Evaluation of the anesthetic efficacy of alfaxalone in oscar fish (Astronotus ocellatus) OBJECTIVE To evaluate effects of alfaxalone on heart rate (HR), opercular rate (OpR), results of blood gas analysis, and responses to noxious stimuli in oscar fish (Astronotus ocellatus).
Alice M. Bugman dvm Peter T. Langer dvm Eva Hadzima dvm, mvdr Anne E. Rivas dvm
ANIMALS 6 healthy subadult oscar fish.
Mark A. Mitchell dvm, phd Received December 30, 2014. Accepted June 1, 2015. From the Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois, Urbana, IL 61802 (Bugman, Langer, Rivas, Mitchell); and the Dewinton Pet Hospital, 412 Pine Creek Rd, Dewinton, AB T0L 0X0, Canada (Hadzima). Address correspondence to Dr. Mitchell (mmitch@ illinois.edu).
PROCEDURES Each fish was immersed in water containing 5 mg of alfaxalone/L. Water temperature was maintained at 25.1°C, and water quality was appropriate for this species. The HR, OpR, response to noxious stimuli, and positioning in the tank were evaluated, and blood samples for blood gas analysis were collected before (baseline), during, and after anesthesia. RESULTS Immersion anesthesia of oscar fish with alfaxalone (5 mg/L) was sufficient for collection of diagnostic samples in all fish. Mean ± SD induction time was 11 ± 3.8 minutes (minimum, 5 minutes; maximum, 15 minutes), and mean recovery time was 37.5 ± 13.7 minutes (minimum, 20 minutes; maximum, 55 minutes). There was a significant difference in OpR over time, with respiratory rates significantly decreasing between baseline and anesthesia and then significantly increasing between anesthesia and recovery. There was no significant difference in HR over time. Median lactate concentrations were significantly increased in all anesthetized fish. Other physiologic or blood gas variables did not change significantly. CONCLUSIONS AND CLINICAL RELEVANCE Alfaxalone should be considered as a readily available and easy-to-use anesthetic for oscar fish. Because it is more likely to be found in veterinary hospitals than other traditional anesthetics for fish, its value as an anesthetic for other species of fish should also be considered. (Am J Vet Res 2016;77:239– 244)
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lobal commerce for aquarium fish is a multibilliondollar industry involving international import and export of fish, invertebrates, and plants.1 With > 11 million aquarium hobbyists, the US sector comprises > $25 billion of the global import economy.2 In addition to their popularity as pets, many tropical freshwater and marine fish are used in biomedical research because of the short duration of their reproductive cycles and the fact that their embryonic development is similar to that of larger vertebrates and in aquarium and zoo collections for conservation and education purposes.3,4 Because of the increasing demand for aquarium fish by hobbyists and institutions, veterinarians will increasingly be tasked to provide care, diagnostic examinations, and surgery to treat various afflictions in ABBREVIATIONS HCO3 Bicarbonate HR Heart rate OpR Opercular rate So2 Oxygen saturation
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these animals. To provide appropriate diagnostic testing and treatment, practitioners must anesthetize fish to decrease stress and facilitate safe handling. Tricaine methanesulfonate (MS-222) is one of the most commonly used fish anesthetics and the only US FDA– approved anesthetic for food fish5; MS-222 is a sodiumchannel blocker used as an immersion anesthetic. A number of other immersion agents, including clove oil (eugenol or isoeugenol) and quinaldine sulfate, have also been used as anesthetics for fish, but with variable results.6,7 All these anesthetics are used specifically for fish, so general practitioners may not stock them in their hospitals or clinics. It would be helpful to identify anesthetics that clinicians may routinely stock in their hospitals and clinics and use to safely handle fish. One anesthetic that has recently become available for use in veterinary practice and that could also potentially be used as an anesthetic agent for fish is alfaxalone (3 α-hydroxy-5 α-pregnane-11, 20-dione), a synthetic neurosteroid that has been used for cats,8 dogs,9 amphibians,10,11 marine mammals,a and fish.12,13 Alfaxalone is an agonist of γ–aminobutyric acid type A
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receptors and glycine receptors.14 It leads to hyperpolarization of the postsynaptic cell membrane, which causes inhibition of the propagation of action potentials and decreased activation of pathways related to arousal and awareness.15 Alfaxalone is poorly water soluble; however, a formula of alfaxalone in 2-hydroxypropyl-β cyclodextrin is water soluble. Although multiple studies16–18 have been conducted to examine the efficacy of MS-222 in fish, the authors are aware of only 3 studies13,19,20 conducted to evaluate the efficacy of alfaxalone as an immersion anesthetic for fish, and all 3 involved cold-water species of fish. The objective of the study reported here was to determine clinical and physiologic effects of alfaxalone for a warm-water cichlid species, oscar fish (Astronotus ocellatus). Specific aims included evaluation of the effects of alfaxalone on HR, OpR, results of blood gas analysis, and responses to noxious stimuli. The hypotheses tested were that oscar fish anesthetized with alfaxalone would reach a surgical anesthetic plane, OpR and HR would decrease when fish reached a surgical anesthetic plane, and Pco2 and lactate concentration would increase and pH, HCO3, and So2 would decrease.
Materials and Methods Animals
Six subadult oscar fish were obtained from a commercial supplierb for use in the study. Sex of the fish could not be established. The sample size (n = 6) was determined a priori; the calculation had the assumptions of α = 0.05, power = 0.80, and an expectation that anesthesia could be induced with alfaxalone within a mean ± SD of 15 ± 12 minutes against a null hypothesis that the fish could not be anesthetized. This study was approved by the Institutional Animal Care and Use Committee at the University of Illinois.
