'The Ballista': a penetrative spring-powered captive bolt (CB) gun used for the euthanasia of guinea pigs in this research. Image used with permission [51]

**Figure 2.** (**Left**): '*The Ballista*' spring-powered non-penetrating captive bolt. (**Right**): The red cross denotes where the captive bolt was positioned on the guinea pigs.

Parameters to determine humane euthanasia: There are currently no specific recommendations for CB use in guinea pigs. Hence, the livestock parameters of an absence of corneal blink reflex, rhythmic breathing, righting reflexes, vocalization and head/neck tension were utilized for this study.

Experiment: Operators first trialed the device on oranges to gain familiarity with the device. Excess breeding stock of male rat cadavers weighing 250 g or more were used to trial the device prior to use in guinea pigs. Twelve tri-color short-haired American female and male guinea pigs aged 16–22 weeks, weighing 500 g or more, were sourced as excess stock. Guinea pigs were euthanized using CBE. Accurate placement of the CB was first practiced on euthanized rats retrieved from other experiments to decrease the number of guinea pigs needed. All guinea pigs used were part of another animal ethics approved research project (University of Melbourne Animal Ethics ID: 1814500.3) as the researchers agreed to use this method as their humane euthanasia technique. Two personnel were required, with a total of 8 operators performing this method. Prior to the procedure being performed, each guinea pig was moved to a quiet place away from the others. One operator wrapped and held the guinea pig in a towel with its head resting on a rolled-up towel to elevate the head, increase comfort and decrease stress. The second operator pre-loaded the spring prior to positioning the penetrating spring-loaded CB on its head at the intersection between lines drawn from the base of the ears with the contralateral eyes (see Figure 2). When triggered, a retractable bolt was fired with the aim of producing effective and instantaneous unconsciousness. Animals were unwrapped to assess the 5 livestock humane euthanasia parameters. This was determined using five indicators (absence of corneal blink, rhythmic breathing, audible vocalization, righting reflex and head/neck tension) in each individual. Exsanguination was performed within the next 20–30 s. The results were recorded in and reviewed.
