Euthanasia of fish is a common procedure and may be indicated for a variety of reasons, including poor quality of life or individual health concerns, as an endpoint for laboratory or field research, or for the purpose of zoo or aquarium population management. By definition, euthanasia must be rapid and result in minimal pain, stress, and distress. Current guidelines published by the AVMA1 recommend both chemical and physical methods for finfish euthanasia. Chemical methods involve injecting an overdose of anesthetic directly into the fish (eg, into a vessel, muscle, or body cavity) or immersing the fish in a highly concentrated anesthetic solution for uptake by the gills, known as immersion euthanasia. There are currently no FDA-approved drugs for immersion euthanasia of aquatic species; however, several are considered acceptable by the AVMA.1 Among them, tricaine methanesulfonate (MS-222) is one of the most commonly used. It is currently the only FDA-approved anesthetic for use in food fish2 and, thus, is widely used as an immersion anesthetic and euthanasia agent in US aquaculture.3,4 In light of the 21-day withdrawal period following its use, TMS is not recommended for euthanasia of fish intended for human consumption. Tricaine methanesulfonate is classified as a local anesthetic and blocks sodium channels in the peripheral and central nervous systems, decreasing neuronal activity.5 However, despite its wide use as an anesthetic, the exact mechanism by which it produces general anesthesia is currently unknown.6 Similarly, the mechanism by which it causes death in fish has not been evaluated, although TMS has been suggested to cause death as a result of decreased nervous and cardiovascular function.1 Although current AVMA euthanasia guidelines recommend that, for immersion euthanasia, “finfish should be left in the anesthetic solution for a minimum of 10 minutes after cessation of opercular movement” and state that a TMS “concentration of 250 to 500 mg/L, or 5 to 10 times the anesthetic dosage, is effective for most species,”1 pilot research in goldfish (Carassius auratus) conducted by the authors of the report presented here questioned this recommendation.
Fish euthanasia is conventionally performed by individuals in the aquatic sector. However, because a tremendous number of fish are maintained as companion pets in US households,7 veterinarians in other sectors, especially those in companion animal practice, may also be requested to perform euthanasia of fish. Despite the widespread use of TMS in aquatic medicine, the product is unlikely to be in the pharmaceutical inventories of general veterinary practices. Thus, alternative methods of immersion euthanasia, particularly for use by companion animal veterinarians, are needed. Propofol, a γ-aminobutyric acid receptor agonist, is widely used as an anesthetic agent in mammalian species8 and is likely to be available in traditional companion animal veterinary practice settings. Propofol's relative accessibility and proven anesthetic efficacy via immersion in fish species,9–11 including goldfish,12 make it a promising immersion euthanasia agent for fish. Because the recommended TMS concentration for immersion euthanasia is 5 to 10 times the concentration used for immersion anesthesia,1,4 the authors speculated that a similar strategy of using propofol at 5 times its immersion anesthesia concentration would be a reasonable alternative for immersion euthanasia of fish.
Determining the success or failure of a euthanasia technique in a clinical setting is contingent on identifying reliable and consistent indicators of systemic cellular death. In mammalian species, cessation of systemic delivery of oxygenated blood results in myocardial failure and global cerebral death within minutes. Thus, markers of respiratory and cardiac arrest in mammalian species are strongly correlated with and used as surrogate indicators for systemic cellular death. In contrast, fish have developed strategies for coping with oxygen deprivation and have evolved anoxia-tolerant tissues, including the myocardium and higher brain centers (telencephalon).13–15 Although the physiology allowing for continued myocardial work and preservation of brain function in oxygen-depleted environments has evolutionary advantages, it challenges the use of respiratory arrest (ie, cessation of operculation) as an indicator of systemic cellular death in fish. Likewise, the hypoxia tolerance of the myocardium in fish may also limit the use of cardiac function as a marker of systemic cellular death. Further, numerous anecdotal reports exist of fish hearts continuing to contract following chemical euthanasia or even physical removal of the organ from the body.16 Although continued myocardial contraction following fish death is a widely accepted phenomenon in the aquatic medicine community, information on the duration of isolated myocardial contraction following immersion euthanasia in fish has not been published to the authors' knowledge.