Procedures
Each fish was maintained in an 18.9-L tank that contained 12 L of tap water dechlorinated with sodium thiosulfate. The water was aerated with an air stone on a mechanical pump,c and the temperature was maintained at 25.1°C with a submersible heater.d Ammonia, nitrite, nitrate, and chlorine concentrations; pH; alkalinity; and general hardness of the water were assessed with a commercial test kit.e Two additional 18.9-L tanks were used as the anesthesia and recovery tanks. The water in both of those tanks was dechlorinated, aerated, tested, and heated as described previously. For the anesthesia tank, alfaxalonef (5 mg/L) was added to 6 L of water, whereas the recovery tank contained 12 L of water. Fresh batches of water for the anesthetic and recovery tanks were prepared between successive anesthetic events. Before each fish was anesthetized (time 0 = start of immersion anesthesia), baseline measurements were recorded for OpR, HR (by use of Doppler ultrasonographyg), response to noxious stimuli, and anesthetic plane. 240
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Response to noxious stimuli was assessed by removing each fish from the water, placing it in lateral recumbency on a moistened paper towel, and using a padded hemostat to apply pressure to the upper lip and caudal peduncle. Reactions to applied pressure were recorded by use of an ordinal scale as follows: 1 = immediate response, 2 = mild response, and 3 = no response. Anesthetic plane was determined by use of a published scale,7 with modifications. Briefly, anesthesia was scored on a scale of 0 to 3 as follows: 0 (not sedated) = freely swimming, > 40 respirations/min, and rapid righting reflex; 1 (lightly sedated) = slow swimming or lower positioning in tank (toward the bottom), decreased respiration rate (< 40 respirations/ min), delayed (> 2 seconds) but appropriate righting reflex, and response to pressure applied to lip or peduncle; 2 (sedated) = slower swimming or lower positioning in tank (toward the bottom), decreased respiration rate (< 40 respirations/min), severely delayed (> 5 seconds) but appropriate righting reflex, and response to pressure applied to lip or peduncle; and 3 (anesthetized) = not swimming and positioning at the bottom of the tank, decreased respiration rate (< 40 respirations/min), no righting reflex, and no response to pressure applied to the lip or peduncle. At time 0, an initial blood sample (0.1 mL; baseline sample) was collected via cardiocentesis by use of a 25-gauge needle attached to a 1-mL syringe.The blood sample was used to analyze pH, Pco2, Po2, HCO3, total carbon dioxide concentration, So2, and lactate concentration; analysis was performed with a commercial portable analyzerh,i immediately (within 2 minutes) after sample collection. Temperature of the water (25.1°C) was entered into the analyzer to adjust the data for the body temperature of the fish. After baseline measurements and the baseline blood sample were collected, each fish was transferred to the anesthetic tank. The OpR, HR, response to noxious stimuli, and anesthetic plane were measured every 5 minutes. When the fish reached an anesthetic plane of 3, it was removed from the anesthetic tank, weighedj (nearest 0.1 g), and measured (fork length [length from snout to fork in caudal fin]; nearest 1 mm). A second blood sample (anesthetized sample) was collected as described previously. After the blood sample was collected, the fish was placed in the recovery tank and manually moved forward and backward in a swimming motion until spontaneous swimming and the righting reflex resumed. The OpR, HR, response to noxious stimuli, and anesthetic plane were measured every 5 minutes until the value reached 0. A final blood sample was collected (recovery sample) as described previously.Thus, a total of 0.3 mL of blood was collected from each fish, which represented 1% of blood volume. Fish were returned to the commercial supplier at the end of the study.
Statistical analysis
Distribution of continuous data was evaluated by use of the Shapiro-Wilk test, skewness, kurtosis, and
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Q-Q plots. Data that were normally distributed were reported as mean ± SD, minimum, and maximum, whereas nonnormally distributed data were reported as median, 25th to 75th percentiles, minimum, and maximum. Data that were not normally distributed were logarithmically transformed for parametric analysis. A general linear model for repeated measures was used to evaluate OpR, HR, and blood gas variables over time. Because of variation in the induction and recovery times, specific comparisons were made for baseline, stage 3 anesthetized, and recovery measurements. Commercial statistical softwarek was used to analyze the data. Values of P < 0.05 were considered significant.
Results Mean ± SD body weight of fish was 74.2 ± 24.6 g (minimum, 30.0 g; maximum, 95.0 g), and mean fork length was 14.5 ± 1.9 cm (minimum, 11.0 cm; maximum, 16.3 cm). Ammonia, nitrite, and nitrate concentrations of the water were 0. General hardness was 120 mg/L, pH was 8.0, and alkalinity was 240 mg/L. Mean ± SD induction time (interval from immersion until achievement of stage 3 anesthesia) was 11 ± 3.8 minutes (minimum, 5 minutes; maximum, 15 minutes), whereas mean recovery time (interval from placement into the recovery tank until achievement of stage 0 anesthesia) was 37.5 ± 13.7 minutes (minimum, 20 minutes; maximum, 55 minutes). The OpR differed significantly (P = 0.01) over time, with respiratory rates decreasing significantly (P = 0.04) between baseline and anesthesia and then increasing significantly (P = 0.01) between anesthesia and recovery (Table 1). The HR did not differ significantly (P = 0.12) over time. Lactate concentration differed significantly (P = 0.02) over time, with concentrations increasing significantly (P = 0.04) between baseline and anesthesia and between baseline and recovery; lactate concentrations did not differ significantly (P = 0.14) between anesthesia and recovery. Other blood variables did not differ significantly over time (pH [P = 0.67], So2 [P = 0.4], Po2 [P = 0.78],
HCO3 [P = 0.9], and Pco2 [P = 0.07]). The post hoc power analysis for Pco2 was low (power, 0.07), which suggested the potential for a type II error.
