# Co and N2 Are No Humane Alternatives to Co2 Euthanasia in Rats.

Petra Seebeck, Charlotte Calvet, Philipp Villiger, Francesco Prisco, Tim Buchholz, Felix Gantenbein, Christa Thöne-Reineke, Boris Gourévitch, Carsten Wagner

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Petra Seebeck, Charlotte Calvet, Philipp Villiger, Francesco Prisco, Tim Buchholz, et al., Co and N2 Are No Humane Alternatives to Co2 Euthanasia in Rats., 2025. hal-05344089

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Distributed under a Creative Commons Attribution 4.0 International License
# Co and N2 Are No Humane Alternatives to Co2 Euthanasia in Rats.

**Petra Seebeck**

petra.seebeck@uzh.ch

Zurich Integrative Rodent Physiology (ZIRP), University of Zurich https://orcid.org/0000-0002-8398-5495

**Charlotte Calvet**

Institute of Physiology, University of Zurich and Zurich Integrative Rodent Physiology (ZIRP)
https://orcid.org/0000-0002-6163-9807

**Philipp Villiger**

University of Zurich

**Francesco Prisco**

Institut für Veterinärpathologie

**Tim Buchholz**

University of Zurich

**Felix Gantenbein**

University of Zurich https://orcid.org/0000-0003-4681-6438

**Christa Thöne-Reineke**

Institut für Tierschutz, Tierverhalten und Versuchstierkunde

**Boris Gourévitch**

Institut de l'Audition

**Carsten Wagner**

University of Zurich https://orcid.org/0000-0002-9874-8898

## Article

**Keywords:** euthanasia, seizures, gasping, refinement, animal welfare, telemetry, plethysmography, distress

**Posted Date:** May 24th, 2024

**DOI:** https://doi.org/10.21203/rs.3.rs-4276908/v1
**License:** © Ⓟ This work is licensed under a Creative Commons Attribution 4.0 International License.

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**Additional Declarations:** There is NO Competing Interest.
**Abstract**

Carbon dioxide (CO₂) is commonly used for euthanasia of laboratory animals but has potential negative side effects. There is an urgent need to identify alternatives causing minimal distress while providing a fast and reliable loss of consciousness (LOC). We evaluated different CO₂ concentrations (30% and 70%) and explored carbon monoxide (CO) and nitrogen (N₂) as alternatives for a more humane euthanasia in rats. Isoflurane anesthesia was included as a reference. We assessed relevant physiological parameters in rats using a multifaceted approach combining EEG, ECG, telemetry, EMG, respiration, behavior, biochemical stress parameters, and lung histopathology. 70% CO₂ resulted in rapid LOC and a faster death compared to 30% CO₂ thus minimizing the time of consciousness in which distress and eventually pain can be experienced. Nevertheless, a higher amount of distress (e.g. gasping, arrhythmia or seizures) experienced until LOC might outweigh the benefits of a faster LOC. CO and N₂ resulted in longer times to LOC, induced seizures before LOC, increased stress levels and caused higher lung damage. Therefore, CO and N₂ are no humane alternatives to CO₂ and should not be used for euthanasia of rodents. However, combining CO₂ with anesthetic gases might be suitable to induce a more humane euthanasia.

**Introduction**

In 2020, approximately 3.8 million mice and 665,000 rats were euthanized in Europe for research purposes¹. In addition, numerous rodents are euthanized to serve as food for zoo or pet animals, for fur production or during pest control. Carbon dioxide (CO₂) is the most commonly used gas for euthanasia of rodents²,³. Ideally, euthanasia should result in a quick and reliable death with minimal pain and distress before loss of consciousness (LOC)⁴. However, various studies suggest that CO₂ euthanasia may not satisfactorily meet all of these criteria²,⁵,⁶. CO₂ is aversive to rodents, potentially leading to pain, anxiety, fear, and breathlessness before LOC⁵,⁷,⁸. As high CO₂ concentrations (between 50 and 80%) are painful for humans⁹⁻¹¹, it has been assumed that rodents experience the same¹⁰,¹². Moreover, even low concentrations of CO₂ cause anxiety in humans¹³. Despite these negative connotations associated with CO₂, no alternatives have been identified so far causing less distress while providing a fast, reliable, easy to handle, inexpensive and user-safe way to induce unconsciousness and death in rodents using non-physical methods.

Different gases have been tested as alternatives to CO₂. Carbon monoxide (CO) inhalation reportedly induces a fast death in different species: rats¹⁴, guinea pigs¹⁵, minks, and chinchillas¹⁶. Only a few studies investigated its impact on animal welfare, reporting CO to be less aversive to rodents than CO₂¹⁴ but also inducing agitation in dogs or pigs¹⁷,¹⁸. Inert gases (Nitrogen (N₂), Helium, Argon and Xenon) have also been tested. However, epileptiform activities in the electroencephalogram (EEG) have been reported in mice exposed to N₂, Helium and Argon, but also half of the animals exposed to CO₂¹⁹. Both
CO₂ and N₂ increased locomotor activity, vertical jumps and freezing episodes, but it was more pronounced with CO₂ suggesting that CO₂ might be more aversive²⁰.

Refinement methods for CO₂ exposure have also been explored. Recently, it has been recommended to anesthetize rodents with isoflurane before CO₂ exposure; however isoflurane could also be aversive²¹⁻²³ and cause excitement²⁴. Additionally, recommendations on the use of CO₂ for euthanasia have recently changed from a low (< 30%) to a high (30–70%) gas displacement rate, without providing a clear rationale for this change⁴,²⁵.

