Introduction

With the development of modern biology and medicine, animal experimentation has become an important means for people to understand the laws of life processes. Gradually, issues concerning animal ethics and welfare have attracted increasing attention1,2. Treating experimental animals roughly can cause them to be in an abnormal state of psychology and physiology, and the resulting stress reaction can interfere with the credibility of experimental data or results3. In the field of animal experimentation, ensuring the welfare of experimental animals, minimizing their suffering, and maintaining the rigor and reliability of experimental data have become an important responsibility and mission for researchers.

Currently, the immobilization methods used in animal experiments are mainly targeted at mammals4, while there are relatively few studies on immobilization and sacrifice methods for reptiles5. Turtles and dinosaurs both lived during the Mesozoic Era6, turtles are a major group within reptiles. Currently, an increasing number of scholars are conducting in-depth studies on this group, some of which involve the immobilization and sacrifice of turtles. Among these methods, “direct decapitation7,8” and “ether immobilization9” are the primary means of sacrifice, while other approaches include “cryogenic freezing10” and “injecting anesthetics11”. Turtles are typically capable of living both on land and in water, and they possess a certain ability to survive under hypoxic conditions12. There is still debate on whether inhalation immobilization is an appropriate method. Additionally, the American Veterinary Medical Association (AVMA) Guidelines for the Sacrifice of Animals, as stated in the 2013 edition, indicate that hypothermia is an unacceptable method for reptile immobilization or sacrifice13. However, some scientists have suggested that hypothermia can also be used for reptile immobilization without causing significant pain to the reptiles14,15. But cryoimmobilization may cause animals to experience unnecessary pain and stress. For example, cryoimmobilization may cause animals to shiver and have their hair stand on end, which is a sign of a drop in core body temperature and may compromise patient safety, especially in older and febrile animals. In addition, cryoimmobilization may need to be used repeatedly, which increases the risk of injury to the animal and may not be in line with the ‘3Rs’ (Replacement, Reduction, Refinement) principles of animal experimentation.

However, among the various methods of sacrificing animals using immobilization, there is seldom any detailed discussion regarding the implementation of the immobilization process, the selection of anesthetic agents, or the recommended dosages. Only Lutvikadic, in 2024, investigated the efficiency of ketamine and medetomidine at two different doses and via two different routes on anesthesia depth and cardiac stability in red-eared sliders5. The lack of research surrounding the animal welfare implications of allowing turtles to die and the subsequent effects on their biological systems remains a gap in current knowledge. When experimental animals are sacrificed, it can induce varying degrees of stress responses5,16. This is a reactive process of the animal’s body to external stimuli, an adaptive mechanism, but it can lead to a series of functional and metabolic changes within the animal’s body17.

Therefore, we have chosen the red-eared sliders (Trachemys scripta elegans) as our subject of study to investigate the effects of various Sacrifice methods on corticosterone (CORT), adrenocorticotropic hormone (ACTH), and markers reflecting brain (serotonin (5-HT), dopamine (DA)), heart (creatine kinase (CK), cardiac troponin (cTn), lactate dehydrogenase (LDH)), and liver (acetylcholinesterase (AchE), aspartate aminotransferase (AST), alanine aminotransferase (ALT)) functions. This research strives to enhance and refine the ethical welfare standards in turtle animal experiments, ensuring that scientific pursuits are conducted with due consideration for animal welfare and rights. By systematically evaluating the impact of different sacrifice methods on the physiological status of red-eared sliders, we aim to gain a more precise understanding of their stress responses during experimentation, enabling the optimization of experimental procedures and minimization of potential harm to the animals. Additionally, this study provides valuable references for subsequent molecular biology and blood biochemistry research on turtles and accumulates experience for other reptile-related research.