Discussion Results of the present study indicated that alfaxalone can be used as an immersion anesthetic to achieve surgical anesthesia in a tropical (warm-water) freshwater fish species. Results of this study are promising for veterinary practitioners who work with these fish because they are more likely to stock alfaxalone in their practices than they are to stock MS-222. In another unpublished study, the authors attempted to anesthetize oscar fish with MS-222 at 125 mg/L, which resulted in incomplete anesthetic induction and an inability to achieve surgical anesthesia at that dose. Although a higher dose of MS-222 would likely result in surgical anesthesia, the dose used (125 mg/L) is considered sufficient for most freshwater and marine species,21,22 and MS-222 at doses > 200 mg/L can result in death.23 Mean induction time for the oscar fish was 11 minutes, which was longer than the induction time for koi (Cyprinus carpio) dosed at 10 mg/L in an immersion bath (mean, 5.4 minutes) but shorter than that for goldfish (Carassius auratus) dosed at 5 mg/L in an immersion bath (mean, 28 minutes).13,19 The more rapid onset for koi dosed at 10 mg/L is not surprising because the dose was twice as high as that for the goldfish; however, goldfish (dosed at 5 mg/L) are typically housed in cold water, and lower water temperatures have been associated with prolonged induction and recovery times, compared with those for fish housed in warm water.21 For koi that received an injection of alfaxalone (5 or 10 mg/ kg, IM), induction time to anesthetic plane was a median of 3 minutes, with a duration of anesthesia of 120 minutes and 300 minutes, respectively.20 Although a surgical anesthetic plane was achieved at a dose of 10 mg/kg in those koi, alfaxalone at that dose administered as an IM injection is an inappropriate anesthetic agent for koi, and presumably oscar fish, because of a substantial mortality rate (33%) and prolonged recovery time. Recov-
Table 1—Results for OpR, HR, and blood gas analyses of 6 oscar fish (Astronotus ocellatus) before (baseline), during, and after immersion anesthesia with alfaxalone (5 mg/L). Variable OpR (/min) HR (/min) Lactate (mmol/L)* pH SO2 (%) Po2 (mm Hg) Pco2 (mm Hg)* HCO3 (mmol/L)
Baseline Mean ± SD 47 ± 12.7a 18.3 ± 7.6 1.4 (1.0–3.8)a 7.15 ± 0.07 89.8 ± 7.7 80.6 ± 23.8 12.6 (12.2–15.3) 4.8 ± 1.1
Anesthesia
Min to max 32–64 12–30 0.8–4.7 7.07–7.26 78.0–95.0 47.0–102.0 12.1–16.6 3.5–5.7
Mean ± SD 33 ± 8.5b 30.6 ± 5.9 3.8 (2.4–4.7)b 7.12 ± 0.08 69.2 ± 35.8 85.8 ± 75.0 14.8 (1.2–18.2) 4.5 ± 0.7
Min to max 20–42 20–36 2.2–5.2 7.02–7.19 20.0–99.0 18.0–207.0 10.8–16.9 3.9–5.6
Recovery Mean ± SD 46 ± 6.9a 27.3 ± 12.7 5.3 (2.4–6.9)c 7.18 ± 0.14 87.8 ± 12.7 108.2 ± 76.0 11.4 (10.2–13.2) 4.6 ± 1.8
Min to max 40–56 12–48 2.2–8.2 6.94–7.31 68.0–99.0 41.0–202.0 9.6–14.3 2.3–7.3
Anesthesia indicated a fish had achieved a surgical plane of anesthesia (3 on a scale of 0 [not sedated] to 3 [not swimming and positioning at the bottom of the tank, decreased respiration rate {< 40 respirations/min}, no righting reflex, and no response to pressure applied to the lip or peduncle]). *Values reported are median (25th to 75th percentiles). Max = Maximum. Min = Minimum. a-cWithin a row, values with different superscript letters differ significantly (P < 0.05).