Currently, the use of CO₂ for euthanasia remains highly non-standardized⁵. Studies conducted so far used different CO₂ displacement rates and mainly collected observational findings and rarely physiological parameters indicating distress or unpleasant pathophysiological events. Additionally, it remains unclear when animals lose consciousness and what they experience until that point. This makes it hard to compare different studies and to get a conclusive overview of the impact of euthanasia on animal welfare¹¹,¹²,²⁴,²⁶⁻²⁸. Therefore, it is necessary to clearly identify LOC and accurately describe what amount of distress or pain the animals experience until then by using suitable parameters with the final goal to better understand how euthanasia can be performed in a fast and ethical way.

In this study, we acquired and analyzed a multitude of physiological and behavioral parameters, using, for the first time, a combination of cardiovascular, respiratory, neural, biochemical, histological and behavior data acquired in parallel in male and female Sprague Dawley rats. With this multi-modal approach we tested isoflurane, CO and N₂ and two CO₂ filling rates for their suitability to provide a more humane euthanasia.

# Results

## CO₂ and isoflurane induce bradycardia while CO and N₂ lead to tachycardia

To understand the impact of different gases on the cardiovascular system, we compared heart rate (HR) and blood pressure (BP) before and after gas exposure (Fig. 1). Isoflurane anesthesia typically induced, after 20 seconds (s), an initial short-term reduction (30s) in HR before HR returned to baseline levels (Fig. 1, Supplementary Fig. 1–2). With CO₂, HR also decreased, first rapidly and then continuously until death. HR decreased faster with 70% CO₂ than 30% CO₂, with a distinct plateau observed in the 30% CO₂ group before a subsequent decrease. On the contrary, exposure to CO and N₂ resulted in a transient HR rise (for 40s). The maximum HR was significantly different between groups and higher with CO and N₂ than other gases. Following the initial rise, HR also decreased for both CO and N₂ until death. At the time of loss of motion (LOM), HR was different between groups and was lowest for the 70% CO₂ group (Table 1).
Following gas exposure, mean blood pressure (MBP) temporarily increased in all groups compared to baseline levels, during periods ranging from a few seconds (CO) to 50s (isoflurane, 70% CO₂), 80s (30% CO₂) up to 100s (N₂). The longest increases of MBP (30% CO₂, N₂) were also delayed by 10s (30% CO₂) or 40s (N₂) while the increase was instantaneous for the other gases (Fig. 1, Supplementary Fig. 1–2). The highest MBP was reached for N₂ (1w-ANOVA, adjusted-p < 0.0001) while the CO-induced MBP increase was very modest (Table 1). At LOM, MBPs were not different from baseline values except for the N₂ group where it remained elevated. All groups exhibited similar dynamics for systolic BP and diastolic BP as observed for MBP (Table 1, Supplementary Fig. 2).

Taken together, our data show that CO₂ and isoflurane led to bradycardia while HR was increased after exposure to CO and N₂. On the other hand, MBP was increased before LOM in all groups even during anesthesia with isoflurane.

## Arrhythmias occur faster with CO than with other gases

We explored the occurrence of arrhythmias and their onset after exposure to different gases (Supplementary Fig. 3). We observed arrhythmias in all groups including second degree atrio-ventricular blocks, sinus pauses, premature ventricular contractions (PVCs) and tachycardia, but also ventricular fibrillation. 70% of animals anesthetized with isoflurane experienced arrhythmias during induction which decreased during maintenance, while 100% of animals in all other groups had arrhythmias during exposure to gases. We noted that animals had isolated arrhythmias during the acclimatization phase with room air (data not shown). After gas exposure with CO₂, the onset of arrhythmias occurred much faster than with isoflurane, CO or N₂ (1w-ANOVA, adjusted-p < 0.0001): within 5 seconds with 70% CO₂ and twice faster with 30% CO₂ (adjusted-p = 0.006, 0.006, 0.002, respectively). In conclusion, the occurrence of arrhythmias was lower with isoflurane and the onset was faster with CO₂ compared to other gases.

## All gases induce an alteration of the respiratory function

Respiratory rate (RR) rapidly increased in all groups after gas exposure for 30s with 70% CO₂, 50s with isoflurane, 60s with CO, 80s with N₂ and 90s with 30% CO₂ (Fig. 1, Supplementary Fig. 2). Maximal RR was not different between groups (1w-ANOVA, adjusted-p ns). Thereafter, RR decreased in an exponential way to reach baseline values at LOM except for 70% CO₂ for which it was already below (Table 1).

Tidal volume (TV) temporarily decreased for 25 seconds with isoflurane but progressively increased for other gases to reach above baseline values at LOM for all gases. For all gases except isoflurane, TV decreased 50-100s after LOM until death (Fig. 1, Supplementary Fig. 2). All four gases had higher
maximal TV than the isoflurane group (1w-ANOVA, adjusted-p < 0.0001) and the N₂ group exhibited higher maximal TV than the 30% CO₂ and CO groups (adjusted-p < 0.05)(Table 1).

Minute volume (MV) increased rapidly after gas exposure in all groups but less strongly with isoflurane. Compared to isoflurane, the maximal MVs were significantly higher with CO₂ and N₂ (adjusted-p < 0.001) (Fig. 1, Supplementary Fig. 2). MV returned to baseline levels at LOM for 70% CO₂ and isoflurane but were still elevated for 30% CO₂, CO and N₂ (Table 1).

In summary, our results show that RR and MV were increased in all groups after gas exposure, but TV transiently decreased during anesthesia and progressively increased during euthanasia.

