Short Communication Journal of Threatened Taxa | www.threatenedtaxa.org | 26 May 2013 | 5(9): 4414–4419 Use of tricaine methanesulfonate (MS-222) to induce anaesthesia in Puntius denisonii (Day, 1865) (Teleostei: Cypriniformes: Cyprinidae), a threatened barb of the Western Ghats, India T.V. Anna Mercy 1, V. Malika 2 & S. Sajan 3 ISSN Online 0974-7907 Print 0974-7893 OPEN ACCESS 1,3 Faculty of Fisheries, College of Fisheries, Kerala University of Fisheries and Ocean Studies (KUFOS), Panangad, Ernakulam, Kerala 682506, India 2 Faculty of Management Studies, College of Fisheries, Kerala University of Fisheries and Ocean Studies (KUFOS), Panangad, Ernakulam, Kerala 682506, India 1 annamercy2002@yahoo.co.in, 2 malikaramankuty@gmail.com, 3 sajanpolayil@gmail.com (corresponding author) Abstract: Anaesthesia is essenial to minimize stress and physical damage during handling of ish in capivity. In the present study, inducion ime in Punius denisonii (Day, 1865), an endangered aquarium ish exposed to four concentraions of MS-222 (50 mg L-1, 100 mg L-1, 150 mg L-1 and 200 mg L-1) was determined. MS-222 appears to be highly efecive as an anaestheic with no side efects to both ish as well as humans. An inducion ime of less than or equal to three minutes, and a complete recovery in ive minutes was used a basis to record the anaesthesia stages for diferent doses. The onsets of individual phases of anaesthesia and recovery stages were also studied. Concentraion of 150mg L -1 (inducion 165±10 seconds and recovery ime 112±10 seconds) was determined as the lowest concentraion that induces anaesthesia in P. denisonii in less than three minutes. Inducion and recovery imes were dose-dependent. An inversely proporional relaionship was observed between concentraions of anaestheic and inducion ime. This is the irst study to invesigate the eicacy of diferent concentraions of MS-222 in Punius denisonii and will be helpful to develop standardised techniques for transportaion, capive breeding and other ex-situ conservaion plans for this endangered and endemic barb. Keywords: Anaestheic, handling, MS-222, Punius denisonii, Redlined Torpedo Barb. Anaestheics in ichthyological research greatly facilitate procedures including inducion of spawning, obtaining body length/weight, conducing gonadal biopsies and transportaion. Anaesthesia and sedaion is usually essenial to minimize stress and physical damage during handling of ish for rouine husbandry operaions (Summerfelt & Smith 1990; Iwama et al. 1997; Ross & Ross 1999). Commonly used anaestheics in ishes include MS-222, benzocaine, quinaldine, chlorobutanol, phenoxyethanol and metomidate. A number of consideraions should be taken care of when choosing an anaestheic including its eicacy, cost, availability, ease of use, and side efects on ish, humans and the environment (Marking & Meyer 1985; Gilderhus & Marking 1987; Mylonas et al. 2005). Overdose of an anaestheic or retaining the ish in an anaestheic bath for too long leads to the fading of venilaion, hypoxia, and inally, respiratory and cardiac collapse (Tytler & Hawkins 1981). The fading of venilaion is an important warning sign suggesing that the exposure should be DOI: htp://dx.doi.org/10.11609/JoTT.o3294.4414-9 Editor: Rajeev Raghavan, St. Albert’s College, Kochi, India. Date of publicaion: 26 May 2013 (online & print) Manuscript details: Ms # o3294 | Received 06 August 2012 | Final received 19 April 2013 | Finally accepted 03 May 2013 Citaion: Mercy, T.V.A., V. Malika & S. Sajan (2013). Use of tricaine methanesulfonate (MS-222) to induce anaesthesia in Punius denisonii (Day, 1865) (Teleostei: Cypriniformes: Cyprinidae), a threatened barb of the Western Ghats, India. Journal of Threatened Taxa 5(9): 4414–4419; htp://dx.doi.org/10.11609/JoTT. o3294.4414-9 Copyright: © Mercy et al. 2013. Creaive Commons Atribuion 3.0 Unported License. JoTT allows unrestricted use of this aricle in any medium, reproducion and distribuion by providing adequate credit to the authors and the source of publicaion. Funding: Marine Products Export Development Authority, Kochi, India. Compeing Interest: None. Acknowledgements: This research is carried out as a part of the Marine Products Export Development Authority (MPEDA) funded Project enitled, “Stock assessment and development of capive breeding technology of Punius denisonii endemic indigenous ornamental ish of Western Ghats of India”. Thanks are due to anonymous reviewers for their criicisms which improved the quality of the manuscript. 