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ery times between goldfish and oscar fish immersed in 5 mg of alfaxalone/L were similar (28 and 37.5 minutes, respectively); however, these are more prolonged than typically observed by the authors when using MS-222.19 A potential reason for the prolonged recovery time in the oscar fish was that 1 fish required 55 minutes to recover. When dealing with small sample sizes (n = 6), 1 outlier can dramatically affect results. Prolonged recovery is an important consideration when selecting an anesthetic for a fish because it can increase the overall time required for case management. All of the fish in the present study reached a plane of anesthesia that was adequate for the collection of basic diagnostic samples (ie, venipuncture). Additional studies to evaluate the efficacy of alfaxalone for a longer duration, such as would be necessary for surgery, are needed. The primary intent of the study reported here was to determine whether alfaxalone immersion at a dose of 5 mg/L would provide a consistent plane of anesthesia; maintenance of surgical anesthesia was not an aim of this study, but it deserves attention. Heart rate increased after immersion in alfaxalone but did not return to baseline values after recovery. High doses of alfaxalone reportedly cause a temporary decrease in mean arterial blood pressure in dogs, which can lead to reflex tachycardia with no change or a minimal increase in cardiac output.15,24 In koi there was no significant difference in HR at induction doses of 5 and 10 mg/L14; however, these koi were not kept in immersion solutions of that concentration once the desired anesthetic plane was achieved and instead were transferred to immersion baths of 2.5 and 1.0 mg/L, respectively. Analysis of the present study suggested that, similar to results for dogs,15,24 alfaxalone administered to oscar fish may increase HR to maintain cardiac output secondary to peripheral vasodilation. Further evaluations to compare induction and maintenance doses of alfaxalone could help delineate the effects of alfaxalone in warm-water fish. The OpR decreased from immersion to anesthesia and then rebounded once fish were allowed to recover from anesthesia. Similar reports of respiratory depression (as defined by OpR) have been noted for MS-222 and eugenol in various fish species, including salmonids and carp.24,25 Respiratory depression is a described adverse effect of alfaxalone that has been reported in multiple species, and ventilatory arrest necessitating resuscitation is a possible severe adverse effect in fish.15 Interestingly, respiratory depression was not reported in goldfish anesthetized via alfaxalone immersion, although it was seen in koi anesthetized with alfaxalone.13 Blood lactate concentrations of oscar fish changed significantly throughout the study, with consistently increasing values through recovery. Similar findings in yellow perch (Perca flavescens) and koi anesthetized with MS-222, particularly from preinduction to anesthesia, have been reported,18 which suggests that a prolonged increase in blood lactate concentration after recovery may be predictive of a decrease in short-term survival. 242
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Lactate concentrations may have increased as a result of the decrease in oxygen delivery to tissues that followed the decrease in respiratory rate. However, stress attributable to capture and removal from water and sequential venipuncture may also have contributed to this increase in lactate concentration. A portable blood gas analyzer was used to process blood samples. When this type of analyzer is used, it is important to consider the methods that provide the results. For poikilotherms, such as tropical fish, a correction is required because analyzers perform the analyses at a set temperature, which is typically appropriate for an endotherm (ie, 37°C for humans).26–28 To adjust for the difference in temperatures between poikilotherms and endotherms, analyzers may have a built-in corrective algorithm, or additional corrective algorithms can be applied.26 In the study reported here, we used the analyzer’s temperature correction algorithm to address this difference; however, limitations may still remain, even with this correction. For sandbar sharks (Carcharrhinus plumbeus), analyses of blood gases such as Pco2, Po2, and So2 are unreliable when this temperature correction is used.26 In the case of Po2, the final values are nearly doubled. This has been attributed to a closedsystem temperature change for Po2 within the analyzer’s cartridge and the fact the corrective algorithm is based on hemoglobin-oxygen binding at the temperature for humans.28 Values of Po2 for oscar fish of the present study were 1.34 times as high as baseline values over the course of the study, with wide SD and minimum-maximum values (Table 1). These patterns over time did not make sense from a physiologic standpoint; thus, it is important to consider the potential limitations of portable analyzers and corrective algorithms when interpreting results. In a study27 conducted to evaluate 2 species of marine teleosts, investigators found that the portable blood gas analyzer was unreliable for Po2. Because of the limitations associated with the portable blood gas analyzer used in the present study and in previous studies,26–28 researchers and clinicians should be cautious when interpreting blood gas results obtained by use of this portable analyzer for fish. Ultimately, validation of equipment for each species is important. To our knowledge, the study reported here represented the first description of blood gas analyses performed on a cichlid species. This makes it challenging to interpret the values with regard to health of the fish. However, when compared with results for other freshwater teleosts such as the moonlight gourami (Trichogaster microlepis; pH, 7.36), koi carp (pH, 7.39 to 7.47), yellow perch (P flavescens; pH, 7.16), and walleye pike (Sander vitreus; pH, 7.34), the mean values for pH were lower in oscar fish, except for perch.13,18,29 The HCO3 concentration was also found to be lower in oscar fish than in koi (10.0 mmol/L).13 Baseline lactate concentrations were similar between oscar fish and koi (0.8
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to 1.43 mmol/L), perch (2.0 mmol/L), and walleye (1.21 mmol/L).13,18 The Pco2 in oscar fish was lower than that reported for perch (16.75 mm Hg), walleye (13.82 mm Hg), and koi (14.2 to 16.93 mm Hg), whereas Po2 was highly variable among all species of freshwater teleosts (perch, 59.9 mm Hg; walleye, 78.6 mm Hg; and koi, 18.0 to 31.95 mm Hg). The variability in Po2 reinforces the limited value that portable analyzers may have for measuring blood gases. Differences detected for oscar fish, compared with results for other fish, reinforced that there is large diversity among teleosts and that physiologic adaptions have occurred over time as these animals evolved to survive in specific habitats. For example, oscar fish are native to South America, where the water pH is rather acidic (pH, 4.5 to 6.6), compared with water acidity in other regions of the world.30 It is likely that these fish adapted to survive in lower pH conditions than was optimal for other species. However, it is also important to recognize that the values derived for the various blood gas variables of the present study were determined on the basis of algorithms derived from the portable blood gas analyzer. Therefore, some of these differences may have been attributable to limitations in the algorithms applied for various species. Regardless, because of these differences, it is important to establish species-specific reference ranges to ensure the most robust data when evaluating various species of fish. In the present study, the results were used to evaluate patterns in oscar fish over time and would not be recommended as stand-alone reference values because of the limited sample size. The cichlid family is a large and diverse group of warm-water fish that continue to be popular among fish hobbyists. Because of their popularity and the growing public awareness of treatment options, veterinarians will be faced with providing appropriate sedation and anesthesia to enable diagnostic testing and surgical treatments to be performed to address the needs of cichlid owners. The study reported here revealed that alfaxalone should be considered as a readily available and easy-to-use anesthetic agent for oscar fish. Future studies are needed to further evaluate efficacy and dosages in other species of fish. Additionally, a direct comparison of various doses of alfaxalone would be beneficial to examine the range of effective doses of alfaxalone for fish.
d.
Acknowledgments
15.