## Histological lung lesions are more pronounced in animals euthanized with CO and N₂

The main histological changes observed in euthanized animals (Fig. 2) were multifocal perivascular and intramural edema of pulmonary arteries, multifocal congestion of alveolar capillaries, occasional alveolar and perivascular hemorrhages and changes in air content including multifocal emphysema and atelectasis. Animals euthanized with CO or N₂ showed higher overall lung inflation than the two CO₂ groups (Kruskal-Wallis, adjusted-p < 0.01). No differences were found between 30% and 70% CO₂ or between CO and N₂. There was no difference in the number of erythrocytes in the lungs. Edema was more severe for animals in the N₂ group compared to the CO group (adjusted-p = 0.02) and higher in females than males from the N₂ group (2w-ANOVA, adjusted-p = 0.03) (Supplementary Fig. 4d-f). In summary, histological changes in the lungs were more pronounced with CO and N₂ compared to CO₂.

**Activity is increased in all groups but only CO₂ induces gasping before LOM.**

Behavioral parameters were compared during baseline and after exposure to the different gases until LOM. During baseline animals showed exploratory behavior (sniffing and circling) (Fig. 3). After gas exposure, all animals sniffed more than during baseline except with CO (Kruskal-Wallis, adjusted-p < 0.05 and ns) (Fig. 3a). However, total duration of sniffing per animal was significantly different between groups. Animals from the 30% CO₂ and N₂ groups spent more time sniffing than animals from the CO group, followed by 70% CO₂ and isoflurane groups (Fig. 3b). After gas exposure, the time spent circling per minute was not different from baseline (Fig. 3c). The total duration of circling per animal was significantly different between groups and longer with 30% CO₂ and N₂ compared to isoflurane and CO (adjusted-p < 0.01) (Fig. 3d). Sniffing and circling durations were positively correlated to the time to LOM (data not shown).

While isoflurane anesthesia did not induce gasping, 89% and 53% of animals from the 70% and 30% CO₂ groups, respectively, experienced gasping. This percentage was significantly lower for CO (10%) and N₂ (20%). The total number of gasping events was significantly higher with 70% CO₂ and 30% CO₂
compared to CO and N₂, and isoflurane (adjusted-p < 0.05) (Fig. 3e). Only 8% of animals in the 70% CO₂ group and 45% of animals in the 30% CO₂ group lost motion before gasping, whereas the animals from the CO and N₂ groups lost motion before gasping. We did not observe significant differences between males and females in terms of sniffing, circling and gasping (2w-ANOVA, adjusted-p ns) (Supplementary Fig. 4a-c).

Activity counts acquired by the ECG transmitters were paralleled to the sniffing responses (Fig. 3a-b). All animals presented a baseline activity which was increased upon gas induction (Fig. 3f-j). However, the increase in activity was lower for isoflurane, CO and 70% CO₂ with a total activity count per animal of 8.1, 6.9 and 5.4 respectively and was higher for 30% CO₂ and N₂ with a total activity count per animal of 16.9 and 32.7 respectively (data not shown).

## LOM precedes EEG isoelectricity, cessation of breathing and asystole

LOM as an indicator of LOC, isoelectricity of EEG, cessation of breathing and asystole were compared for all gases and only LOM during anesthesia with isoflurane (Fig. 4). The time to LOM was significantly longer with N₂ than the other groups (1w-ANOVA, adjusted-p < 0.01) and with 30% CO₂ compared to 70% CO₂ and isoflurane (adjusted-p < 0.0001). The time to LOM was significantly longer for females than males euthanized with N₂ (2w-ANOVA, p = 0.006) (Supplementary Fig. 4g). Animals from the isoflurane group lost motion at an oxygen concentration of approximately 20.9%. For the CO group the oxygen concentration was around 19.5% at the time of LOM. With 30% CO₂ the oxygen concentration was at approximately 12% when the animals lost motion and for 70% CO₂ at an oxygen concentration of 6.3%. With N₂ the animals lost motion at an oxygen concentration of 1.9% (Supplementary Fig. 5a). The chamber temperature was 25-27.5°C and relative humidity around 50% (except for 30% CO₂ for which the air flow was lower, it was 75%), both were constant through the recordings until LOM (Supplementary Fig. 5b-c).

The EEG became isoelectric before the cessation of breathing for all gases (Fig. 4). The time to EEG isoelectricity was significantly longer for 30% CO₂ than 70% CO₂ and CO (1w-ANOVA, adjusted-p < 0.001). Both N₂ and 30% CO₂ led to an increased time until cessation of breathing compared to 70% CO₂ (adjusted-p < 0.0001) and compared to CO (adjusted-p < 0.05). In ECG, the time to asystole was higher for 30% CO₂ than CO (adjusted-p = 0.01). No sex differences were found (Supplementary Fig. 4h-j).

The chronology of events (Fig. 4) revealed that the onset of LOM always preceded the other events although it occurred faster with 70% CO₂ and isoflurane, then CO and 30% CO₂ and finally N₂. Secondly, the EEG signal exhibited a faster flattening with 70% CO₂ and CO followed by N₂ and finally 30% CO₂. Following this, cessation of breathing occurred, and notably, the duration was significantly extended for the N₂ and 30% CO₂ groups followed by CO and faster with 70% CO₂. Lastly, asystole occurred within 5–
7 minutes in all groups, marking the loss of the last vital parameter and therefore, definite death. Interestingly, we observed tachycardia in all groups, albeit less pronounced with 30% CO₂, during the last seconds of the recording (Supplementary Fig. 1).

## EEG waves increase upon gas induction and decrease at LOC

To analyze the dynamics of EEG oscillations leading to LOC, we computed their time-varying spectra (spectrograms) (Supplementary Fig. 6). Comparing the percentage of each EEG wave revealed delta being dominant through the whole recordings (Supplementary Fig. 6d). EEG waves normalized to baseline levels showed a transient increase in all EEG waves post-gas-induction in all groups, followed by a subsequent decrease below baseline levels after LOM (Supplementary Fig. 7). We hypothesize that this might indicate the onset of LOC. This time point varied between groups, suggesting that LOC occurred faster for 70% CO₂ and isoflurane groups than for 30% CO₂ and CO (1w-ANOVA, adjusted-p < 0.05). Notably, this time was doubled with N₂ compared to 70% CO₂ and isoflurane (adjusted-p < 0.0001) and also longer with N₂ compared to 30% CO₂ and CO (adjusted-p < 0.001) (Fig. 4). This change in the EEG pattern (Supplementary Fig. 6e) coincided well with the time to LOM. We did not observe any differences within each group between the time to LOM and the time to LOC (unpaired-t-tests, p > 0.2) (Fig. 4).

