Evaluation of efficacy of selected anesthetic agents on blood-spotted crab (Portunus sanguinolentus).
KEY WORDS: anesthesia, immobilization, Portunus sanguinolentus, xylazine, clove oil
Crustaceans have received little attention regarding anesthesia and analgesia. The live export market for crustaceans, nature photography, and laboratory research has increased the interest in anesthesia of crustaceans. Crustaceans, however, respond differently to certain commonly used anesthetics possibly because their postsynaptic receptor sites do not respond to commonly used drugs (Ross & Ross 2008). Those anesthetic agents depending on the animal's size (body weight) and procedure (intramuscular injections), MS-222, isobutyl alcohol, and intramuscular (IM) injections of lidocaine, ketamine, or xylazine have been reported (Oswald 1977, Brown et al. 1996, Ferraro & Pressacco 1996, Gardner 1997). According to the study by Chou and Heath (2000), the choice of anesthetic generally depends on availability, cost-effectiveness, ease of use, nature of the study, and user safety. Some anesthetics conventionally used for aquatic vertebrates, such as MS-222 (Sandoz), are ineffective when used by the immersion method for decapods (Oswald 1977).
Gardner (1997) tested various treatments like hypercapnia seawater bath [carbon dioxide (COz) addition], magnesium sulfate (MgS[O.sub.4]) bath, clove oil bath, and xylazine-HCl by injection. He found that crabs could be paralyzed within 30 min with clove oil at a dose of 0.125 ml/1, indicating that the time required for induction and recovery with clove oil is relatively useful in crustaceans. Keene et al. (1998) mentioned clove oil as a low-cost, ready-made, natural, safe, and most important anesthetic oil. Three Pacific Coast crab species, Cancer Magister, Hemigrapsus oregonensis, and Pugettia producta, were evaluated with clove oil as an anesthetic. Induction was stress free and dose dependent, with effective ranges of 0.5-1.5 ml/1, 1-3 ml/1, and 0.015-0.25 ml/1, respectively, in the three species (Morgan et al. 2001). Injectable anesthesia is possible in Decapoda and the most comprehensive study of the crabs, Cancer and Carcinus, was by Oswald (1977). Xylazine has shown good anesthetic effects in adult giant crabs when used at doses between 16 and 22 mg/kg (Gardner 1997) or in common shore crabs at 70 mg/kg (Oswald 1977). Carbon dioxide remains a popular agent to immobilize insects in entomological research, although multiple side effects, including convulsion and excitation at induction, are well recognized and mortality is high (Nicolas & Sillans 1989). Carbon dioxide is an effective anesthetic for fish (McFarland & Klontz 1969) but had, until recently, only been occasionally used as a sedative for transportation (Leitritz & Lewis 1980). Anderson et al. (1997) noted that induction and recovery times are relatively long and anesthesia is shallow in comparison with other drugs. When necessary, over dosage of immobilization drugs is an acceptable means of euthanasia (Harms & Bakal 1995). Inhalant drugs at 5-10 times the anesthetic concentration of a particular species are usually chosen, although injectable agents can also be used (Ross & Ross 1984). Although anesthesia minimizes handling stress, it is inherently stressful (Bressler & Ron 2004). A long recovery period is essential when animals are handled for an extended duration; otherwise, rapid recovery is preferred (Marking & Meyer 1985, Stoskopf 1993).
MATERIALS AND METHODS
The effects of clove oil, xylazine, C[O.sub.2], crush ice, and MgS[O.sub.4] were evaluated separately. Normal, mature crabs (n = 55) of Portunus sanguinolentus with mean body weight of 69.98 [+ or -] 3.56 g (mean [+ or -] SE) were used for independent experiments.
Blood-spotted crabs were collected from commercial fishers from the tangle fishery harbor during September 2013 to April 2014. Crabs were acclimated in two separated 20-1 glass tanks with thickness (7 cm), sand layer 1,800 [cm.sup.2] bottoms, used seawater filtration and aerated sea water, at the temperature of 28 [+ or -] 1[degrees]C, and fed with commercial raw fish food. Crabs were acclimatized to laboratory conditions for 3 days before the experiment in 20-1 glass tanks supplied with aerated sea water at 28 [+ or -] 1[degrees]C and under natural light conditions. Experimental tank was partly covered with black polyvinyl chloride foil and provided natural conditions.
Several parameters were considered for acclimated crabs. They were temperature (28 [+ or -] 1[degrees]C), pH (7.5), turbidity (2.35), C[O.sub.2] concentration (40 mg/1), and salinity (4.4). The Crabs were fed with commercial raw fish food and were starved for 24 h before the experiment.
