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 malikaramankutty@gmail.com, 3 sajanpolayil@gmail.com (corresponding author)
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 anaesthesia and 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. denisonii in 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, Redlined Torpedo Barb.
Anaesthetics in ichthyological research greatly
facilitate procedures including induction of spawning,
obtaining body length/weight, conducting gonadal
biopsies and transportation. Anaesthesia and 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 anaesthetic including 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
anaesthetic or 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
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 Unported License. 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.
4414
Anaesthesia in Puntius 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 anaesthetics in 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 (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).
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.
Image 1. Red-lined Torpedo Barb Puntius denisonii
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. denisonii during
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 solubilised in 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
anaesthetic bath.
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
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Anaesthesia in Puntius denisonii
Mercy et al.
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 anaesthetic tank 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 anaesthetic administered. The
lowest effective concentration is the concentration that
produces general anaesthesia within 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).
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 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 (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 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 denisonii decreased
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
Journal of Threatened Taxa | www.threatenedtaxa.org | 26 May 2013 | 5(9): 4414–4419
Anaesthesia in Puntius denisonii
Mercy et al.
Table 2. Stages of anaesthetic induction (after Bowser 2001)
Stages
Descriptor
General Behaviour response of fish
0
Normal
Reactive to external stimuli; opercular rate and muscle tone normal
I
Light sedation
Slight loss of reactivity to external stimuli; opercular rate slightly decreased; equilibrium normal
II
Deep sedation
Total loss of reactivity to all but strong external stimuli; Slight decrease in opercular rate; equilibrium normal
III
Partial loss of equilibrium
Partial loss of muscle tone; swimming erratic; increased opercular rate; reactivity only to strong tactile and vibration
stimuli
IV
Total loss of equilibrium
Total loss of muscle tone and equilibrium; slow but regular opercular rate; loss of spinal reflexes
V
Medullary collapse
Respiratory movement ceases
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
behaviour and/or mortality.
Discussion
The definition of efficacy with regard to anaesthetics
is more or less subjective (Gilderhus & Marking 1987).
560
Table 3. Timing (seconds) of anaesthesia and recovery phases
in Puntius denisonii exposed to various MS-222 concentrations
(mean±S.D)
Stages of anaesthesia
MS 222 concentrations [mg L-1]
50
100
150
200
Light sedation (I)
746±56
59±4
16± 1
10±1
Deep sedation(II)
---
192±5
46±2
31±2
Partial 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 time
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 (Pramod et 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 anaesthetic concentration (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;
R2 = 0.9537
180
160
140
400
120
320
100
240
80
60
160
Recovery time (s)
Induction time (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. Induction time and recovery time in relation to MS-222 concentrations
(mg L ) in Puntius denisonii (P<0.05, n=20)
-1
Journal of Threatened Taxa | www.threatenedtaxa.org | 26 May 2013 | 5(9): 4414–4419
4417
Anaesthesia in Puntius denisonii
Mercy et al.
Heo & Shin 2010; Pramod et al. 2010; Pawar et al. 2011;
Sajan et al. 2012). The effective concentration of MS222 causing anaesthesia to P. denisonii was 150mg L-1,
similar to the observations made by Pawar et al. (2011)
in Hippocampus kuda (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. denisonii at 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 denisonii is
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 haematological
profile will considerably advance our understanding of
anaesthesia in the husbandry of this threatened and
endemic freshwater fish.
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