Supported in part by Fluker Farms, Sailfin Pets, and Abaxis Inc. Presented in abstract form at the 46th Annual Conference of the American Association of Zoo Veterinarians, Orlando, Fla, October 2014.
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Alice M. Bugman dvm Peter T. Langer dvm Eva Hadzima dvm, mvdr Anne E. Rivas dvm
ANIMALS 6 healthy subadult oscar fish.
Mark A. Mitchell dvm, phd Received December 30, 2014. Accepted June 1, 2015. From the Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois, Urbana, IL 61802 (Bugman, Langer, Rivas, Mitchell); and the Dewinton Pet Hospital, 412 Pine Creek Rd, Dewinton, AB T0L 0X0, Canada (Hadzima). Address correspondence to Dr. Mitchell (mmitch@ illinois.edu).
PROCEDURES Each fish was immersed in water containing 5 mg of alfaxalone/L. Water temperature was maintained at 25.1°C, and water quality was appropriate for this species. The HR, OpR, response to noxious stimuli, and positioning in the tank were evaluated, and blood samples for blood gas analysis were collected before (baseline), during, and after anesthesia. RESULTS Immersion anesthesia of oscar fish with alfaxalone (5 mg/L) was sufficient for collection of diagnostic samples in all fish. Mean ± SD induction time was 11 ± 3.8 minutes (minimum, 5 minutes; maximum, 15 minutes), and mean recovery time was 37.5 ± 13.7 minutes (minimum, 20 minutes; maximum, 55 minutes). There was a significant difference in OpR over time, with respiratory rates significantly decreasing between baseline and anesthesia and then significantly increasing between anesthesia and recovery. There was no significant difference in HR over time. Median lactate concentrations were significantly increased in all anesthetized fish. Other physiologic or blood gas variables did not change significantly. CONCLUSIONS AND CLINICAL RELEVANCE Alfaxalone should be considered as a readily available and easy-to-use anesthetic for oscar fish. Because it is more likely to be found in veterinary hospitals than other traditional anesthetics for fish, its value as an anesthetic for other species of fish should also be considered. (Am J Vet Res 2016;77:239– 244)
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lobal commerce for aquarium fish is a multibilliondollar industry involving international import and export of fish, invertebrates, and plants.1 With > 11 million aquarium hobbyists, the US sector comprises > $25 billion of the global import economy.2 In addition to their popularity as pets, many tropical freshwater and marine fish are used in biomedical research because of the short duration of their reproductive cycles and the fact that their embryonic development is similar to that of larger vertebrates and in aquarium and zoo collections for conservation and education purposes.3,4 Because of the increasing demand for aquarium fish by hobbyists and institutions, veterinarians will increasingly be tasked to provide care, diagnostic examinations, and surgery to treat various afflictions in ABBREVIATIONS HCO3 Bicarbonate HR Heart rate OpR Opercular rate So2 Oxygen saturation
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these animals. To provide appropriate diagnostic testing and treatment, practitioners must anesthetize fish to decrease stress and facilitate safe handling. Tricaine methanesulfonate (MS-222) is one of the most commonly used fish anesthetics and the only US FDA– approved anesthetic for food fish5; MS-222 is a sodiumchannel blocker used as an immersion anesthetic. A number of other immersion agents, including clove oil (eugenol or isoeugenol) and quinaldine sulfate, have also been used as anesthetics for fish, but with variable results.6,7 All these anesthetics are used specifically for fish, so general practitioners may not stock them in their hospitals or clinics. It would be helpful to identify anesthetics that clinicians may routinely stock in their hospitals and clinics and use to safely handle fish. One anesthetic that has recently become available for use in veterinary practice and that could also potentially be used as an anesthetic agent for fish is alfaxalone (3 α-hydroxy-5 α-pregnane-11, 20-dione), a synthetic neurosteroid that has been used for cats,8 dogs,9 amphibians,10,11 marine mammals,a and fish.12,13 Alfaxalone is an agonist of γ–aminobutyric acid type A
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receptors and glycine receptors.14 It leads to hyperpolarization of the postsynaptic cell membrane, which causes inhibition of the propagation of action potentials and decreased activation of pathways related to arousal and awareness.15 Alfaxalone is poorly water soluble; however, a formula of alfaxalone in 2-hydroxypropyl-β cyclodextrin is water soluble. Although multiple studies16–18 have been conducted to examine the efficacy of MS-222 in fish, the authors are aware of only 3 studies13,19,20 conducted to evaluate the efficacy of alfaxalone as an immersion anesthetic for fish, and all 3 involved cold-water species of fish. The objective of the study reported here was to determine clinical and physiologic effects of alfaxalone for a warm-water cichlid species, oscar fish (Astronotus ocellatus). Specific aims included evaluation of the effects of alfaxalone on HR, OpR, results of blood gas analysis, and responses to noxious stimuli. The hypotheses tested were that oscar fish anesthetized with alfaxalone would reach a surgical anesthetic plane, OpR and HR would decrease when fish reached a surgical anesthetic plane, and Pco2 and lactate concentration would increase and pH, HCO3, and So2 would decrease.
Materials and Methods Animals
Six subadult oscar fish were obtained from a commercial supplierb for use in the study. Sex of the fish could not be established. The sample size (n = 6) was determined a priori; the calculation had the assumptions of α = 0.05, power = 0.80, and an expectation that anesthesia could be induced with alfaxalone within a mean ± SD of 15 ± 12 minutes against a null hypothesis that the fish could not be anesthetized. This study was approved by the Institutional Animal Care and Use Committee at the University of Illinois.