Before the experiment, the crabs were observed on a daily basis to evaluate their health conditions as indicated by their activities. Three days before the experiment, crabs were randomly selected from each tank and sampled for behavior and morphological examinations. Microsoft Excel software and Graph Prism software were used for the data analysis.
Clove Oil Test
Clove oil is not completely soluble in sea water below 28[degrees]C. When the clove oil was used without any solvent, the oil required vigorous shaking and produced an oily layer on the sea water surface. When absolute ethanol was used as a solvent, a completely dissolved light yellowish brown mixture was received without an oily layer that was very easy to use for experimental purposes. Therefore, absolute ethanol has been used as a solvent in this experiment, even though the sea water temperature was 28[degrees]C. Twenty-six crabs were individually tested in each of the four concentrations, 0.125, 0.15, 0.2, and 0.25 ml/1 clove oil with the desired concentration of clove oil dissolved in absolute ethanol at a ratio of 1:7,1:5,1:4, and 1:3 to make it sea water soluble. The crabs were exposed (immersed in clove oil) for 30 min to the anesthetic bath. After the exposure, crabs were introduced to natural conditions in the experimental container.
Xylazine (50, 60, and 70 mg/kg) was injected into the hemocoels of a separate group of normal, mature crabs (n = 13), through the arthrodial membrane between the carapace and the walking leg 4. Crab was injected by a syringe (1 cc) with 25 gauge needles (Figure 1).
Carbon Dioxide/Lovv pH
NaHC[O.sub.3(8)] = [Na.sub.2]C[O.sub.3(s)] + [H.sub.2][O.sub.(l)] +C[O.sub.2(g)] [up arrow]
When burn sodium bicarbonate releases C[O.sub.2] gas, saturated sea water (165 mg/l) will do so much more rapidly if the pH is decreasing. One-liter sea water bath was used without aeration and crabs were immersed for 15 min. During immersed bath, C[O.sub.2] concentration (165 mg/l) and sea water pH (5.5) were maintained. The effects of C[O.sub.2] were evaluated separately, in normal, mature crabs (n = 5) of Portunus sanguinolentus. The anesthetic properties of C[O.sub.2] were used as anesthetic with blood-spotted crabs. It is a colorless, odorless, nonflammable gas, which is extremely soluble in sea water making it a useful alternative for crabs. The use of this C[O.sub.2] as an anesthetic is a relatively inexpensive technique. Exposure of carbon dioxide in sea water causes acidosis and a reduction in blood pH and it was effected in anesthetic on blood-spotted crabs.
Magnesium Sulfate Solution
A group of individually separated crabs (n = 4) was kept immersed in a 21 saturated MgS[O.sub.4] solution mixed with an equal volume of seawater and another group was kept immersed in a mixture containing 75% saturated MgS[O.sub.4] and 25% sea water. Crabs were observed for signs of anesthesia.
The individually separated crabs (n = 5) were kept immersed in a used cooling with fresh water crush ice. Experimental container (2 l) crabs were introduced to anesthetic for 10 min. Cooling caused anesthetic effect on all the crabs within few seconds of introduction to the experimental container, and recovery took shorter time. In most cases, blood-spotted crabs' four walking legs were contracted.
Behavior and response (such as moving, walking legs, taking a defensive posture or an ability to use claws) to visual and tactile stimuli were assessed. Crabs were considered anesthetized when they became immobilized, that is, they lost the ability to regain the position when placed on their back. The time required for the crabs to reach a total loss of equilibrium and not respond to stimuli was determined using a time recorder (stop watch). The behavioral changes were recorded and were considered as the beginning of the recovery period, and total recovery was reached when the crabs turned right side up and stayed in one position in the container. Anesthetic and recovery times were judged visually and measured to the nearest second. A long recovery period is essential when animals are handled for an extended duration; otherwise, rapid recovery is preferred (Marking & Meyer 1985, Stoskopf 1993). Anesthetic and recovery times are expressed as means ([+ or -]SE). Recovered crabs were reared in natural conditions in the tanks for 1 week to observe the posttreatment survival and released into their natural environment. The anesthetic and recovery times, behavioral changes during anesthesia, and survival rate are important factors to consider while anesthetizing animals.
Cooling crush ice and clove oil at the concentration of 0.15, 0.2, and 0.25 ml/l sea water showed clear anesthetic effect. Clove oil 0.25 ml/l sea demonstrated a stress-free, smooth induction and took approximately 5 min. Anesthesia was maintained for 100.6 [+ or -] 8.1 (mean [+ or -] SE) min (Figures 2 and 3).