4414 Anaesthesia in Punius denisonii Mercy et al. © S. Sajan terminated (Hajek & Klyszejko 2004; Dziaman et al. 2005). Tricaine methanesulfonate (MS-222) is one of the most widely used anaestheics in ish research and husbandry (Ross & Ross 1999). MS-222 is a benzocaine derivaive that is absorbed across the gills, bio transformed in the liver and probably kidney, and cleared primarily through the gills, with addiional metabolites eliminated in urine and bile (Maren et al. 1968; Harms 1999). Several studies have evaluated the eicacy of MS-222 in various ish species (Roubach et al. 2001; Walsh & Pease 2002; Iversen et al. 2003; King et al. 2005; Mylonas et al. 2005; Hajek et al. 2006; Pramod et al. 2010; Pawar et al. 2011). Punius denisonii (Teleostei: Cypriniformes: Cyprinidae) popularly known as the Red-lined Torpedo Barb or Miss Kerala (Image 1) is a small to medium sized barb endemic to the rivers lowing through the Western Ghats. The species is much sought ater in the internaional ornamental ish trade and contributes to around 60% of India’s ornamental ish exports (Mital 2009). However, due to indiscriminate exploitaion from the wild, the species is listed as Endangered in the IUCN Red List of Threatened Species (Ali et al. 2011). Capive breeding is considered to be one of the soluions for ensuring sustainability and conserving wild populaions of endangered species (Fraser 2008). Although P. denisonii is well adapted to capive condiions (Mercy 2009), it is very sensiive to handling and transportaion, which frequently results in high mortality (Ramachandran et al. 2005). Eforts to develop capive breeding technology for P. denisonii have revealed that the species is very diicult to handle for ariicial propagaion (Mercy et al. Image 1. Red-lined Torpedo Barb Punius denisonii 2010). When handled out of water, ish were observed to experience stress, oten leading to death within a very short period. Therefore, atempts were made to use anaestheics to handle the ish during capive breeding. Using clove oil, handling stress was minimized and P. denisonii was bred successfully under hatchery condiions (Sajan et al. 2012). In the present paper, we determine the efecive concentraion of Tricaine methanesulfonate (MS-222) that can be used as an anaestheic for P. denisonii during capive breeding. Materials and Methods Experimental animals:Twenty individuals of capive bred P. denisonii (Image 1) of uniform age (approximately two years old) and mean weight of 16.5±3.5 g (13.0– 20.0 g) were used for the study. Prior to staring the experiment, ish were reared in outdoor cement tanks (2000L) for a period of 14 days to get acclimaized with the controlled rearing condiions. Water quality condiions such as temperature, pH, alkalinity, hardness and ammonia were monitored and maintained within a narrow range of values. A photoperiod of 12L: 12D cycle (light period from 06.00–18.00 hr) was maintained throughout the duraion of the experiment. Fish were fed with a commercial formulated diet with crude protein (38%), crude fat (4.0%), crude ibre (3.0%), ash (16%) and moisture (11%) twice a day (09.00 and 17.00 hr). All ish were healthy prior to, and throughout the duraion of the study. Anaestheic: Tricaine methanesulfonate (MS-222) (Argent Laboratories, Redmond WA, United States of America) was used as the anaestheic agent. MS-222 is an isomer of benzocaine with the amine group in the meta posiion of the benzene ring rather than the para posiion. MS-222 was solubilised in deionized water and bufered with sodium bicarbonate, using a raio of 1:1 (sodium bicarbonate: MS-222), providing a inal concentraion of 10 mg mL-1 (pH 7.4). MS-222 dissolves well in water and was therefore added directly to the anaestheic bath. Experiment design: The experiment was carried out at the indigenous ish breeding hatchery of the Kerala University of Fisheries and Ocean Studies, Panangad, Ernakulam, Kerala (India), where techniques for the standardizaion of capive breeding and larval rearing of P. denisonii are being standardised (Mercy et al. 2010; Sajan et al. 2011). Preliminary studies were conducted to evaluate the efect of MS-222 on the behaviour and anaestheic performance on P. denisonii. Dosages of anaesthesia for various teleosts provided in Weber et Journal of Threatened Taxa | www.threatenedtaxa.org | 26 May 2013 | 5(9): 4414–4419 4415 Anaesthesia in Punius denisonii Mercy et al. al. (2009) were used as base informaion and diferent concentraions of MS-222 (50 mg L-1, 100 mg L-1, 150 mg L-1 and 200 mg L-1) were selected for the experiment. Each concentraion was added to the experiment tank ive minutes before the introducion of ish (Charoendat et al. 2009). Both