Procedures
Each fish was maintained in an 18.9-L tank that contained 12 L of tap water dechlorinated with sodium thiosulfate. The water was aerated with an air stone on a mechanical pump,c and the temperature was maintained at 25.1°C with a submersible heater.d Ammonia, nitrite, nitrate, and chlorine concentrations; pH; alkalinity; and general hardness of the water were assessed with a commercial test kit.e Two additional 18.9-L tanks were used as the anesthesia and recovery tanks. The water in both of those tanks was dechlorinated, aerated, tested, and heated as described previously. For the anesthesia tank, alfaxalonef (5 mg/L) was added to 6 L of water, whereas the recovery tank contained 12 L of water. Fresh batches of water for the anesthetic and recovery tanks were prepared between successive anesthetic events. Before each fish was anesthetized (time 0 = start of immersion anesthesia), baseline measurements were recorded for OpR, HR (by use of Doppler ultrasonographyg), response to noxious stimuli, and anesthetic plane. 240
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Response to noxious stimuli was assessed by removing each fish from the water, placing it in lateral recumbency on a moistened paper towel, and using a padded hemostat to apply pressure to the upper lip and caudal peduncle. Reactions to applied pressure were recorded by use of an ordinal scale as follows: 1 = immediate response, 2 = mild response, and 3 = no response. Anesthetic plane was determined by use of a published scale,7 with modifications. Briefly, anesthesia was scored on a scale of 0 to 3 as follows: 0 (not sedated) = freely swimming, > 40 respirations/min, and rapid righting reflex; 1 (lightly sedated) = slow swimming or lower positioning in tank (toward the bottom), decreased respiration rate (< 40 respirations/ min), delayed (> 2 seconds) but appropriate righting reflex, and response to pressure applied to lip or peduncle; 2 (sedated) = slower swimming or lower positioning in tank (toward the bottom), decreased respiration rate (< 40 respirations/min), severely delayed (> 5 seconds) but appropriate righting reflex, and response to pressure applied to lip or peduncle; and 3 (anesthetized) = not swimming and positioning at the bottom of the tank, decreased respiration rate (< 40 respirations/min), no righting reflex, and no response to pressure applied to the lip or peduncle. At time 0, an initial blood sample (0.1 mL; baseline sample) was collected via cardiocentesis by use of a 25-gauge needle attached to a 1-mL syringe.The blood sample was used to analyze pH, Pco2, Po2, HCO3, total carbon dioxide concentration, So2, and lactate concentration; analysis was performed with a commercial portable analyzerh,i immediately (within 2 minutes) after sample collection. Temperature of the water (25.1°C) was entered into the analyzer to adjust the data for the body temperature of the fish. After baseline measurements and the baseline blood sample were collected, each fish was transferred to the anesthetic tank. The OpR, HR, response to noxious stimuli, and anesthetic plane were measured every 5 minutes. When the fish reached an anesthetic plane of 3, it was removed from the anesthetic tank, weighedj (nearest 0.1 g), and measured (fork length [length from snout to fork in caudal fin]; nearest 1 mm). A second blood sample (anesthetized sample) was collected as described previously. After the blood sample was collected, the fish was placed in the recovery tank and manually moved forward and backward in a swimming motion until spontaneous swimming and the righting reflex resumed. The OpR, HR, response to noxious stimuli, and anesthetic plane were measured every 5 minutes until the value reached 0. A final blood sample was collected (recovery sample) as described previously.Thus, a total of 0.3 mL of blood was collected from each fish, which represented 1% of blood volume. Fish were returned to the commercial supplier at the end of the study.
Statistical analysis
Distribution of continuous data was evaluated by use of the Shapiro-Wilk test, skewness, kurtosis, and
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Q-Q plots. Data that were normally distributed were reported as mean ± SD, minimum, and maximum, whereas nonnormally distributed data were reported as median, 25th to 75th percentiles, minimum, and maximum. Data that were not normally distributed were logarithmically transformed for parametric analysis. A general linear model for repeated measures was used to evaluate OpR, HR, and blood gas variables over time. Because of variation in the induction and recovery times, specific comparisons were made for baseline, stage 3 anesthetized, and recovery measurements. Commercial statistical softwarek was used to analyze the data. Values of P < 0.05 were considered significant.
Results Mean ± SD body weight of fish was 74.2 ± 24.6 g (minimum, 30.0 g; maximum, 95.0 g), and mean fork length was 14.5 ± 1.9 cm (minimum, 11.0 cm; maximum, 16.3 cm). Ammonia, nitrite, and nitrate concentrations of the water were 0. General hardness was 120 mg/L, pH was 8.0, and alkalinity was 240 mg/L. Mean ± SD induction time (interval from immersion until achievement of stage 3 anesthesia) was 11 ± 3.8 minutes (minimum, 5 minutes; maximum, 15 minutes), whereas mean recovery time (interval from placement into the recovery tank until achievement of stage 0 anesthesia) was 37.5 ± 13.7 minutes (minimum, 20 minutes; maximum, 55 minutes). The OpR differed significantly (P = 0.01) over time, with respiratory rates decreasing significantly (P = 0.04) between baseline and anesthesia and then increasing significantly (P = 0.01) between anesthesia and recovery (Table 1). The HR did not differ significantly (P = 0.12) over time. Lactate concentration differed significantly (P = 0.02) over time, with concentrations increasing significantly (P = 0.04) between baseline and anesthesia and between baseline and recovery; lactate concentrations did not differ significantly (P = 0.14) between anesthesia and recovery. Other blood variables did not differ significantly over time (pH [P = 0.67], So2 [P = 0.4], Po2 [P = 0.78],
HCO3 [P = 0.9], and Pco2 [P = 0.07]). The post hoc power analysis for Pco2 was low (power, 0.07), which suggested the potential for a type II error.