As already mentioned, the actual anesthetic effect achieved using clove oil will depend on the dose or level of agent used and the duration of exposure. When clove oil was inducted as an anesthetic agent, recovery times were relatively long and anesthesia was shallow in comparison with other drugs. The induction of anesthesia with clove oil was smooth and the time taken for induction appeared to be decreasing with increasing doses. Morgan et al. (2001) have also evaluated clove oil as an anesthetic in three other crab species and have identified clove oil as a smooth inducer of anesthesia. Cho and Heath (2000) mentioned that clove oil is preferable for field-based research because, currently, no withdrawal period is necessary and it poses no known environmental hazard.
The induction of anesthesia with xylazine was not smooth and the induction time seemed to be decreasing with increasing doses.
When 0.20 ml/l clove oil concentration was used, the crab's induction time was 13.5 [+ or -] 1.12 (mean [+ or -] SE) min and recovery required about 41.7 [+ or -] 7.3 (mean [+ or -] SE) min. And when a lower dose of 0.15 ml/l clove oil concentration was used, the crab's induction time was 20 [+ or -] 2.26 (mean [+ or -] SE) min and recovery required about 26.6 [+ or -] 2.7 (mean [+ or -] SE) min. Female blood-spotted crab was not anesthetic with 0.15 ml dosage. High doses showed effect (greater than 0.20 and 0.25 ml/l) with very slow induction time and the recovery time prolonged.
Xylazine, when used at the rate of 70 mg/kg dose, consistently caused rapid death and most of the blood-spotted crabs have not been recovered. Injection of 50 and 60 mg/kg solution of xylazine resulted in partial sedation. Crabs that were injected with 70 mg/kg xylazine did not fully recover and after the experiment they died. Some high dosage-injected blood-spotted crabs died during the experiment (Figures 4 and 5).
The induction occurred in 9.33 [+ or -] 2.19 (mean [+ or -] SE) with the dose rate of 50 mg/kg, whereas it took 6.33 [+ or -] 2.33 (mean [+ or -] SE) at the dose rate of 70 mg/kg. Anesthesia lasted for 21 [+ or -] 4.04 (mean [+ or -] SE) and 27 [+ or -] 9.45 (mean [+ or -] SE) with dose rates of 50 and 60 mg/kg, respectively. Oswald (1977) has also shown that xylazine resulted in a sleeping time of 45 min, when used at the rate of 70 mg/kg when used at the rate of 70 mg/kg that were injected with Blood-spotted crabs. Also injected 50 and 60 mg/kg of xylazine rapidly lost consciousness, but were not completely anesthetic also there are walking legs slowly move with spasms, reach recovery time. Side effects such as bradycardia, extra systoles, and dysrhythmias have been reported with high dosages (70 mg/kg) (Oswald 1977). Crabs were more effectively anesthetic when in sea water with a slight 5.5 pH. Carbon dioxide concentrations of 165 mg/l at pH 5.5 were most effective, inducing induction time of 233.33 [+ or -] 24.77 sec (mean [+ or -] SE) and anesthetic time of 100.67 [+ or -] 21.4 sec (mean [+ or -] SE). With MgS[O.sub.4], the induction was smooth and slow. Complete anesthesia, however, was not observed. The crabs appeared sedated and were immobile. The duration of sedation and immobilization lasted for 22-25 min. for both 50% and 75% MgS[O.sub.4] concentrations. The 50% and 75% MgS[O.sub.4] concentration had no anesthetic effect on crabs for 30 min of exposure. Cooling experiment anesthetic of all the crabs was 59.25 [+ or -] 4.09 sec and recovery took 87.25 [+ or -] 11.50 sec. In most cases, regaining the right body position was sudden, not commenced by walking legs and hand movement. Leakage of a brownish green fluid was observed in some blood-spotted crabs during the induction.
To conclude, clove oil and other anesthetic agents induced anesthesia in blood-spotted crabs, in this study. However, the induction was smooth with clove oil and C[O.sub.2] compared to other anesthetic agents. The duration of anesthesia was also longer with clove oil compared to other anesthetic agents. Recovery was time related, increasing exponentially with dose, and anesthetic time was prolonged. Crabs, Portunus sanguinolentus, can be anesthetized with clove oil depending on the clove oil dose used. Xylazine is generally accepted as a noneffective agent to produce anesthesia in blood-spotted crabs and not suitable as agents for anesthesia and euthanasia. Seventy mg/kg xylazine also has been used in crabs and those crabs were not obtaining good anesthesia with a very short induction time and anesthetized. Crabs that were injected with 70 mg/kg xylazine died after the experiment. Xylazine is not a suitable anesthetic for blood-spotted crabs. Clove oil and its extracts have become better anesthetic agents for blood-spotted crabs because of their wide availability, low cost, and long anesthetic times when compared to xylazine. Inhaling overdoses of carbon dioxide consistently caused rapid death, which can be used effectively for euthanasia. Controlled carbon dioxide anesthesia, however, is more difficult to achieve than other drugs and has short recovery time. Cooling method is a relatively inexpensive technique to be used as an anesthetic. Similar to C[O.sub.2], cooling obtained short induction time and short anesthetic time. Complete anesthesia was not observed with MgS[O.sub.4]. Thus, in this study, clove oil is identified as the most suitable anesthetic agent for P. sanguinolentus.