The objectives of the study reported here were to substantiate current AVMA guidelines1 for immersion euthanasia of goldfish with TMS, determine whether immersion in propofol at 5 times its immersion anesthesia concentration for 30 minutes would be sufficient for euthanasia of goldfish, and quantify the duration of myocardial contractions following immersion of goldfish in TMS at a concentration currently recommended for euthanasia followed by decapitation.
The authors hypothesized that 15 minutes of immersion in TMS at a concentration of 500 or 1,000 mg/L or 30 minutes of immersion in TMS at a concentration of 500 mg/L would be sufficient to euthanize all exposed goldfish, that 30 minutes of immersion in propofol at a concentration of 25 mg/L would be sufficient to euthanize all exposed goldfish, and that 15 minutes of immersion in TMS at a concentration of 500 mg/L followed by decapitation would terminate myocardial contractions in less than 10 minutes in all exposed goldfish.
Materials and Methods
Animals and husbandry
Thirty-six adult red comet goldfish, approximately 10 to 15 cm in length, weighing 20 to 40 g (0.7 to 1.4 oz), and of unspecified sex, were obtained from a commercial fish hatchery.a Fish were housed in a 284-L (75-gallon) tank equipped with a mesh cover, sponge filter, air stone, drain, and calibrated digital thermometer. The room was maintained on a 12-hour photoperiod, and the tank water temperature was allowed to equilibrate with the climate-controlled room temperature (23.1° to 23.3°C [73.6° to 74.0°F]). Daily water quality assessments were made, and 50% water changes were conducted as needed to maintain water quality within the following parameters: pH between 6.9 and 7.4, total ammonia nitrogen concentration ≤ 0.5 mg/L, and nitrite concentration ≤ 0.04 mg/L. Commercial aquarium salt was used to maintain water salinity between 2 and 3 parts per thousand (2 to 3 g/L). Fresh water was obtained from a reservoir of city tap water treated with sodium thiosulfate and allowed to incubate for a minimum of 3 weeks. Dissolved oxygen content of > 5 ppm (> 5 mg/L) was confirmed with a portable dissolved oxygen meterb in both tank and fresh dechlorinated water. A commercial pelleted goldfish dietc was fed every Monday, Wednesday, and Friday. Fish had no known history of disease and were considered healthy on the basis of visual and parasite examinations. Fish had been maintained under these husbandry conditions for 18 months preceding the present study. The North Carolina State University Institutional Animal Care and Use Committee reviewed and approved all procedures.
Study design
Fish were randomly assigned to be immersed in 1 of 6 test solution treatments, with equal numbers (n = 6) of fish in each group: TMSd at a concentration of 500 mg/L for 15 minutes followed by placement in anesthetic agent–free water (T15W), placement out of water (T15A), or decapitation (T15D); TMS at a concentration of 1,000 mg/L for 15 minutes followed by placement in anesthetic agent–free water (T15XW); TMS at a concentration of 500 mg/L for 30 minutes followed by placement in anesthetic agent–free water (T30W); and propofole at a concentration of 25 mg/L for 30 minutes followed by placement in anesthetic agent–free water (P30W). A 10-g/L stock solution of TMS was prepared by dissolving powdered TMS in fresh water and buffering with an equal weight of sodium bicarbonate/ Fresh stock solution was made on each test day and used for preparation of individual dilutions.