In summary, our data show that the time to LOM was associated with a specific EEG pattern that could reflect LOC, and that EEG profiles were similar until LOM independently of the anesthesia or euthanasia method used.

## Only CO and N elicit seizures

We investigated the incidence of epileptiform events caused by each gas by examining video recordings, EEG and electromyography (EMG) data (Fig. 5). We noted myoclonic jerks characterized by brief involuntary twitching, in all rats euthanized with CO and N₂ (data not shown). Importantly, this was not observed with CO₂ and isoflurane. Furthermore, we found an association between myoclonic jerks and EEG epileptic activity with CO and N₂ (Fig. 5a-b). The total number of spikes (including isolated and train spikes) was twice higher with N₂ than CO (Wilcoxon-test, adjusted-p = 0.04). The number of spikes per second in spike trains was not different between the two groups. However, the total duration of seizure events was 3 times higher with N₂ than CO (adjusted-p = 0.02). Importantly, for 90% of animals the seizures happened before LOM and therefore LOC (Fig. 5c). We observed higher number of spikes and longer seizures in females euthanized with N₂ compared to males (2w-ANOVA, adjusted-p = 0.04 and 0.048, respectively) (data not shown).

## Noradrenaline and ACTH levels are increased in all groups
Plasma noradrenaline levels were increased in 30% CO₂, CO and N₂ groups but not in 70% CO₂ group compared to baseline (Kruskal-Wallis tests, adjusted-p = 0.003, < 0.0001, < 0.0001, 0.054 respectively) (Fig. 6). Furthermore, we found that both CO and N₂ induced a more pronounced rise in noradrenaline levels compared to the 70% CO₂ group (adjusted-p < 0.02). Adrenocorticotropic hormone (ACTH) levels were elevated in all groups compared to baseline (adjusted-p = 0.01 to < 0.0001). Corticosterone levels were not significantly increased compared to baseline levels. However, animals in the 70% CO₂ group had higher values than those in the 30% CO₂ group (adjusted-p = 0.04). We did not observe differences between males and females (2w-ANOVA, adjusted-p ns) (Supplementary Fig. 4k-m). Our results suggest that all gases used for euthanasia elicit a rapid stress response as shown by noradrenaline and ACTH levels (Fig. 6a). Importantly, this response was more pronounced with CO and N₂ compared to 70% CO₂.

Discussion

In this study, we evaluated the suitability of CO and N₂ and two different concentrations of CO₂ for humane euthanasia. Isoflurane, the most frequently used anesthetic gas, served as the "gold standard", assuming that the amount of distress associated with the induction of isoflurane anesthesia is an acceptable reference for humane euthanasia. We employed a multimodal approach to assess the overall impact of the tested gases on male and female rats. With all gases used for euthanasia, animals underwent the following sequence of events: alteration of cardiovascular parameters and behavior, LOM-LOC, isoelectric EEG, cessation of breathing and asystole, as previously reported²⁸. However, distinct differences were found between gases including the time to LOC, signs of breathlessness/gasping, seizure episodes, alteration of lung morphology and increased biochemical stress markers.

Increase in low frequency waves and a decrease in high frequencies is often used to evaluate LOC²⁹ although increase in low frequency waves may occur during prolonged wakefulness similarly to non-rapid eye movement sleep³⁰. In our study, the low-frequency delta wave was dominant through the entire recordings possibly because experiments were performed during the resting phase of the rats³⁰. Post-LOM, EEG showed a decrease in all waves compared to pre-LOM. This specific EEG change observed at LOM reflects a decrease in cortical activity and possibly the transition to unconsciousness (LOC). Thus, we believe that LOM can be used as clinically accessible marker/ indicator for LOC. Nevertheless, care has to be taken to precisely determine LOM as involuntarily movements can also occur in unconscious animals if anesthesia is not deep enough²⁸,³¹,³².

Consistent with previous studies, we can conclude that the lower the CO₂ displacement rate, the longer the time until LOM and LOC ¹²,²⁸. Time to LOM/LOC was the shortest for isoflurane and 70% CO₂ but longer for CO and 30% CO₂. These results are consistent with previous studies performed in rats¹⁴, guinea pigs¹⁵, mink and chinchillas¹⁶. Finally, we confirmed that N₂ extends the time to LOM and LOC compared to the other gases²⁶. In this regards, 70% CO₂ and isoflurane minimize the time of potential stress or discomfort.
To ensure a humane euthanasia, animals should also experience a minimum of distress until LOC. CO₂ euthanasia could induce pain and/or distress by irritation of mucosae and gasping²,⁵. Both with 30% and 70% CO₂, gasping occurred when the majority of rats was still conscious¹¹,³³,³⁴ but it was more frequent with 70% CO₂. This suggests that rats exposed to both CO₂ displacement rates experience distress prior to LOC. In contrast, only few animals in the CO and N₂ groups experienced gasping after LOM. Gasping due to hypercapnia commonly occurs prior to LOM, while gasping due to inert gas hypoxia like N₂ is often considered agonal³⁵. Additionally, animals from the 70% CO₂ and N₂ groups experienced severe hypoxia before LOM/LOC while with 30% CO₂ it was less severe. Hypoxia has been reported to induce anxiety or panic attacks in humans³⁶,³⁷ and anxiety-like behaviors have been reported in mice and rats after exposure to hypoxic environments³⁸,³⁹.