Materials and methods

Experimental animals and acclimation

Red-eared sliders were purchased from Hongwang Turtle Farm in Dongshan Town, Haikou, and housed in a 107 cm × 77 cm × 30 cm tank within an ecological garden. Before the experiment, they were fed every 4 days with a ration equal to 2% of their body weight, and the water was changed within 24 h after feeding. A 2-weeks acclimation period was implemented to ensure the turtles were fully adapted to the experimental conditions. Seventy-eight healthy individuals with an average weight of 302.94 ± 22.96 g were randomly assigned to 13 groups, including decapitation sacrifice, the − 20 °C cryoimmobilization sacrifice (divided into 45 min, 60 min, 75 min groups), the pentobarbital sodium intraperitoneal injection immobilization sacrifice group (divided into 0.02 mg/g, 0.025 mg/g, 0.03 mg/g concentration groups) and the pentobarbital sodium intramuscular injection immobilization sacrifice group (divided into 0.02 mg/g, 0.025 mg/g, 0.03 mg/g concentration groups), and the ether inhalation immobilization sacrifice group (divided into 60 min, 90 min, 120 min groups), with six turtles in each group, ensuring no significant difference in body weight among the groups (P > 0.05). During the experiment, the conditions were kept the same as those during domestication. Before the experiment, 12 red-eared turtles were randomly selected and blood was drawn from the brachial venous sinus before death. During the experiment, blood was drawn via decapitation and centrifuged at 4 °C, 3000 r/min for 10 min to obtain plasma for standby use.

Sacrifice methods and indicator measurement

In our study, we selected six red-eared turtles from the Venous blood collection group for blood collection. We referred to the Guidelines for Blood Collection for Common Laboratory Animals by the IQ 3Rs Leadership Group, as well as the blood collection methods from Diehl18, the Laboratory Animal Center of Nankai University and Zhejiang University. We controlled the single blood collection volume to within 15% of the animal’s total blood volume (3 ml, 12.5%). When experimental samples are sufficient, to better safeguard the health and welfare of the animals, it is recommended to keep the blood collection volume below 10%. Approximately 3 ml of venous blood was drawn from each red-eared turtle. In the decapitation group, post-decapitation, blood was extracted directly from the neck. For the − 20 °C cryoimmobilization group, we established three cohorts of red-eared turtles, each comprising six individuals, with freezing times set at 45, 60, and 75 min (Fig. 1A). The procedure involved placing each cohort of turtles into a freezer, covering them with pre-cooled crushed ice, leaving only their heads exposed, and ensuring the freezer door remained closed until the specified freezing duration had passed. Once the − 20 °C cryoimmobilization was complete, the turtles were immediately removed and decapitated to collect blood from the neck area. Additionally, we prepared a pentobarbital sodium solution at a concentration of 5 mg/ml by mixing 0.1 g of pentobarbital sodium with 20 ml of a 0.65% saline solution. The animals were then allocated into groups based on three dosage gradients: 0.02 mg/g, 0.025 mg/g, and 0.03 mg/g for intraperitoneal or intramuscular injection. Fresh anesthetic solutions were prepared immediately before the experiment and administered according to the body mass of each turtle and the selected dosage gradient. The administration was guided by the following formula:

$${\text{The}}\;{\text{ injection}}\;{\text{ volume}} = {\text{turtles }}\;{\text{weight }}\left( {\text{g}} \right) \times {\text{gradient }}\left( {{\text{mg}}/{\text{g}}} \right) \div {5 }({\text{mg}}/{\text{ml}})$$
Fig. 1
figure 1

Sacrifice methods to the different groups of red-eared sliders. (A) − 20 °C cryoimmobilization sacrifice; (B) the pentobarbital sodium intraperitoneal injection immobilization sacrifice; (C) the pentobarbital sodium intramuscular injection immobilization sacrifice; (D) the ether inhalation immobilization sacrifice.

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Prefemoral fossa injection (Fig. 1B,C). After the injection is completed, the turtle is placed in a transparent acrylic box, and the surrounding environment is kept quiet. The state of the red-eared slider is observed, and its limbs are pricked with a needle. Once the red-eared slider shows no response at all, it is immediately decapitated, and blood is collected from the neck area. We prepared a transparent acrylic box with a lid for the experiment. Inside the box, we positioned two small beakers, each filled with cotton balls, and secured their bases using a hot glue gun to prevent movement. We then introduced ether into the beakers to create a vaporizing environment. After adding the ether, we sealed the box tightly to allow the vapor to diffuse throughout the interior (Fig. 1D). For the ether inhalation, we established three durations: 60, 90, and 120 min. For each duration, we placed six red-eared sliders into the box. Upon completion of the ether inhalation period, we promptly removed the sliders, performed decapitation, and collected blood from the neck area. To isolate the plasma, these blood samples were centrifuged at 3000 r/min for 10 min.