For groups T15W, T15A, T15XW, T30W, and P30W, each fish was moved via fishnet from the main housing tank to an individual 1,000-mL glass beaker containing 500 mL of the test solution. Fresh, individual dilutions were prepared for each fish with a new 1-mL syringe (for propofol) or a 25-mL graduated cylinder (for TMS). Immediately after transfer, fish were observed for signs of anesthesia, characterized by loss of the righting reflex and lack of response to stimulation, and when noted, an anesthetic induction time and OR (OR1) were recorded. Each fish was then observed for loss of operculation, defined as at least 15 seconds without gross gill movement, and when noted, a time to loss of operculation was recorded. For all groups, except group T15XW, HR (HR1) was recorded at this time. Heart rates were obtained with a Doppler crystalg primed with ultrasound transmission gelh that was submerged in each beaker and placed over the cranioventral aspect of each fish. Fish were then left unstimulated in the test solution for the remainder of the prescribed total immersion time, either 15 or 30 minutes. After the immersion time had elapsed, an OR (OR2) and HR (HR2) were recorded. Fish were then manually removed from the test solution, weighed, measured in length, and immediately placed in individual 1,000-mL glass beakers containing 500 mL of anesthetic agent–free fresh water (T15W, T15XW, T30W, P30W) or out of water in left lateral recumbency (T15A). The presence of myocardial contractions and HR, as detected by Doppler ultrasonography, were recorded every 5 minutes until either cessation of Doppler ultrasounds or resumption of operculation. In the former situation, time to loss of Doppler ultrasounds was recorded, whereas in the latter situation, time to resumption of operculation was recorded and Doppler ultrasonography was discontinued. Any fish in group T15A that resumed operculation was immediately moved to an individual 1,000-mL glass beaker containing 500 mL of anesthetic agent–free fresh water. Operculating fish were observed for signs of complete recovery, evidenced by an intact righting reflex when pushed into a lateral orientation, and when noted, recovery time was recorded. Following the final experiment of the day, recovered fish were returned to the main housing tank and monitored daily for at least 1 month for latent morbidity and death.
For group T15D, each fish was moved via fishnet from the main housing tank to an individual 1,000-mL glass beaker containing TMS at a concentration of 500 mg/L. Fresh, individual dilutions were made for each fish with a 25-mL graduated cylinder. Immediately after transfer, each fish was observed for signs of anesthesia, characterized by loss of the righting reflex and lack of response to stimulation, and when noted, an anesthetic induction time was recorded. Fish were then left unstimulated in the test solution for a total immersion time of 15 minutes. After the immersion time had elapsed, an OR (OR2) and HR (HR2) were recorded. Fish were then manually removed from the test solution, weighed, measured in length, and decapitated cranial to the gills, with a new No. 22 scalpel blade used for each fish. The body was positioned in left lateral recumbency out of water. The presence of Doppler ultrasounds and associated HR were recorded every 5 minutes with a Doppler crystal placed over the cranioventral aspect of the fish, and time to cessation of Doppler ultrasounds was recorded.
Results
Overall, 5 of 6, 6 of 6, 6 of 6, 6 of 6, and 5 of 6 fish survived in groups T15W, T15A, T15XW, T30W, and P30W, respectively. The 2 fish that died had brief periods of operculation 4 minutes (group T15W) and 10 minutes (group P30W) after removal from the test solution followed by permanent cessation of operculation, at which time periodic Doppler ultrasonographic assessments were resumed. Time to loss of Doppler ultrasounds in these 2 fish was 248 minutes (group T15W) and 780 minutes (group P30W). Median times to anesthetic induction, loss of operculation, resumption of operculation, and complete recovery and median ORs and HRs for groups T15W, T15A, T15XW, T30W, and P30W were calculated (Table 1). All fish exceeded the AVMA recommendation1 of immersion for 10 minutes following loss of operculation. No latent morbidity or death was noted in any surviving fish. Median times to anesthetic induction and cessation of Doppler ultrasounds and median ORs and HRs for group T15D were also calculated (Table 2).