Discussion Results of the present study indicated that alfaxalone can be used as an immersion anesthetic to achieve surgical anesthesia in a tropical (warm-water) freshwater fish species. Results of this study are promising for veterinary practitioners who work with these fish because they are more likely to stock alfaxalone in their practices than they are to stock MS-222. In another unpublished study, the authors attempted to anesthetize oscar fish with MS-222 at 125 mg/L, which resulted in incomplete anesthetic induction and an inability to achieve surgical anesthesia at that dose. Although a higher dose of MS-222 would likely result in surgical anesthesia, the dose used (125 mg/L) is considered sufficient for most freshwater and marine species,21,22 and MS-222 at doses > 200 mg/L can result in death.23 Mean induction time for the oscar fish was 11 minutes, which was longer than the induction time for koi (Cyprinus carpio) dosed at 10 mg/L in an immersion bath (mean, 5.4 minutes) but shorter than that for goldfish (Carassius auratus) dosed at 5 mg/L in an immersion bath (mean, 28 minutes).13,19 The more rapid onset for koi dosed at 10 mg/L is not surprising because the dose was twice as high as that for the goldfish; however, goldfish (dosed at 5 mg/L) are typically housed in cold water, and lower water temperatures have been associated with prolonged induction and recovery times, compared with those for fish housed in warm water.21 For koi that received an injection of alfaxalone (5 or 10 mg/ kg, IM), induction time to anesthetic plane was a median of 3 minutes, with a duration of anesthesia of 120 minutes and 300 minutes, respectively.20 Although a surgical anesthetic plane was achieved at a dose of 10 mg/kg in those koi, alfaxalone at that dose administered as an IM injection is an inappropriate anesthetic agent for koi, and presumably oscar fish, because of a substantial mortality rate (33%) and prolonged recovery time. Recov-
Table 1—Results for OpR, HR, and blood gas analyses of 6 oscar fish (Astronotus ocellatus) before (baseline), during, and after immersion anesthesia with alfaxalone (5 mg/L). Variable OpR (/min) HR (/min) Lactate (mmol/L)* pH SO2 (%) Po2 (mm Hg) Pco2 (mm Hg)* HCO3 (mmol/L)
Baseline Mean ± SD 47 ± 12.7a 18.3 ± 7.6 1.4 (1.0–3.8)a 7.15 ± 0.07 89.8 ± 7.7 80.6 ± 23.8 12.6 (12.2–15.3) 4.8 ± 1.1
Anesthesia
Min to max 32–64 12–30 0.8–4.7 7.07–7.26 78.0–95.0 47.0–102.0 12.1–16.6 3.5–5.7
Mean ± SD 33 ± 8.5b 30.6 ± 5.9 3.8 (2.4–4.7)b 7.12 ± 0.08 69.2 ± 35.8 85.8 ± 75.0 14.8 (1.2–18.2) 4.5 ± 0.7
Min to max 20–42 20–36 2.2–5.2 7.02–7.19 20.0–99.0 18.0–207.0 10.8–16.9 3.9–5.6
Recovery Mean ± SD 46 ± 6.9a 27.3 ± 12.7 5.3 (2.4–6.9)c 7.18 ± 0.14 87.8 ± 12.7 108.2 ± 76.0 11.4 (10.2–13.2) 4.6 ± 1.8
Min to max 40–56 12–48 2.2–8.2 6.94–7.31 68.0–99.0 41.0–202.0 9.6–14.3 2.3–7.3
Anesthesia indicated a fish had achieved a surgical plane of anesthesia (3 on a scale of 0 [not sedated] to 3 [not swimming and positioning at the bottom of the tank, decreased respiration rate {< 40 respirations/min}, no righting reflex, and no response to pressure applied to the lip or peduncle]). *Values reported are median (25th to 75th percentiles). Max = Maximum. Min = Minimum. a-cWithin a row, values with different superscript letters differ significantly (P < 0.05).
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ery times between goldfish and oscar fish immersed in 5 mg of alfaxalone/L were similar (28 and 37.5 minutes, respectively); however, these are more prolonged than typically observed by the authors when using MS-222.19 A potential reason for the prolonged recovery time in the oscar fish was that 1 fish required 55 minutes to recover. When dealing with small sample sizes (n = 6), 1 outlier can dramatically affect results. Prolonged recovery is an important consideration when selecting an anesthetic for a fish because it can increase the overall time required for case management. All of the fish in the present study reached a plane of anesthesia that was adequate for the collection of basic diagnostic samples (ie, venipuncture). Additional studies to evaluate the efficacy of alfaxalone for a longer duration, such as would be necessary for surgery, are needed. The primary intent of the study reported here was to determine whether alfaxalone immersion at a dose of 5 mg/L would provide a consistent plane of anesthesia; maintenance of surgical anesthesia was not an aim of this study, but it deserves attention. Heart rate increased after immersion in alfaxalone but did not return to baseline values after recovery. High doses of alfaxalone reportedly cause a temporary decrease in mean arterial blood pressure in dogs, which can lead to reflex tachycardia with no change or a minimal increase in cardiac output.15,24 In koi there was no significant difference in HR at induction doses of 5 and 10 mg/L14; however, these koi were not kept in immersion solutions of that concentration once the desired anesthetic plane was achieved and instead were transferred to immersion baths of 2.5 and 1.0 mg/L, respectively. Analysis of the present study suggested that, similar to results for dogs,15,24 alfaxalone administered to oscar fish may increase HR to maintain cardiac output secondary to peripheral vasodilation. Further evaluations to compare induction and maintenance doses of alfaxalone could help delineate the effects of alfaxalone in warm-water fish. The OpR decreased from immersion to anesthesia and then rebounded once fish were allowed to recover from anesthesia. Similar reports of respiratory depression (as defined by OpR) have been noted for MS-222 and eugenol in various fish species, including salmonids and carp.24,25 Respiratory depression is a described adverse effect of alfaxalone that has been reported in multiple species, and ventilatory arrest necessitating resuscitation is a possible severe adverse effect in fish.15 Interestingly, respiratory depression was not reported in goldfish anesthetized via alfaxalone immersion, although it was seen in koi anesthetized with alfaxalone.13 Blood lactate concentrations of oscar fish changed significantly throughout the study, with consistently increasing values through recovery. Similar findings in yellow perch (Perca flavescens) and koi anesthetized with MS-222, particularly from preinduction to anesthesia, have been reported,18 which suggests that a prolonged increase in blood lactate concentration after recovery may be predictive of a decrease in short-term survival. 242