We thankfully acknowledge. Dr. Shyamalie D Senadheera, who is Academic Head of Department of Fisheries and Marine Science, Ocean University of Sri Lanka. Especially, we would like to acknowledge Dr. M. P. Kumara, senior lecturer of Ocean University of Sri Lanka. Also, we would like to thank Tangalle fisheries sector for their cooperation during the research works and the analysis.
Anderson, W. G., R. S. Mckinley & M. Colavecchia. 1997. The use of clove oil as an anesthetic for rainbow trout and its effects on swimming performance. N. Am. J. Fish. Manage. 17:301-307.
Bressler, K. & B. Ron. 2004. Effect of anesthetics on stress and the innate immune system of gilthead bream (Sparusaurata). Isr. J. Aquacult. 56:5-13.
Brown, P. B., M. R. White, J. Chaille, M. Russell & C. Oseto. 1996. Evaluation of three anesthetic agents for crayfish (Orconectesvirilis). J. Shellfish Res. 15:433-435.
Chou, G. K. & D. D. Heath 2000. Comparison of tricane methane sulphonate (MS 222) and clove oil anaesthesia effects on the physiology of juvenile Chinook salmon Oncorhynchus tshawytscha. Aquacult. Res. 31:537-546.
Ferraro, E. A. & L. Pressacco. 1996. Anesthetic procedures for Crustacea. An assessment of isobutanol and xylazine as general anesthetics for Squiila mantis (Stomatopoda). Mem. Biol. Mar. Oceanogr. 12:471-475. Gardner, C. 1997. Options of humanely immobilizing and killing crabs. J. Shellfish Res. 16:219-224.
Harms, C. A. & R. S. Bakal. 1995. Techniques in fish anesthesia. J SmExotAnim Med 3:19-25.
Keene, J. L., D. L. Noakes, R. D. Moccia & C. G. Soto. 1998. The efficacy of clove oil asan anesthetic for rainbow trout, Oncorhynchusmykiss (Walbaum). Aquacult. Res. 29:89-101.
Leitritz, E. & R. C. Lewis. 1980. Trout and salmon culture (hatchery methods). Calif. Dep. Fish Game Fish Bull. 164:197.
Marking, L. L. & F. P. Meyer. 1985. Are better anesthetics needed in fisheries? Fisheries 10:2-5.
McFarland, W. N. & G. W. Klontz. 1969. Anesthesia in fishes. Fed. Proc. 28:1535-1540.
Morgan, J., C. Cargill & E. Groot. 2001. The efficacy of clove oil as an anesthetic for decapod crustaceans, shellfish health-risks and management: proceedings of a special session held at Aquaculture Canada 2001, Halifax. Bull. Aquacult. Assoc. Can. 101 103:27-31.
Nicolas, G. & D. Sillans. 1989. Immediate and latent effects of carbon dioxide on insects. Annu. Rev. Entomol. 34:97-116.
Oswald, R. L. 1977. Immobilization of decapod crustacean for experimental procedures. J. Mar. Biol. Ass. U.K. 57:715-721.
Ross, L. G. & B. Ross. 1984. Anaesthetic and sedative techniques for fish. Glasgow, United Kingdom: Nautical Press.
Ross, L. G. & B. Ross. 2008. Anaesthesia of aquatic invertebrates. In: Ross, L.G. & B. Ross, editors. Anesthetic and sedative techniques for aquatic animals, 3rd edition. United Kingdom: Blackwell Publishers.
Stoskopf, M. 1993. Anaesthesia. In: Brown, L., editor. Aquaculture for veterinarians, pp. 161-167.
AMAL D. PREMARATHNA, (1) * IDUNIL PATHIRANA, (2) R. P. V. JAYANTHA RAJAPAKSE (3) AND ERANDI PATHIRANA (1)
(1) Faculty of Fisheries & Marine Science, Ocean University of Sri Lanka, Mahawela Road, Tangalle, Sri Lanka 82200; (2) Department of Animal Science, University of Ruhuna, Kamhurupitiya, Sri Lanka; (3) Department of Veterinary Pathobiology, Faculty of Veterinary Medicine and Animal Science, University of Peradeniya, Peradeniya, Sri Lanka
* Corresponding author. E-mail: email@example.com
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|Author:||Premarathna, Amal D.; Pathirana, Idunil; Rajapakse, R.P.V. Jayantha; Pathirana, Erandi|
|Publication:||Journal of Shellfish Research|
|Date:||Apr 1, 2016|
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