Time from onset of immersion to induction of anesthesia, cessation of operculation, resumption of operculation, and recovery from anesthesia as well as physiologic variables of goldfish (Carassius auratus) immersed in TMS at a concentration of 500 mg/L for 15 minutes followed by placement in anesthetic agent–free water (T15W; n = 6) or placement out of water (T15A; 6), TMS at a concentration of 1,000 mg/L for 15 minutes followed by placement in anesthetic agent–free water (T15XW; 6), TMS at a concentration of 500 mg/L for 30 minutes followed by placement in anesthetic agent–free water (T30W; 6), or propofol at a concentration of 25 mg/L for 30 minutes followed by placement in anesthetic agent–free water (P30W; 6).
| Variable | T15W | T15A | T15XW | T30W | P30W |
|---|---|---|---|---|---|
| Induction of anesthesia (min) | 1.4 (0.8–1.5) | 1.4 (1.2–2.4) | 1.0 (0.7–1.1) | 1.5 (1.1–1.7) | 1.6 (0.9–1.9) |
| Cessation of operculation (min) | 2.5 (0.8–2.7) | 2.2 (2.0–3.7) | 1.0 (0.7–4.1) | 1.9 (1.6–4.2) | 1.9 (1.5–2.4) |
| OR1 (operculations/min) | 70 (0–100) | 113 (48–180) | 0 (0–64) | 1-rapid, shallow‡ | 24 (0–60)∥ |
| HR1 (beats/min) | 52 (50–72) | 70 (60–78) | NR | 63 (30–66) | 12 (6–24) |
| OR2 (operculations/min) | 0 (0–3) | 0 (0–0) | 0-intermittent† | 0-intermittent§ | 0.5 (0–1) |
| HR2 (beats/min) | 50 (44–64) | 56 (40–64) | 56 (40–60) | 63 (54–66) | 24 (18–36) |
| Resumption of operculation (min) | 6.5 (2.1–15.3) | 2.4 (0.7–9.2) | 6.4 (0.9–27.9) | 1.0 (0.4–8.5) | 0.4 (0.3–14.0) |
| Recovery time (min) | 24.1 (4.4–32.5)* | 11.6 (3.8–22.0) | 45.9 (36.6–127.4) | 41.3 (21.0–87.2) | 54.3 (20.3–331.0)* |
Data are reported as median (range), unless otherwise indicated.
Data represent values for only 5 fish; the remaining fish died.
Operculation rate ranged from 0/min (n = 5) to intermittent (< 1/min; 1).
Operculation rate ranged from 1/min (n = 1) to rapid and shallow (5).
Operculation rate ranged from 0/min (n = 3) to intermittent (< 1/min; 3).
Data represent values for only 5 fish because the opaque nature of the solution prevented reliable observation of OR.
HR1 = HR at the time of cessation of operculation. HR2 = HR at the end of the immersion period. NR = Not recorded. OR1 = OR at the time of anesthetic induction. OR2 = OR at the end of the immersion period.
Time from onset of immersion to induction of anesthesia and cessation of Doppler ultrasounds as well as physiologic parameters of 6 goldfish immersed in TMS at a concentration of 500 mg/L for 15 minutes followed by decapitation (T15D).
| Variable | T15D |
|---|---|
| Induction of anesthesia (min) | 1.4 (0.9–1.8) |
| OR2 (operculations/min) | 0 (0–24) |
| HR2 (beats/min) | 58 (48–68) |
| Cessation of Doppler ultrasounds (min) | 77.5 (30–240) |
All data reported as median (range). See Table 1 for remainder of key.
Discussion
Current AVMA euthanasia guidelines1 recommend that, for immersion euthanasia, “finfish should be left in the anesthetic solution for a minimum of 10 minutes after cessation of opercular movement” and state that a TMS “concentration of 250 to 500 mg/L, or 5 to 10 times the anesthetic dosage, is effective for most species.” However, results of the present study did not support these recommendations because immersion in TMS at a concentration of 500 mg/L for 15 or 30 minutes, TMS at a concentration of 1,000 mg/L for 15 minutes, or propofol at a concentration of 25 mg/L for 30 minutes did not reliably euthanize goldfish. Twenty-three of 24 (96%) goldfish immersed in TMS at concentrations and for durations that exceeded the recommendations in the AVMA guidelines1 made complete recoveries with no latent morbidity or mortality. These fish were arguably in a deep plane of anesthesia and recovered without additional treatment. Although anecdotal reports exist of fish recovering following immersion euthanasia with TMS at the currently recommended concentrations, the present study is the first, to the authors' knowledge, to report the unreliability of this protocol for an ornamental fish species, goldfish. Euthanasia methods for any species should be nearly 100% reliable, thus, a > 95% failure rate is unacceptable.