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Lactate concentrations may have increased as a result of the decrease in oxygen delivery to tissues that followed the decrease in respiratory rate. However, stress attributable to capture and removal from water and sequential venipuncture may also have contributed to this increase in lactate concentration. A portable blood gas analyzer was used to process blood samples. When this type of analyzer is used, it is important to consider the methods that provide the results. For poikilotherms, such as tropical fish, a correction is required because analyzers perform the analyses at a set temperature, which is typically appropriate for an endotherm (ie, 37°C for humans).26–28 To adjust for the difference in temperatures between poikilotherms and endotherms, analyzers may have a built-in corrective algorithm, or additional corrective algorithms can be applied.26 In the study reported here, we used the analyzer’s temperature correction algorithm to address this difference; however, limitations may still remain, even with this correction. For sandbar sharks (Carcharrhinus plumbeus), analyses of blood gases such as Pco2, Po2, and So2 are unreliable when this temperature correction is used.26 In the case of Po2, the final values are nearly doubled. This has been attributed to a closedsystem temperature change for Po2 within the analyzer’s cartridge and the fact the corrective algorithm is based on hemoglobin-oxygen binding at the temperature for humans.28 Values of Po2 for oscar fish of the present study were 1.34 times as high as baseline values over the course of the study, with wide SD and minimum-maximum values (Table 1). These patterns over time did not make sense from a physiologic standpoint; thus, it is important to consider the potential limitations of portable analyzers and corrective algorithms when interpreting results. In a study27 conducted to evaluate 2 species of marine teleosts, investigators found that the portable blood gas analyzer was unreliable for Po2. Because of the limitations associated with the portable blood gas analyzer used in the present study and in previous studies,26–28 researchers and clinicians should be cautious when interpreting blood gas results obtained by use of this portable analyzer for fish. Ultimately, validation of equipment for each species is important. To our knowledge, the study reported here represented the first description of blood gas analyses performed on a cichlid species. This makes it challenging to interpret the values with regard to health of the fish. However, when compared with results for other freshwater teleosts such as the moonlight gourami (Trichogaster microlepis; pH, 7.36), koi carp (pH, 7.39 to 7.47), yellow perch (P flavescens; pH, 7.16), and walleye pike (Sander vitreus; pH, 7.34), the mean values for pH were lower in oscar fish, except for perch.13,18,29 The HCO3 concentration was also found to be lower in oscar fish than in koi (10.0 mmol/L).13 Baseline lactate concentrations were similar between oscar fish and koi (0.8
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to 1.43 mmol/L), perch (2.0 mmol/L), and walleye (1.21 mmol/L).13,18 The Pco2 in oscar fish was lower than that reported for perch (16.75 mm Hg), walleye (13.82 mm Hg), and koi (14.2 to 16.93 mm Hg), whereas Po2 was highly variable among all species of freshwater teleosts (perch, 59.9 mm Hg; walleye, 78.6 mm Hg; and koi, 18.0 to 31.95 mm Hg). The variability in Po2 reinforces the limited value that portable analyzers may have for measuring blood gases. Differences detected for oscar fish, compared with results for other fish, reinforced that there is large diversity among teleosts and that physiologic adaptions have occurred over time as these animals evolved to survive in specific habitats. For example, oscar fish are native to South America, where the water pH is rather acidic (pH, 4.5 to 6.6), compared with water acidity in other regions of the world.30 It is likely that these fish adapted to survive in lower pH conditions than was optimal for other species. However, it is also important to recognize that the values derived for the various blood gas variables of the present study were determined on the basis of algorithms derived from the portable blood gas analyzer. Therefore, some of these differences may have been attributable to limitations in the algorithms applied for various species. Regardless, because of these differences, it is important to establish species-specific reference ranges to ensure the most robust data when evaluating various species of fish. In the present study, the results were used to evaluate patterns in oscar fish over time and would not be recommended as stand-alone reference values because of the limited sample size. The cichlid family is a large and diverse group of warm-water fish that continue to be popular among fish hobbyists. Because of their popularity and the growing public awareness of treatment options, veterinarians will be faced with providing appropriate sedation and anesthesia to enable diagnostic testing and surgical treatments to be performed to address the needs of cichlid owners. The study reported here revealed that alfaxalone should be considered as a readily available and easy-to-use anesthetic agent for oscar fish. Future studies are needed to further evaluate efficacy and dosages in other species of fish. Additionally, a direct comparison of various doses of alfaxalone would be beneficial to examine the range of effective doses of alfaxalone for fish.
d.
Acknowledgments
15.
Supported in part by Fluker Farms, Sailfin Pets, and Abaxis Inc. Presented in abstract form at the 46th Annual Conference of the American Association of Zoo Veterinarians, Orlando, Fla, October 2014.
e. f. g. h. i. j. k.
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