treatment and recovery water were taken from the tank, where the ish were maintained and both bath systems were aerated throughout the procedure. Water quality parameters monitored are listed in Table 1. During the experiment, a number of guidelines recommended by Hicks (1989) were followed. Measures of anaesthesia: Stages of anaestheizaion include inducion, maintenance and recovery. A maximum duraion from iniial anaestheic exposure to inducion (stage IV) and the inducion stage achieved usually depends on the dose and the length of exposure. Generally, an ideal anestheic should produce anesthesia rapidly (e.g., less than 3 or 5 min), allow a speedy recovery, not be toxic to ish and users, leave low issue residues and be inexpensive (Marking & Meyer 1985; Gilderhus & Marking 1987). The anaestheic inducion ime is the period from the ime when an experimental ish is placed in the anaestheic tank unil the ime it does not respond to external simuli. The recovery ime is the period from the ime when an anaestheized ish is placed in a recovery tank unil it recovers from anaestheizaion with full equilibrium moion. Iniial recovery ime may vary from a few seconds to minutes, depending on the anaestheic administered. The lowest efecive concentraion is the concentraion that produces general anaesthesia within three minutes and allows the recovery within 10 minutes (Gilderhus 1990; Weyl et al. 1996). An inducion ime of three minutes or less with complete recovery in ive minutes suggested by Marking & Meyer (1985) was used to record the anaesthesia-inducion stages for diferent dosages presented in this experiment (Table 2). Table 1. Water Quality parameters in the experimental tanks Parameters Values pH 7.0±0.3 65±8.0 Alkalinity (mg L-1) Hardness ( mg L ) 70±5.0 Dissolved Oxygen ( mg L-1) 6.5±0.5 Temperature (0C) 27±0.5 -1 <0.01 Nitrite (mg L-1) Total ammonia ( mg L ) -1 4416 <0.00 Experimental procedure: Each ish was randomly assigned to a paricular anaestheic concentraion. Water used for the experiment was obtained from the same water system used in the tanks in which the ish were held prior to the experiment. The ish was then placed in 2L experimental water bath equipped with an air stone and the stages of anaesthesia were recorded. When ish reached stage IV of anaesthesia (complete lack of voluntary movement), they were removed from the anaestheic bath and returned to the recovery tank. Experiments were repeated four imes. The inducion and recovery imes were measured using an electronic stopwatch (Casio India). Each ish was subjected to monitoring for any behavioural and/or health related changes for another seven days. Post-treatment mortality: Ater the experiment, ish were transferred to circular cement tanks kept in outdoor facility (1000L) for seven days to assess the post recovery mortality (Bambang 2003; Charoendat et al. 2009; Pawar et al. 2011). During the post-treatment period, 50% of the tank water was exchanged daily and the ish were fed twice a day ad libitum with the commercial formulated feed given during pre-anaestheic maintenance. Data analysis: One way ANOVA was used to explain the signiicance between dosage and inducion ime, as well as dosage and recovery ime. Inducion ime of anaesthesia was recorded as the interval from iniial exposure to the anaestheic, unil the end of anaesthesia (stage IV). Duraion for each recovery stage was also recorded, as the interval from reintroducion of the ish to the recovery tank. All data were reported as mean±S.D. Signiicant diference was tested at 95% conidence interval, represented as P<0.05. The results were processed and analysed with the SPSS (Windows, Version 15.0). Results The inducion ime of Punius denisonii decreased with increasing concentraions of MS-222. The inducion ime was less than three minutes for a dose of 150mg L-1 and therefore this was considered as the best efecive concentraion of MS-222 for the inducion of anaesthesia in P. denisonii. At 150mg L-1, the ime to reach a complete anesthesia (stage IV) (165±10 seconds) was signiicantly diferent (P<0.05) from the other dosages (50, 100 and 200 mg L-1) (Table 3). At lower concentraions (50mg L-1 and 100mg L-1 ), more ime (746±56 seconds and 506±20 seconds) was required to reach stage I and stage IV, respecively. There was a clear linear patern of decreasing inducion ime with increasing concentraion of the Journal of Threatened Taxa | www.threatenedtaxa.org | 26 May 2013 | 5(9): 4414–4419 Anaesthesia in Punius denisonii Mercy et al. Table 2. Stages of anaestheic inducion (ater Bowser 2001) Stages 0 Descriptor General Behaviour