Use of tricaine
methanesulfonate (MS-222) to induce anaesthesia in Puntiusdenisonii (Day, 1865) (Teleostei: Cypriniformes: Cyprinidae), a
threatened barb of the Western Ghats, India
T.V. Anna Mercy 1, V. Malika 2 & S. Sajan 3
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 -malikaramankutty@gmail.com,3 sajanpolayil@gmail.com (corresponding author)
doi: http://dx.doi.org/10.11609/JoTT.o3294.4414-9
Editor: Rajeev Raghavan,
St. Albert’s College, Kochi, India. Date
of publication: 26 May 2013 (online & print)
Manuscript details: Ms # o3294 | Received 06 August 2012 | Final received
19 April 2013 | Finally accepted 03 May 2013
Citation: Mercy, T.V.A., V. Malika & S. Sajan (2013). 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. Journal of Threatened Taxa 5(9):
4414–4419; http://dx.doi.org/10.11609/JoTT.o3294.4414-9
Copyright: © Mercy et al. 2013. Creative Commons Attribution 3.0 UnportedLicense. JoTT allows unrestricted use of this
article in any medium, reproduction and distribution by providing adequate
credit to the authors and the source of publication.
Funding: Marine Products Export Development
Authority, Kochi, India.
Competing Interest: None.
Acknowledgements: This
research is carried out as a part of the Marine Products Export Development
Authority (MPEDA) funded Project entitled, “Stock assessment and development of
captive breeding technology of Puntius denisonii endemic indigenous ornamental fish of Western
Ghats of India”. Thanks are due to anonymous reviewers for their criticisms which improved the quality of the manuscript.
Abstract: Anaesthesia is essential to minimize
stress and physical damage during handling of fish in captivity. In the present
study, induction time in Puntius denisonii (Day, 1865), an endangered aquarium fish
exposed to four concentrations 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 effective as
an anaesthetic with no side effects to both fish as
well as humans. An induction time
of less than or equal to three minutes, and a complete recovery in five minutes
was used a basis to record the anaesthesia stages for
different doses. The onsets of individual phases of anaesthesiaand recovery stages were also studied. Concentration of 150mg L -1 (induction 165±10 seconds and
recovery time 112±10 seconds) was determined as the lowest concentration that
induces anaesthesia in P. denisoniiin less than three minutes. Induction and recovery times were
dose-dependent. An inversely
proportional relationship was observed between concentrations of anaesthetic and induction time. This is the first study to investigate
the efficacy of different concentrations of MS-222 in Puntius denisonii and will be helpful to develop standardised techniques for transportation, captive
breeding and other ex-situ conservation plans for this endangered and endemic
barb.
Keywords: Anaesthetic,
handling, MS-222, Puntius denisonii,
Red-lined Torpedo Barb.
For figures, images, tables -- click here
Anaesthetics in ichthyologicalresearch greatly facilitate procedures including induction of spawning,
obtaining body length/weight, conducting gonadal biopsies and
transportation. Anaesthesiaand sedation is usually essential to minimize stress and physical damage during
handling of fish for routine husbandry operations (Summerfelt& Smith 1990; Iwama et al. 1997; Ross & Ross
1999). Commonly used anaesthetics in fishes include MS-222, benzocaine, quinaldine, chlorobutanol, phenoxyethanol and metomidate.
A number of considerations
should be taken care of when choosing an anaestheticincluding its efficacy, cost, availability, ease of use, and side effects on
fish, humans and the environment (Marking & Meyer 1985; Gilderhus& Marking 1987; Mylonas et al. 2005). Overdose of an anaestheticor retaining the fish in an anaesthetic bath for too
long leads to the fading of ventilation, hypoxia, and finally, respiratory and
cardiac collapse (Tytler & Hawkins 1981). The fading of ventilation is an
important warning sign suggesting that the exposure should be terminated (Hajek & Klyszejko 2004; Dziaman et al. 2005).
Tricaine methanesulfonate(MS-222) is one of the most widely used anaestheticsin fish research and husbandry (Ross & Ross 1999). MS-222 is a benzocaine derivative that
is absorbed across the gills, bio transformed in the liver and probably kidney,
and cleared primarily through the gills, with additional metabolites eliminated
in urine and bile (Maren et al. 1968; Harms
1999). Several studies have
evaluated the efficacy of MS-222 in various fish species (Roubachet 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).
Puntius 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
flowing through the Western Ghats. The species is much sought after in the international ornamental fish
trade and contributes to around 60% of India’s ornamental fish exports (Mittal
2009). However, due to
indiscriminate exploitation from the wild, the species is listed as Endangered
in the IUCN Red List of Threatened Species (Ali et al. 2011).