There are several potential reasons for the lack of efficacy of TMS as a euthanasia agent in the present study. First, TMS may not be an appropriate drug for immersion euthanasia of fish. The mechanism by which TMS produces general anesthesia and results in euthanasia in fish is currently unknown. Its anesthetic effects and effects as a euthanasia agent are thought to be a result of neuronal depressant activity,1,6 but this may misrepresent the cerebral effects of this drug. Because practical markers of cerebral death in fish are virtually nonexistent, confirmation of true cerebral debilitation following immersion in a TMS solution in a clinical setting is extremely difficult. Considering that in the present study 96% (23/24) of fish that were immersed in TMS at concentrations and for durations meeting or exceeding those recommended by euthanasia guidelines1 made complete recoveries, cerebral death did not occur.
The AVMA euthanasia guidelines1 suggest that TMS causes death through a combination of decreased nervous and cardiovascular function, with nervous function decreased as a result of blockage of voltage-sensitive sodium channels and cardiovascular function decreased by accumulation of TMS in the ventricular myocardium. In mammals, local anesthetic overdose can lead to direct myocardial depression, rapidly ensuing cardiovascular collapse, and death.17 In vivo studies have identified TMS-induced reductions in myocardial contractility of 75% and 85% in Chinook salmon18 and rainbow trout,19 respectively; however, to the authors' knowledge, no studies have definitively linked myocardial depression and cardiovascular collapse in fish. Furthermore, in the present study, the myocardium of fish that were immersed in TMS at a concentration of 500 mg/L for 15 minutes and then decapitated continued to contract for at least 30 minutes to as long as 240 minutes. Thus, development of cardiovascular collapse in a rapid and clinically relevant time frame during immersion in TMS seems unlikely in goldfish.
Insufficient immersion concentration is a second possible reason for the failure of TMS as an immersion euthanasia agent in the present study. Currently recommended concentrations of TMS for immersion euthanasia are 250 to 500 mg/L.1 In the present study, a concentration twice this high (1,000 mg/L) was used, but was not effective in that all 6 goldfish tested at this concentration survived. Although the recommended concentration range has been widely published in numerous sources, no scientific research forming the basis for the recommendation could be identified by the authors. It is unknown whether TMS immersion concentrations higher than that used in the present study (ie, > 1000 mg/L) would result in euthanasia of goldfish.
Finally, insufficient immersion duration is a third potential reason for the ineffectiveness of TMS in the present study. The AVMA guidelines suggest that immersion “for 10 minutes following loss of rhythmic opercular movement is sufficient for euthanasia of most finfish.”1 However, even though all the fish in the present study exceeded this criterion, 96% survived. In fact, fish in group T30W had a median time of exposure after loss of operculation of 28.1 minutes, almost triple the suggested exposure duration, yet had a 100% (6/6) survival rate. The authors could not identify published literature providing evidence-based support for the currently recommended immersion duration. Considering the mechanism of TMS uptake (extraction from water moved over the gills during operculation) and the similar time to loss of operculation (approx 2 minutes) for the 15-minute and 30-minute immersion groups in the present study, it seemed unlikely that additional exposure time could have resulted in a clinically meaningful increase in drug uptake. That is, if drug uptake relies on operculation, then once operculation ceases, extending the immersion time may not result in greater drug uptake or greater drug effect.