Subsequently, ELISA kits were used to measure stress-related indicators such as 5-hydroxytryptamine (5-HT), dopamine (DA), corticosterone (CORT), and adrenocorticotropic hormone (ACTH). Functional indicators such as creatine kinase (CK), cardiac troponin (cTn), lactate dehydrogenase (LDH), acetylcholinesterase (AchE), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were measured using biochemical methods, with all procedures strictly following the manufacturer’s instructions.

Data processing and analysis

The experimental data underwent normality assessment with the Kolmogorov–Smirnov (K-S) test and homogeneity of variance evaluation with the F-test. Both tests yielded P-values exceeding 0.05, indicating that the data met the assumptions of normality and variance homogeneity. Subsequently, a one-way analysis of variance (ANOVA) was employed for statistical analysis. If the calculated F-value surpasses the critical value, the least significant difference (LSD) method is then applied for multiple comparisons among groups. A P-value below 0.05 for the LSD test signifies statistically significant differences between the groups.

Ethics approval

The animal study protocol was approved by the Animal Ethics Committee of the Hainan Ecological Environment Education Centre (No. HNECEE-2023-005). All methods are reported in accordance with ARRIVE guidelines (https://arriveguidelines.org) for the reporting of animal experiments.

Result

Approximately 3 ml of venous blood is drawn from the brachial venous sinus of the red-eared slider to measure 10 biochemical indicators in the blood. These indicators reflect the levels of stress hormones and blood biochemical indicators under normal conditions of the red-eared slider, providing a certain degree of reference.

Effects of different immobilization methods on CORT and ACTH

The effects of different immobilization methods on the levels of CORT and ACTH in red-eared turtles are shown in Table 1. There were no significant differences in CORT levels among all groups. The ACTH level in the ether inhalation immobilization 90 min group was significantly lower than that of the venous blood collection group, the − 20 °C cryoimmobilization 75 min group, the ether inhalation immobilization 60 min group, the ether inhalation immobilization 120 min group, the − 20 °C cryo-immobilization 45 min group, and the 75 min group. The ACTH level in the − 20 °C cryo-immobilization 75 min group was significantly higher than that of the pentobarbital sodium intraperitoneal injection immobilization 0.025 mg/g group and the pentobarbital sodium intramuscular injection immobilization 0.025 mg/g group. There were no significant differences in ACTH levels among the other groups.

Table 1 Comparison of stress levels of Trachemys scripta elegans under different death methods.
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Effects of different immobilization methods on brain function indicators in red-eared turtles

The effects of different immobilization methods on the levels of 5-HT and DA in red-eared turtles are shown in Table 2. The 5-HT content of the pentobarbital sodium intraperitoneal injection immobilization 0.025 mg/g group was significantly lower compared to the 0.02 mg/g group and the − 20 °C cryoimmobilization 60 min group. Similarly, the 5-HT content of the pentobarbital sodium intramuscular injection immobilization 0.025 mg/g group was significantly lower than that of the pentobarbital sodium intraperitoneal injection immobilization 0.02 mg/g group. Regarding dopamine (DA) levels, the venous blood collection group exhibited significantly lower DA levels compared to the pentobarbital sodium intraperitoneal injection immobilization 0.03 mg/g group, as well as the − 20 °C cryoimmobilization 45 min and 60 min groups. Additionally, the DA level in the group where turtles were sacrificed by decapitation was significantly lower than those in the pentobarbital sodium intraperitoneal injection immobilization 0.03 mg/g group and the − 20 °C cryoimmobilization 45 min and 60 min groups. Lastly, the DA level in the ether inhalation immobilization 90 min group was significantly lower than those in the pentobarbital sodium intraperitoneal injection immobilization 0.03 mg/g group and all the − 20 °C cryoimmobilization groups.