Moreover, EEG analysis revealed that both N₂ and CO induced seizures in rats, most often before LOC. We also observed myoclonic jerks during the seizures. Convulsions were previously reported in rats exposed to CO¹⁴ and with inert gases¹²,²⁶. In dogs exposed to CO, EEG abnormalities were reported, potentially indicative of seizures¹⁷. Seizures observed on EEG were also reported with N₂ in rats prior to LOC²⁶ and with CO₂, N₂, Helium and Argon in mice¹⁹. Contrary to reports in mice¹⁹, we observed no seizures with CO₂ or isoflurane.

Our study shows that HR and MBP of rats euthanized with CO and N₂, increased rapidly after gas exposure contrary to previous observations with different experimental settings²⁶. This is consistent with the activation of the sympathetic nervous system and the measured increase of noradrenaline⁴⁰. Respiratory acidosis in presence of CO₂ contributes to a decrease in blood pH and bradycardia like observed in our study and by others¹¹,¹²,²⁶,²⁸,³³,⁴¹. The activation of the sympathetic nervous system in response to stressors can lead to peripheral vasoconstriction, thereby increasing systemic vascular resistance and blood pressure⁴⁰ like observed here and by others¹¹,²⁶,⁴¹. Apnea, bradycardia and an increase in MBP were also observed during exposure of the nasal mucosa to high CO₂ concentrations in rats⁴². While a study reported a stronger increase in MBP at low CO₂ concentrations (10% CO₂)¹¹, our study reveals that 30% CO₂ extended the duration of elevated MBP compared to 70% CO₂, likely attributed to the longer time until LOM.

Additionally, animals might experience discomfort due to bradycardia which occurred with CO₂ and isoflurane before LOM. Animals in all groups presented arrhythmias before LOC⁴³, which was faster with CO₂. Chest pain has been reported in humans experiencing certain types of arrhythmias⁴⁴.

The influx of all gases induced an immediate increase in activity combined with sniffing and circling which was more pronounced with 30% CO₂, CO and N₂. An increase in activity was reported with CO₂¹¹,²⁶,³³ and N₂ in rats²⁶, with CO in dogs¹⁷, and with isoflurane in rats²⁴. The increase in activity could be a response to the odor of the gas as relative humidity and temperature were constant through the
experiment. However, while isoflurane and CO₂ are clearly associated with an unpleasant smell or sensation⁴⁵,⁴⁶, CO and N₂, which are odorless (and N₂ is even part of our breathing air)⁴⁷, also induced a similar activity increase. Thus, we can assume that the activity increase must be caused by not only the smell of the gas, but most likely by an unpleasant sensation.

High noradrenaline and ACTH levels are indicative of a fast stress response⁴⁸⁻⁵⁰. The noradrenaline levels were higher with CO and N₂, thus supporting that CO and N₂ are inducing more stress than CO₂ which is also suggested by increases in HR and MBP. Corticosterone levels were highly variable and not increased compared to baseline levels. It can be assumed that the time until sampling was too short to raise the animals' corticosterone levels, since it is known that corticosterone – compared to ACTH and noradrenaline - shows a delayed increase in response to stress⁴⁸,⁵⁰.

Moreover, we demonstrated that pulmonary lesions occurred with all euthanasia methods. It can be assumed that histologic signs of at least mild lung damage will be observed even with the most humane death, simply due to labored breathing or poor ventilation before death. In addition, all histologic reference data originate from euthanized animals and it has been shown that euthanasia can cause lung damage, including congestion of alveolar capillaries, hemorrhages, and intramural and perivascular edema of pulmonary arteries probably associated with alteration of vascular permeability²,⁷,⁹,¹²,⁵¹⁻⁵⁴. Therefore, the most relevant finding from our study is that both CO and N₂ caused more pronounced lung damage than CO₂.

From a biological point of view, death can be defined by the total and permanent cessation of all vital functions including the complete cessation of brain function⁵⁵. In our study, brain death always occurred faster than the cessation of cardiorespiratory functions. Compared to 70% CO₂, the time to isoelectric EEG was increased with 30% CO₂ displacement rates which is in agreement with data reported for 20% CO₂²⁸. For CO and N₂ the time to achieve EEG isoelectric was in between 30% and 70% CO₂. The cessation of breathing rapidly followed or was concomitant to EEG isoelectricity and was faster with 70% CO₂ and CO compared to 30% CO₂ and N₂. Finally, the time to asystole was longer for 30% CO₂ compared to CO, the other groups were in the middle, faster with CO than N₂ and 70% CO₂. No differences were found between the two CO₂ groups, given the variability within the 70% CO₂ group, there was a tendency towards the lower the CO₂ displacement rate, the longer the time until death, which is consistent with previous studies¹²,²⁸.

The ideal euthanasia gas should cause a fast LOC with minimal distress before. Additionally, potentially unpleasant, uncomfortable, or even painful sensations like breathlessness, arrhythmias or seizures should not occur before the animal loses consciousness. Ideally, the gas should have no (unpleasant) smell or other aversive sensations adding to distress before LOC. Furthermore, it can be assumed that experiencing hypoxia before LOC is causing distress as well.
CO and N₂ resulted in longer times to LOC, induced seizures before LOC, increased stress levels and caused higher lung damage. Therefore, CO and N₂ are no humane alternatives to CO₂ and should not be used for euthanasia of rodents alone (without prior sedation). For CO₂, the higher displacement rate provided a more rapid LOC, thus minimizing the time of consciousness in which distress and eventually pain can be experienced. Nevertheless, a higher amount of distress or even pain (due to gasping, arrhythmia, or seizures) experienced until LOC might outweigh the benefits of a faster LOC. Therefore, we rather cannot conclude that one CO₂ displacement rate was superior to the other, but believe that an additional effort should be made to better avoid unpleasant sensations before LOC. Our data provide additional evidence that CO₂ euthanasia can be further refined by adding previous anesthesia. Isoflurane induced a fast LOC and in contrast to others²¹,²³,⁵⁶ we did not observe adverse reactions. Therefore, a combination of CO₂ with anesthetic gases like isoflurane might be suitable to induce a more humane euthanasia by compensating the adverse effects of CO₂ and ensuring unpleasant events like gasping to occur definitively after LOC.