Captive breeding is
considered to be one of the solutions for ensuring sustainability and
conserving wild populations of endangered species (Fraser 2008). Although P.denisonii is well adapted to captive conditions
(Mercy 2009), it is very sensitive to handling and transportation, which
frequently results in high mortality (Ramachandran et
al. 2005).
Efforts to develop captive
breeding technology for P. denisonii have
revealed that the species is very difficult to handle for artificial
propagation (Mercy et al. 2010). When handled out of water, fish were observed to experience stress,
often leading to death within a very short period. Therefore, attempts were made to use anaesthetics to handle the fish during captive breeding.Using clove oil, handling stress was minimized and P. denisonii was bred successfully under hatchery
conditions (Sajan et al. 2012).
In the present paper, we
determine the effective concentration of Tricaine methanesulfonate (MS-222) that can be used as an anaesthetic for P. denisoniiduring captive breeding.
Materials and Methods
Experimental animals:Twenty individuals of captive 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 starting the
experiment, fish were reared in outdoor cement tanks (2000L) for a period of 14
days to get acclimatized with the controlled rearing conditions. Water quality conditions 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 duration of the experiment. Fish were fed with a commercial formulated diet with crude protein
(38%), crude fat (4.0%), crude fibre (3.0%), ash
(16%) and moisture (11%) twice a day (09.00 and 17.00 hr). All fish were healthy prior to, and
throughout the duration of the study.
Anaesthetic: Tricaine methanesulfonate(MS-222) (Argent Laboratories, Redmond WA, United States of America) was used
as the anaesthetic agent. MS-222 is an isomer of benzocaine with
the amine group in the meta position of
the benzene ring rather than the para position. MS-222 was solubilisedin deionized water and buffered with sodium bicarbonate, using a ratio of 1:1
(sodium bicarbonate: MS-222), providing a final concentration of 10 mg mL-1(pH 7.4). MS-222 dissolves well in
water and was therefore added directly to the anaestheticbath.
Experiment design: The experiment was carried
out at the indigenous fish breeding hatchery of the Kerala University of
Fisheries and Ocean Studies, Panangad, Ernakulam, Kerala (India), where techniques for the
standardization of captive 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 effect of MS-222 on the behaviour and anaesthetic performance on P. denisonii.
Dosages of anaesthesia for various teleosts provided in Weber et al. (2009) were used as base
information and different concentrations 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 concentration was added to the experiment
tank five minutes before the introduction of fish (Charoendat et al. 2009). Both treatment
and recovery water were taken from the tank, where the fish 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 anaesthetization include induction, maintenance and
recovery. A maximum duration from
initial anaesthetic exposure to induction (stage IV)
and the induction stage achieved usually depends on the dose and the length of
exposure. Generally, an ideal
anesthetic should produce anesthesia rapidly (e.g., less than 3 or 5 min),
allow a speedy recovery, not be toxic to fish and users, leave low tissue
residues and be inexpensive (Marking & Meyer 1985; Gilderhus& Marking 1987). The anaesthetic induction time is the period from the time when
an experimental fish is placed in the anaesthetictank until the time it does not respond to external stimuli. The recovery time is the period from the
time when an anaesthetized fish is placed in a recovery tank until it recovers
from anaesthetization with full equilibrium motion. Initial recovery time may vary from a
few seconds to minutes, depending on the anaestheticadministered. The lowest effective
concentration is the concentration that produces general anaesthesiawithin three minutes and allows the recovery within 10 minutes (Gilderhus 1990; Weyl et al.
1996). An induction time of three
minutes or less with complete recovery in five minutes suggested by Marking
& Meyer (1985) was used to record the anaesthesia-induction
stages for different dosages presented in this experiment (Table 2).
Experimental procedure: Each fish was randomly assigned
to a particular anaesthetic concentration. Water used for the experiment was
obtained from the same water system used in the tanks in which the fish were
held prior to the experiment. The
fish was then placed in 2L experimental water bath equipped with an air stone
and the stages of anaesthesia were recorded. When fish reached stage IV of anaesthesia (complete lack of voluntary movement), they
were removed from the anaesthetic bath and returned
to the recovery tank. Experiments were repeated four times. The induction and recovery times were
measured using an electronic stopwatch (Casio India). Each fish was subjected to monitoring
for any behavioural and/or health related changes for
another seven days.
Post-treatment mortality: After the experiment, fish
were transferred to circular cement tanks kept in outdoor facility (1000L) for
seven days to assess the post recovery mortality (Bambang2003; Charoendat et al. 2009; Pawar et al. 2011). During the post-treatment period, 50%
of the tank water was exchanged daily and the fish were fed twice a day
ad libitum with the commercial formulated feed given during pre-anaesthetic maintenance.