Table 2 Comparison of brain function indexes of Trachemys scripta elegans under different death methods.
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Effects of different immobilization methods on cardiac function indicators in red-eared turtles

The effects of different immobilization methods on the levels of CK, cTn and LDH in red-eared turtles are shown in Table 3. The CK level in the ether inhalation immobilization 90 min group was significantly lower than that in the decapitation group, t pentobarbital sodium intraperitoneal injection immobilization 0.025 mg/g and 0.02 mg/g group, all the pentobarbital sodium intramuscular injection immobilization group, the ether inhalation immobilization 120 min group, and the − 20 °C cryoimmobilization 60 min and 90 min groups. No significant differences were observed in the cTn levels between all groups. The LDH level in the − 20 °C cryoimmobilization 60 min group was significantly lower than that in the pentobarbital sodium intramuscular injection immobilization 0.025 mg/g and 0.02 mg/g group, as well as the − 20 °C cryoimmobilization 45 min group.

Table 3 Comparison of cardiac function indexes of Trachemys scripta elegans under different death methods.
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The impact of different anesthetic methods on liver function indices in red-eared turtles

The effects of different immobilization methods on the levels of AchE、AST and ALT in red-eared turtles are shown in Table 4. There were no significant differences in AchE indices among all groups. The AST level in the pentobarbital sodium intraperitoneal injection immobilization 0.02 mg/g group was significantly lower than that in the pentobarbital sodium intramuscular injection immobilization 0.03 mg/g group. The AST level in the pentobarbital sodium intramuscular injection immobilization 0.03 mg/g group was significantly higher than that in the ether inhalation immobilization 60 min group. The ALT level in venous blood samples was significantly higher than that in the − 20 °C cryoimmobilization 45 min and 60 min groups. The ALT level in the pentobarbital sodium intraperitoneal injection immobilization 0.03 mg/g group was significantly higher than that in the pentobarbital sodium intraperitoneal injection immobilization 0.02 mg/g group, as well as the − 20 °C cryoimmobilization 45 min and 60 min groups. The ALT level in the − 20 °C cryoimmobilization 45 min groups was significantly lower than that in the ether inhalation immobilization 60 and 90 min groups.

Table 4 Comparison of liver function indexes of Trachemys scripta elegans under different death methods.
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Discussion

Although pentobarbital sodium has a relatively long anesthetic maintenance time, according to the literature, it has a strong inhibitory effect on the respiratory center, which can easily cause laryngeal spasms and bronchial spasms, leading to the death of animals19. On the other hand, ether immobilization is known to have a significant stimulatory impact on the respiratory tract, increasing mucus secretion, which poses a significant risk of asphyxia and hypoxia for turtles, triggering hypoxic stress20,21. Freezing immobilization at − 20 °C can induce acute cold stress, causing myocardial hypoxia, ischemia, and consequently, impairment of cardiac function. This is corroborated by Yu’s study, which demonstrated acute cold stress-induced cardiac dysfunction in experimental dogs22. Furthermore, Cha Sen’s research revealed that cold stress can detrimentally impact liver function in grazing beef cattle23. The varying immobilization treatments employed exhibit diverse degrees of impact on animals. Currently, there is no unified standard for anesthetizing turtles, as different researchers use different methods. The impact of various immobilization methods on turtles is unknown, and each immobilization method may affect the brain, heart, liver, and other related physiological and biochemical indicators to a certain extent.

When an animal faces stress, the hypothalamus–pituitary–adrenal axis (HPA axis) is activated through the paraventricular nucleus of the hypothalamus, releasing corticotropin-releasing hormone (CRH) to the adenohypophysis (anterior pituitary). CRH binds to receptors on the adenohypophysis to stimulate the secretion of adrenocorticotropic hormone (ACTH), which then travels through the blood circulation to reach the adrenal cortex cells. ACTH binds to receptors on the adrenal cortex cells, promoting the secretion of glucocorticoids24. Corticosterone is a type of glucocorticoid, thus the change directions of ACTH and corticosterone are the same. It takes several minutes for the levels of ACTH and corticosterone in the blood to rise, and they can remain elevated for a relatively long period. In this experiment, the rapid decapitation method may not allow enough time for ACTH and corticosterone levels to increase in the blood. Other anesthetic euthanasia methods, however, can take longer, potentially exceeding the duration of the elevated ACTH and corticosterone levels, meaning that as the body gradually enters a state of immobilization, the stress hormone levels return to normal.