The AVMA euthanasia guidelines1 do not provide recommendations for placement of fish following TMS immersion euthanasia; however, this is not surprising because if immersion euthanasia results in death, then placement following immersion would be irrelevant. Because placement of euthanized fish out of water would seem to be the most common clinical scenario, a subset of fish in the present study were exposed to room air following TMS immersion. Conversely, because of concerns related to continued function of the myocardium and higher brain centers despite loss of operculation, the remaining fish were placed in anesthetic agent–free fresh water following TMS immersion in the present study. Placement of goldfish in or out of water following TMS immersion had no effect on overall survival rate, and all 6 fish in the T15A group resumed operculation out of water. Resumption of operculation is a result of anesthetic drug metabolism or excretion and subsequent transition to a lighter plane of anesthesia. Gill excretion of the nonmetabolized parent compound is the primary route of elimination of TMS in fish.6,20 An argument may be made that if a fish is anesthetized when it is removed from the water and, thus, is deprived of its primary means to excrete TMS (ie, gill excretion into water), the fish will remain anesthetized and not regain consciousness. However, results of the present study challenged this reasoning because all 6 fish in group T15A resumed operculation. For humane reasons, these fish were placed in anesthetic agentfree water for the remainder of the recovery period. However, it is not unreasonable to suggest that these fish could have continued to recover and regain full consciousness even out of water. Because most fish euthanized via TMS immersion are then placed out of water (eg, in a freezer or trash bag or on a necropsy table), results of the present study would appear to call for reexamination of the humane implications of the currently recommended TMS immersion euthanasia protocols for fish. If fish are only in a deep plane of anesthesia following TMS immersion and have the ability to regain consciousness out of water, then death may be a result of asphyxiation while conscious, which would be unacceptable and inhumane.1 Interestingly, median time to resumption of operculation was faster when fish were placed out of water (group T15A) than when they were placed in anesthetic agent–free water (group T15W) following TMS immersion (median, 2.4 and 6.5 minutes, respectively). Known secondary routes of TMS elimination in fish include hepatic biotransformation and urinary excretion.6,21 For fish in group T15A, stress associated with being out of water may have contributed to increased cardiac output and subsequent increased hepatic blood flow and drug metabolism.
Similar to TMS immersion, immersion in propofol at a concentration of 25 mg/L for 30 minutes did not reliably euthanize goldfish in the present study, even though previous research22,23 has confirmed the presence of γ-aminobutyric acid receptors in the CNS of teleost fish. The propofol immersion concentration and duration in the present study were extrapolated from TMS immersion euthanasia guidelines; however, a higher propofol concentration or longer immersion duration could potentially result in reliable euthanasia of goldfish. Additional research is warranted.
It was unknown why propofol immersion resulted in pronounced bradycardia in the fish of the present study. One potential reason might have been a response to an overdose of propofol. Koi exposed to propofol via immersion demonstrated bradycardia, with more pronounced effects as concentrations increased up to 10 mg/L.10 Alternatively, bradycardia may have been a protective mechanism, decreasing myocardial work in fish that ceased operculation. Further investigation is needed to determine the cause of the bradycardia.
Although it may be tempting to extrapolate from responses by mammalian species and assume that prolonged cessation of operculation correlates with and will result in death in fish, this was not the case in the present study. Fish in the present study that had periods without operculation of almost 30 minutes still made full recoveries. Blood gas analyses were not performed to confirm the presence of hypoxemia; however, it could be reasoned that some degree of hypoxemia was present in these fish because the gills are the primary source of oxygen uptake. The hypoxia tolerance of goldfish and other fish species is well-known and attributed to high glycogen stores in the brain and liver, low metabolic rates, extensive buffering capacities, and ability to convert lactate, the potentially toxic metabolite of anaerobic glycolysis, into an easily excreted by-product.24,25 In addition, hypoxia tolerance of fish myocardium and brain tissue has been reported.13 In the present study, goldfish made complete recoveries despite prolonged periods of presumed hypoxia. Although this may not hold true for all fish species, because hypoxia tolerance varies, we caution that prolonged cessation of operculation should not be equated with myocardial or cerebral death, at least in goldfish.