Data analysis: One way ANOVA was used to explain
the significance between dosage and induction time, as well as dosage and
recovery time. Induction time of anaesthesia was
recorded as the interval from initial exposure to the anaesthetic,
until the end of anaesthesia (stage IV). Duration for each recovery stage was
also recorded, as the interval from reintroduction of the fish to the recovery
tank. All data were reported as mean±S.D. Significant difference was tested at 95% confidence interval,
represented as P<0.05. The
results were processed and analysed with the SPSS
(Windows, Version 15.0).
Results
The induction time of Puntius denisoniidecreased with increasing concentrations of MS-222. The induction time was less than three
minutes for a dose of 150mg L-1 and therefore this was considered as
the best effective concentration of MS-222 for the induction of anaesthesia in P. denisonii. At 150mg L-1, the time
to reach a complete anesthesia (stage IV) (165±10 seconds) was significantly
different (P<0.05) from the other dosages (50, 100 and 200 mg L-1)
(Table 3). At lower concentrations
(50mg L-1 and 100mg L-1 ), more time (746±56
seconds and 506±20 seconds) was required to reach stage I and stage IV,
respectively.
There was a clear linear
pattern of decreasing induction time with increasing concentration of the anaesthetic, with the longest induction times for fish in
the group exposed to 100mg L-1 of MS-222 (506±20 seconds) and the
shortest for fish exposed to 200mg L-1 (97±5 seconds). Induction times generally decreased
significantly with increasing doses for MS-222 (Fig. 1).
The induction and recovery
stages at different concentrations of the MS-222 showed significant differences
(P<0.05). Induction time decreased with increasing concentration of MS-222
(P<0.05). All fish subjected to
the experiment recovered within three minutes. Recovery times increased with
increasing concentrations of MS-222 (P<0.05). At higher concentrations the time taken
to reach stage IV decreased, but the recovery time was extended. The study on
induction times in terms of fish weight was conducted on 20 fish weighing
between 13.0–20.0 g. No
significant correlation was observed between induction times and weight of the
fish (P>0.05). The recovered, P.denisonii that were observed in the post-
treatment period of seven days did not show any abnormal behaviourand/or mortality.
Discussion
The definition of efficacy
with regard to anaesthetics is more or less
subjective (Gilderhus & Marking 1987). Because stress responses vary widely
between species, it is often necessary to screen dosages of different anaesthetic agents for each cultured species (Ross &
Ross 1999). MS-222 is a water soluble anaesthetic and the
only one approved for use on fish in the United States (Pramodet al. 2010). This study
demonstrated that MS-222 is efficient in anaesthetizing P. denisonii, an important freshwater fish species in the
pet trade.
Induction times decreased
significantly with the increase in anaestheticconcentration (P<0.05), which are consistent with previous studies in
teleost fishes (Mattson & Riple 1989; Hseu et al. 1998; Mylonas et al.
2005; Gullian & Villanueva 2009; Weber et al.
2009; Heo & Shin 2010; Pramodet al. 2010; Pawar et al. 2011; Sajanet al. 2012). The effective
concentration of MS-222 causing anaesthesia to P. denisonii was 150mg L-1, similar to the
observations made by Pawar et al. (2011) in Hippocampuskuda (175mgL-1) and Donald et al.
(2009) in Oreochromis niloticus (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). Puntius denisonii exposed to 50mg L-1 of
MS-222 reached stage 1 in a maximum time of 746±56 seconds, indicating that none
of the fish exposed to this concentration of MS-222 was induced. Similar results were
reported by Sladky et al. (2001) in Piaractus brachypomus. Overall, the concentration of anaesthetic to induce fish varies with the concentration of
chemical required to bring them to a given level of anaesthesia,
their tolerance of a given chemical and their recovery time (Summerfelt & Smith 1990).
Statistical analysis showed
that the time of induction and recovery of P. denisoniiat different concentrations of MS-222 differ significantly (P<0.05). All fishes used in the experiment
recovered within three minutes. We
observed that if the exposure time was prolonged, the recovery also becomes
extended. Similar
observations were made by Grzegorz et al. (2006) on Cyprinus carpio L.
and Inoue et al. (2003) on juveniles of matrinxa Brycon cephalus. Prolonged recovery time with increased anaesthetic dosage has been reported in seven species of
tropical reef teleosts (Cunha & Rosa 2006), Oncorhynchus nerka(Woody et al. 2002), Rachycentron canadum (Gullian &
Villanueva 2009) and Dawkinsia filamentosus (Pramod et
al. 2010).
The effective dose of MS-222
for Puntius denisoniiis 150mg L-1. This dosage induced the fish through all stages of anaesthesia, without any mortality. Further studies on the effects of anaesthetics on the haematologicalprofile will considerably advance our understanding of anaesthesiain the husbandry of this threatened and endemic freshwater fish.
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