The rapid decapitation method is quick in operation and does not cause significant changes in functional indicators such as the brain, heart, and liver. Therefore, selecting the indicator levels under this method as the baseline has a high degree of credibility. There were no significant differences in brain, heart, and liver function indicators between the pentobarbital sodium intramuscular injection immobilization sacrifice group and decapitation sacrifice group. When compared to the decapitation sacrifice group, there were significant differences in brain, heart, and liver function indicators in the ether inhalation immobilization 90 min group, the pentobarbital sodium intraperitoneal injection immobilization 0.03 mg/g group, − 20 °C cryoimmobilization 45 and 60 min group. This is consistent with Simmons’ 1975 study, which verified through experiments on mice that ether immobilization can alter enzyme activities in mice25. Similarly, the difference between the − 20 °C cryoimmobilization 45 and 60 min groups and the decapitation sacrifice group may be caused by cold stress, as some studies have shown that cold stress can change the dopamine level in mice’s bodies, causing it to rise to adapt to the cold26. Wang’s 2020 study on mice demonstrated that pentobarbital sodium intraperitoneal injection immobilization can cause pain in mice, and this pain can lead to changes in DA levels27.

When the degree of stress reaction caused by different immobilization and sacrifice methods, and the level of difference in functional indicators such as brain, heart, and liver, are not significant, we should adhere to the principles of minimizing immobilization duration, minimizing immobilization dosage, and minimizing animal discomfort. We believe that although decapitation is widely used in animal experiments related to turtle research, it is cruel due to the lack of measures to ensure animal welfare, thus it is not suitable as a method of immobilization and sacrifice in animal experiments. − 20 °C cryoimmobilization takes a long time, and long-term cold stress occurs before the state of immobilization is reached, which can lead to abnormal physiological indicators and discomfort in animals. In experiments, we observed that red-eared turtles struggled and escaped under the cover of crushed ice, therefore, it is not suitable as a method of immobilization and sacrifice in animal experiments. Ether is a stimulating gas that may cause damage to the respiratory tract, and turtles can hold their breath, resisting the anesthetic effect of ether to a certain extent, which can prolong the immobilization time and make the effect unstable. Meanwhile, long-term experiments conducted in a closed box may cause hypoxia stress in red-eared turtles, so it is not suitable as a method of sacrifice in animal experiments. Pentobarbital sodium injection immobilization may cause laryngeal and bronchial spasms leading to animal death, but none of the immobilization gradients used in this experiment resulted in the death of red-eared turtles. During the immobilization process, the operation is relatively simple and fast, with a short induction period, long immobilization period, and good immobilization effect, making it a viable option for immobilization and sacrifice in animal experiments. However, it is worth noting that injecting medication into red-eared turtles may cause contamination of certain tissues and organs in the body, which requires judgment and selection based on the needs of the research. Our findings have significant implications for immobilization methods in turtles and reptiles, as we have provided a method that minimizes potential harm to the animals. Of course, physiological and metabolic differences that may exist between species could affect their response to anesthetic drugs. In the future, we plan to broaden the range of species studied to provide more extensive guidance for clinical practice.

Conclusion

Among various methods of immobilization and sacrifice for turtles, injecting pentobarbital sodium solution is a worthy consideration. Intramuscular injection has a shorter induction immobilization period compared to intraperitoneal injection, and it provides more accurate functional indicators. The recommended injection dose is 0.02 mg/g. Our findings advocate for a humane and effective approach to anesthesia that aligns with the ethical considerations of animal welfare. This study has not only contributed a valuable reference for future anesthetic practices in turtles and amphibians but also holds significant instructional value in the field of veterinary anesthesia.