Systemic cellular death is extremely difficult to confirm in fish species, and standard markers of death in mammals overlap with markers of a deep plane of anesthesia in fish. According to the AVMA guidelines,1 indicators of death in fish include loss of rhythmic opercular activity, a loss of reactivity to any stimulus, and loss of physiologic reflexes. Although response to stimulation and loss of physiologic reflexes were not evaluated in the present study, from a practical standpoint, all fish were motionless, lacked rhythmic operculation, and did not respond to stimulation when manipulated for Doppler ultrasonographic assessment. Because 96% of the fish in the present study survived, results of the study emphasize the need for better and more reliable clinical markers of systemic death in fish species and highlight the overlap between markers of anesthesia and death.
The duration of Doppler ultrasounds following TMS immersion and decapitation in the current study documented the hypoxia tolerance of the goldfish myocardium. A study26 of cervical dislocation in anesthetized and conscious mice reported cessation of myocardial contractions in < 4 minutes in both groups. In contrast, myocardial contractions continued for 30 to 240 minutes after decapitation for fish in the present study. Although cerebral death was not objectively quantified in the present study, given the known hypoxia tolerance of fish brain tissue combined with the complete recovery of fish following a period of opercular arrest in the present study, the potential exists for prolonged (> 10 minutes) cerebral function, a potential that has humane implications for placement of fish in effectively hypoxic environments following immersion euthanasia if direct confirmation of cerebral death cannot be obtained. Although electroencephalography, the classic method for assessing brain function, has been used successfully in fish research,27 it is currently not a feasible tool for clinical use.
Results of the present study indicated that currently recommended TMS immersion concentration and duration are insufficient for euthanasia of goldfish. Although some fish in the present study had prolonged periods of respiratory arrest, most were in a deep plane of anesthesia and made complete recoveries without any resuscitative efforts. In theory, immersion euthanasia should be the least invasive, least challenging, and least stressful euthanasia technique for goldfish; however, results of the present study suggested that TMS immersion may not be successful as a sole method of euthanasia and that use of this method in other hypoxia-tolerant species should be reconsidered. Until definitive, clinically applicable markers of cerebral death in fish can be identified, we recommend that TMS immersion for the purpose of euthanasia of fish be followed by a second, cerebraldisrupting method.
Limitations of the study include the use of a single fish species and the challenges associated with assessing systemic cellular and cerebral death in fish. Future research on the effect of species, life stage, health status, and water quality (temperature, pH, and salinity) among other factors, on the effectiveness of attempted immersion euthanasia with TMS or propofol is warranted.
Acknowledgments
Presented in abstract form at the 2016 International Association for Aquatic Animal Medicine Conference, Virginia Beach, Va, May 2016.
No third-party funding or support was received in connection with this study or the writing or publication of the manuscript. The authors did not have any financial interests with companies that manufactured products that were the subject of the present research or with companies that manufactured competing products.
ABBREVIATIONS
| HR | Heart rate |
| OR | Opercular rate |
| TMS | Tricaine methanesulfonate |
Footnotes
Blue Ridge Fish Hatchery, Kernersville, NC.
Dissolved Oxygen Meter, Sper Scientific Ltd, Scottsdale, Ariz.
Koi and Goldfish Food, Blue Ridge Fish Hatchery, Kernersville, NC.
Tricaine-S, Syndel USA, Ferndale, Wash.
PropoFlo (10 mg/mL), Abbott Laboratories, Abbott Park, Ill.
Arm & Hammer, Baking Soda, Church & Dwight Co Inc, Ewing, NJ.
Ultrasonic Doppler Flow Detector, Model 811-B, Parks Medical Electronics Inc, Aloha, Ore.
Aquasonic 100, Parker Laboratories Inc, Fairfield, NJ.
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