# Material and methods

## Animals

31 females and 27 males Sprague Dawley rats, 50-56 days old at the time of surgery were randomized in 5 groups: 30% CO₂, 70% CO₂, isoflurane, CO and N₂. 20 animals were used for isoflurane anesthesia and then euthanized with CO or N₂. Isoflurane anesthesia was performed 8 days post-surgery and euthanasia 15 days post-surgery, between 9:30am and 3pm. All experiments were according to Swiss Animal Welfare Laws and have been approved by the local authority (Kantonales Veterinäramt Zürich, ZH 129/2021).

## Surgery

Implantation of HD-S11 and HD-X02 transmitters (Data Science International (DSI), New Brighton, Minnesota, USA) was performed under isoflurane anesthesia and aseptic surgical conditions. Preoperatively, the following analgesic regime and fluid support was administered: 5 to 10 mL Ringer-Lactate solution, carprofen 5mg/kg, fentanyl 0.01 mg/kg, Emla-crème into the ears. A BP catheter was inserted into the abdominal artery and electrocardiogram (ECG) leads were placed at the left caudal rib and under the right pectoral muscle regions. On a stereotaxic frame, a craniotomy was performed using predefined coordinates (+ ve electrode: AP: +3.2mm, ML: +2.5mm ; -ve electrode: AP: -3.4mm, ML: +3.0mm). EEG leads were fixed to screws, secured and insulated with tissue adhesive and dental cement (Els Unibond and Els cem, SAREMCO dental). EMG leads were placed at the neck muscles. Postoperatively, animals were administered with 5 to 10 mL Ringer-Lactate solution and carprofen 5mg/kg, twice daily for 3 days.

## Euthanasia and anesthesia protocols
Pulmonary function was measured using a 3.48L whole-body plethysmography (WBP) chamber (Buxco®, DSI) and FinePointe 2.9. Each rat was acclimatized (baseline) for 15 minutes to the chamber using room air, then the euthanasia protocol was started according to the group: the chamber was ventilated with an air flow of 1.24 L/min for 30% CO₂ and 4.18 L/min for 70% CO₂, CO and N₂. We used concentrations of 100% CO₂, 100% N₂ and 8.6% CO in air. Isoflurane anesthesia was delivered in compressed air, data were recorded for 10 minutes before induction at a flow of 4.18L/min at 6% (4.2% of isoflurane) and maintenance at 2.1% (1.5% of isoflurane) after LOM. Media recorder 4.0 (Noldus) was used for video recordings. Two Logitech Brio 4K cameras were used per animal. The behavior was analyzed using BORIS 8.2. Ponemah 6.51 (DSI) was used to record and analyze telemetry data. EMG, EEG and blood pressure sample rates were set to 500 Hz. ECG was sampled at 1000 Hz.

## Whole body plethysmography data

Whole body plethysmography (WBP) data were filtered to remove aberrant values during baseline. RR was filtered for values under 90 breaths/minute and above 250 breaths/minute. TV was filtered for values above 3.5 mL. MV was filtered for values above 350 mL/minute. Cessation of breathing was defined as occurring when the tidal volume was inferior to 0.2 mL. Oxygen concentration in the WBP chamber was calculated as follows: for $\frac{dc}{dt} = -Q(\frac{c}{V})$, $c(t) = c_f + (c_i - c_f) * e^{-Q*t/V}$

With $c_i$ the initial oxygen concentration in air (20.95%), $c_f$ the final oxygen concentration for CO (19.15%) and isoflurane (19.65%) only, Q the air flow pushed into the WBP chamber set to 1.4 or 4.18 L/min and V the WBP chamber volume (3.48L).

## Telemetry data

The activity count is a relative measure used to determine the movement status of an animal in x and y relatively to the receiver plate, the number of counts generated is dependent on both distance and speed of movement. The EEG was defined as isoelectric when the threshold was similar to the death state of the animal (last seconds of recordings after the last ECG signal). Asystole was defined as the last ECG peak. For isoflurane, only LOM and LOC are reported as the animals were only anesthetized.

## Assessment of LOM and LOC

LOM was assessed by video recordings and defined as the last movement or position of the animal. LOC was assessed with the EEG wave profile and defined as the return of all waves under baseline levels (Fig. supplementary 6). Baseline levels were assessed for each animal individually and a time frame of 200 seconds was set as baseline.

## Stress hormones

Blood was collected by cardiac puncture after death or vena saphena in awake rats into heparinized collection tubes. Blood samples were centrifuged at 1500g for 15 minutes at 4°C and plasma was stored at -80°C until analysis. The following assays were used: Mouse/Rat ACTH ELISA Kit (ab263880, Abcam, UK) at a 1:7 dilution, Corticosterone ELISA Kit (ADI-900-097, Enzo Life Sciences Inc, USA) at a 1:40
dilution after steroid replacement treatment, and a Noradrenaline High Sensitive ELISA Assay Kit (NOU39-K01, Eagle Biosciences Inc, USA) at a dilution of 1:14.