response of ish Normal Reacive to external simuli; opercular rate and muscle tone normal I Light sedaion Slight loss of reacivity to external simuli; opercular rate slightly decreased; equilibrium normal II Deep sedaion Total loss of reacivity to all but strong external simuli; Slight decrease in opercular rate; equilibrium normal III Parial loss of equilibrium Parial loss of muscle tone; swimming erraic; increased opercular rate; reacivity only to strong tacile and vibraion simuli IV Total loss of equilibrium Total loss of muscle tone and equilibrium; slow but regular opercular rate; loss of spinal relexes V Medullary collapse Respiratory movement ceases anaestheic, with the longest inducion imes for ish in the group exposed to 100mg L-1 of MS-222 (506±20 seconds) and the shortest for ish exposed to 200mg L-1 (97±5 seconds). Inducion imes generally decreased signiicantly with increasing doses for MS-222 (Fig. 1). The inducion and recovery stages at diferent concentraions of the MS-222 showed signiicant diferences (P<0.05). Inducion ime decreased with increasing concentraion of MS-222 (P<0.05). All ish subjected to the experiment recovered within three minutes. Recovery imes increased with increasing concentraions of MS-222 (P<0.05). At higher concentraions the ime taken to reach stage IV decreased, but the recovery ime was extended. The study on inducion imes in terms of ish weight was conducted on 20 ish weighing between 13.0–20.0 g. No signiicant correlaion was observed between inducion imes and weight of the ish (P>0.05). The recovered, P. denisonii that were observed in the post- treatment period of seven days did not show any abnormal behaviour and/or mortality. Discussion The deiniion of eicacy with regard to anaestheics is more or less subjecive (Gilderhus & Marking 1987). 560 Table 3. Timing (seconds) of anaesthesia and recovery phases in Punius denisonii exposed to various MS-222 concentraions (mean±S.D) Stages of anaesthesia MS 222 concentraions [mg L-1] 50 100 150 200 Light sedaion (I) 746±56 59±4 16± 1 10±1 Deep sedaion(II) --- 192±5 46±2 31±2 Parial loss of equilibrium (III) --- 318±8 122±7 57±4 --- 506±20 165±10 97±5 42±5 84±6 114±6 154±7 Total loss of equilibrium (IV) Recovery ime Because stress responses vary widely between species, it is oten necessary to screen dosages of diferent anaestheic agents for each cultured species (Ross & Ross 1999). MS-222 is a water soluble anaestheic and the only one approved for use on ish in the United States (Pramod et al. 2010). This study demonstrated that MS-222 is eicient in anaestheizing P. denisonii, an important freshwater ish species in the pet trade. Inducion imes decreased signiicantly with the increase in anaestheic concentraion (P<0.05), which are consistent with previous studies in teleost ishes (Matson & Riple 1989; Hseu et al. 1998; Mylonas et al. 2005; Gullian & Villanueva 2009; Weber et al. 2009; R2 = 0.9537 180 160 140 400 120 320 100 240 80 60 160 Recovery ime (s) Inducion ime (s) 480 40 80 20 R2 = 0.9301 0 0 0 50 100 150 200 200 150 100 50 0 MS-222 dose (mg L-1) MS-222 dose (mg L-1) Figure 1. Inducion ime and recovery ime in relaion to MS-222 concentraions (mg L ) in Punius denisonii (P<0.05, n=20) -1 Journal of Threatened Taxa | www.threatenedtaxa.org | 26 May 2013 | 5(9): 4414–4419 4417 Anaesthesia in Punius denisonii Mercy et al. Heo & Shin 2010; Pramod et al. 2010; Pawar et al. 2011; Sajan et al. 2012). The efecive concentraion of MS222 causing anaesthesia to P. denisonii was 150mg L-1, similar to the observaions made by Pawar et al. (2011) in Hippocampus kuda (175mgL-1) and Donald et al. (2009) in Oreochromis niloicus (100–200 mg L-1) Cyprinus carpio and Carrassius auratus (60–300 mgL-1), but higher than those obtained for temperate species such as Salmo gairdneri, Cyprinus carpio and Pimephales promelas (50–100 mg L-1) by Ross & Ross (1999), and Sylvester & Holland (1982). Punius denisonii exposed to 50mg L-1 of MS-222 reached stage 1 in a maximum ime of 746±56 seconds, indicaing that none of the ish exposed to this concentraion of MS-222 was induced. Similar results were reported by Sladky et al. (2001) in Piaractus brachypomus. Overall, the concentraion of anaestheic to induce ish varies with the concentraion of chemical required to bring them to a given level of anaesthesia, their tolerance of a given chemical and their recovery ime (Summerfelt & Smith 1990). Staisical analysis showed that the ime of inducion and recovery of P. denisonii at diferent concentraions of MS-222 difer signiicantly (P<0.05). 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