## Histology

Immunohistochemistry was performed using the horseradish peroxidase (HRP) method to detect Albumin⁵⁷. After deparaffination, sections underwent antigen retrieval in citrate buffer (pH 6.0) for 20 min at 98°C, followed by incubation with polyclonal goat anti-albumin antibody (A80-129A, ThermoFisher Scientific, Carlsbad, CA, USA) at 1:600 (diluted in dilution buffer, Agilent Dako, Santa Clara, CA, USA) overnight at 4°C. This was followed by blocking of endogenous peroxidase (peroxidase block, Agilent Dako) for 10 minutes at room temperature and incubation with HRP-conjugated polyclonal rabbit anti-goat IgG (ab6741, Abcam, Cambridge, UK) at 1:100 for 30 min at room temperature and followed by 3,3'-diaminobenzidine (DBA) in an autostainer (Dako Agilent or Ventana, Oro Valley, AZ, USA). Sections were subsequently counterstained with hematoxylin.

The morphometric analysis on hematoxylin and eosin stained sections served to quantify the area in each lung section occupied by erythrocytes, as an estimation of hemorrhages and congestion, and by air spaces⁵⁸, as an estimation of lung inflation. For each section, the lung was manually outlined and annotated as a region of interest (ROI), manually excluding artefactually altered areas. An APP (Analysis Protocol Package), based on a threshold method, was designed in Visiopharm software (Version 2023.01.3.14018; Hoersholm, Denmark) and run on each ROI to measure its total area (µm²) as well as the area of tissue classified as erythrocytes or airspace (classified area; µm²). The percentage of positive area, expressed as the ratio between the classified area and the total area, was obtained for each animal according to the following formula: ([classified area (µm²)]/[total area (µm²)]) × 100.

Sections stained with immunohistochemistry for albumin served to assess the perivascular and intramural edema of the pulmonary arteries by measuring the perivascular and intramural albumin (serum) stained area in pulmonary arteries. For each animal, the lumens of 10 randomly selected transversally sectioned pulmonary arteries were manually outlined and an APP, based on a threshold method, was used to measure the intraluminal albumin-positive area. To measure the albumin-positive area in the vessel wall and perivascular area, the lumen outline was enlarged by 50 pixels at 1× (230 microns) and the albumin-positive area was assessed with the APP. The intramural and perivascular albumin-positive area was assessed as the difference between the perivascular albumin-positive area and the intraluminal albumin-positive area. The percentage of positive area, expressed as the ratio between the intramural and perivascular albumin-positive area and the total perivascular area, was obtained for each animal according to the following formula: ([intramural and perivascular albumin-positive area (µm²)]/[total perivascular area (µm²)]) × 100 (Supplementary Fig. 8).

## EEG data processing

EEG spectrogram was computed on a moving time window of 4s and for 1024 frequencies logarithmically scaled between 1 and 150Hz. To obtain the time course of the power by frequency band,
the absolute value of the log-spectrogram was then summed over frequencies for each of the following frequency bands: delta (1-4Hz), theta (4-8Hz), alpha (8-12Hz), beta (13-30Hz), gamma (30-80Hz).
Relative percentage of power for each band was then computed by dividing the value for each band by the sum for all bands. To normalize EEG relative to pre-induction (Baseline) values, we also used the log10 ratio of the power band to the power in the baseline activity (pre-induction time interval was [-200 0]s before the induction). In Supplementary Fig. 7, power band was considered in the following time intervals: pre-induction [-60:0]s, post-induction [0:60]s, pre-LOM [LOM-60:LOM]s and post-LOM [LOM:LOM + 60]s.

# Data analysis

To analyze EEG data, we used Neuroscore 3.4.0 including the seizure detection module and Matlab R2023a. Statistics were performed with GraphPad Prism 10.1.1 using the appropriate test for each type of data (e.g. ANOVA for normally distributed data) and post-hoc tests. Statistical significance is indicated in the figures as follows: ns. not significant; \* p < 0.05; \*\* p < 0.01; \*\*\* p < 0.001, \*\*\*\* p < 0.0001. The following abbreviations are used 2w-ANOVA for Two-way-ANOVA, 1w-ANOVA for One-way-ANOVA. Graphics were generated using Matlab R2023a, R 4.3.1, Biorender and GraphPad Prism 10.1.1.

# Declarations

## Author contributions

C.C conceived, designed and performed experiments (surgeries, anesthesia, euthanasia, organ collection), analyzed the data (ECG, EEG, WBP, behavior, statistics) and wrote the manuscript.

P.V performed experiments (anesthesia, euthanasia, organ collection, ELISA) and analyzed data (ECG, behavior, ELISA).

F.P performed experiments (organ collection, histopathology) designed the APPs for morphometry and analyzed data (histopathology).

T.B performed experiments (surgeries).

F.G performed experiments (organ collection).

C.T.R obtained funding.

B.G wrote the Matlab code for EEG analysis and edited the manuscript.

C.A.W designed experiments, obtained funding, edited the manuscript.

P.S conceived and designed experiments, obtained funding, and edited the manuscript.

All authors saw and approved the final manuscript.
## Funding

The study was funded by the Swiss Federal Food Safety and Veterinary Office (FSVO): Humanely ending the life of laboratory rodents (2.21.03TSCH).

## Acknowledgments

We would like to thank the DSI company, in particular Cristina Baciu, for their support and Meredith Halbeisen for the verification of behavior annotations.

## Competing interests

The authors declare no competing interests.

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## Table

Table 1 is available in the Supplementary Files section

## Figures
<figure><img src="image_3.png" /><figcaption>Fig. 1: Cardiorespiratory parameters in rats before and after exposure to different gases. Cardiorespiratory parameters over time plotted for 30% CO₂ (blue, n=18-19), 70% CO₂ (black, n=18-19), isoflurane (pink, n=18-20), CO (yellow, n=10) and N₂ (green, n=9-10): a) Heart rate (HR) derived from the blood pressure, b) mean blood pressure (MBP), c) respiratory rate (RR), d) tidal volume (TV) and e) minute volume (MV). The shaded areas show the mean ± SEM of each parameter as a function of time. The average at baseline was estimated at the level shown in the red dashed line. For the sake of readability, when values of two groups during the baseline are close, only one red dashed line is represented. The dotted lines represent the gas influx at time point 0. The arrows represent the time to loss of motion (LOM) for each group.</figcaption></figure>

Figure 1

See image above for figure legend.
<figure><img src="image_4.png" /><figcaption>Fig. 2: Histopathological examination of the lungs after euthanasia with CO₂, CO or N₂. a and b) Subgross 1.25× (a) and 10× (b) picture of hematoxylin and eosin-stained lung sections showing changes in air content; c) Hematoxylin and eosin-stained lung sections showing congestion. The scale bars are 1 mm. Magnification: 20×. d) Lung sections stained for albumin (brown) with immunohistochemistry showing perivascular and intramural edema. Magnification: 20×. Scale bars depict 100 µm. e) Comparison of 70% CO₂ (black, n=10-14), 30% CO₂ (blue, n=10-12), CO (yellow, n=10) and N₂ (green, n=10) after morphometric quantification of air spaces, number of erythrocytes and perivascular and intramural edema spaces. Results are plotted as medians ± range (Kruskal-Wallis tests). Statistical significance is indicated as follows: * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001.</figcaption></figure>

Figure 2

See image above for figure legend.
<figure><img src="image_5.png" /><figcaption>Fig. 3: Responses to different gas influx in rats. Rats were euthanized with 70% CO₂ (black, n=19), 30% CO₂ (blue, n=15), CO (yellow, n=10) or N₂ (green, n=10) or anesthetized with isoflurane (pink, n=20). a) Sniffing duration adjusted to duration per minute and compared to baseline (grey, n=34). b) Total sniffing duration until loss of motion (LOM). c) Circling duration adjusted to duration per minute and compared to baseline. d) Total circling duration until LOM. e) Total number of gasping events (before or after LOM). f-j) Activity counts average over time recorded from ECG transmitter for all groups: 70% CO₂ (black, n=19), 30% CO₂ (blue, n=19), isoflurane (pink, n=20), CO (yellow, n=10) and N₂ (green, n=10). The smooth line represents the estimated activity trend for each gas. The dotted line, at time point 0, represents the gas influx. The arrow represents the LOM averaged for each group. a-e) Statistical analysis was performed with Kruskal-Wallis tests with Dunn's multiple comparisons tests. Values are plotted as medians ± range. Statistical significance is indicated as follows: * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001.</figcaption></figure>

Figure 3

See image above for figure legend.

<figure><img src="image_6.png" /><figcaption>Fig. 4: Assessment of physiological functions during euthanasia or anesthesia with different gases. Animals were attributed to one of the following groups: 70% CO₂ (black, n=16-19), 30% CO₂ (blue, n=15-19), isoflurane (pink, n=17-20), CO (yellow, n=9-10) or N₂ (green, n=8-10). a) For each gas, the times until loss of motion (LOM) (1), loss of consciousness (LOC) (2), isoelectric electroencephalogram (EEG) (3), cessation of breathing (4) and asystole-death (5) are plotted. b) Mean times ± standard deviations for each gas until LOM, LOC, isoelectric EEG, cessation of breathing and asystole.</figcaption></figure>

Statistical analysis was performed with 1-way-ANOVA with Tukey's multiple comparisons tests.

Statistical significance is indicated as follows: * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001.

Times to LOM and LOC within each group are not significantly different (unpaired t-tests, p>0.23).

<figure><img src="image_7.png" /></figure>
Figure 4

See image above for figure legend.

<figure><img src="image_8.png" /></figure>

Fig. 5: Seizures events in rats after exposure to carbon monoxide (CO) and nitrogen ($N_2$). a) EMG and EEG data after exposure to $N_2$ (green, n=9), insets on the EEG periodogram over 1 epoch (10 seconds) and EEG spikes during a seizure episode. b) EMG and EEG data after exposure to CO (yellow, n=9), inset on the EEG periodogram over 1 epoch (10 seconds) and EEG spikes during a seizure episode. c) total number of spikes, number of spikes per second and duration of seizures during exposure to CO and $N_2$. The seizure quantification was performed with the seizure module in Neuroscore (DSI). Statistical analysis was performed with Wilcoxon tests. Values are plotted as medians ± range. Statistical significance is indicated as follows: \* p < 0.05; \*\* p < 0.01; \*\*\* p < 0.001; \*\*\*\* p < 0.0001.

Figure 5

See image above for figure legend.
<figure><img src="image_9.png" /><figcaption>Fig. 6: Stress parameters measured in the blood of rats after euthanasia with 70% CO₂, 30% CO₂, CO or N₂. a) Hypothalamic–pituitary–adrenal axis implicated in the release of adrenaline, noradrenaline and corticosterone by the adrenal glands in response to stress (created on Biorender). b-d) noradrenaline, ACTH, and corticosterone levels after euthanasia with 70% CO₂ (black, n=16-18), 30% CO₂ (blue, n=16-18), CO (yellow, n=10) or N₂ (green, n=10) and compared to baseline levels (grey, n=13-17). CRH = Corticotropin-releasing hormone. ACTH = Adrenocorticotropic hormone. Statistical analysis was performed with Kruskal-Wallis with Dunn's multiple comparisons tests. Values are plotted as medians ± range. Statistical significance is indicated as follows: * p< 0.05; ** p< 0.01; *** p< 0.001, **** p< 0.0001.</figcaption></figure>

Figure 6

See image above for figure legend.

**Supplementary Files**

This is a list of supplementary files associated with this preprint. Click to download.

* GraphicalAbstract.png

* Supplementaryfigures.docx

* Table1.docx