Aquaculture 311 (2011) 155161

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Aquaculture
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / a q u a - o n l i n e

Comparative efcacy of four anaesthetic agents in the yellow seahorse, Hippocampus

kuda (Bleeker, 1852)
H.B. Pawar, S.V. Sanaye, R.A. Sreepada , V. Harish, U. Suryavanshi, Tanu, Z.A. Ansari
Aquaculture Laboratory, National Institute of Oceanography (NIO), Council of Scientic & Industrial Research (CSIR), Dona Paula, Goa-403 004, India

a r t i c l e

i n f o

Article history:
Received 16 September 2010
Received in revised form 1 December 2010
Accepted 1 December 2010
Available online 9 December 2010
Keywords:
MS-222
Benzocaine
Clove oil
2-phenoxyethanol
Induction time
Recovery time

a b s t r a c t
In modern aquaculture, anaesthetics play an important role in reducing handling stress and mortality. In this
investigation, the efcacy of four anaesthetic agents (MS-222, benzocaine, clove oil and 2-phenoxyethanol) was
compared in captive-bred yellow seahorse, Hippocampus kuda (Bleeker, 1852). The lowest effective
concentrations based on the efcacy criteria of complete anaesthetic induction within 180 s and recovery
within 300 s were determined to be 125 mg L1 (induction 115 16 s and recovery time 246 36 s) for MS222, 175 mg L1 (induction 175 19 and recovery time 354 55 s) for benzocaine, 50 mg L1 (induction 115
28 and recovery time 385 37 s) for clove oil, 1000 l L1 (induction 176 22 and recovery time 271 37 s) for
2-phenoxyethanol. Induction and recovery times for adult H. kuda anaesthetised with anaesthetic agents were
dose-dependent (P b 0.05). The onset of individual phases of anaesthesia and recovery times depended
signicantly on the concentration of the anaesthetic used (P b 0.05). An inverse exponential relationship was
observed between concentrations of anaesthetic and induction time, whereas exponential relationships were
observed between concentrations and recovery times for all anaesthetic agents evaluated. Amongst all tested
anaesthetics, MS-222 and clove oil were proven to be most effective and the latter appears to meet many of the
criteria of an ideal sh anaesthetic. The study has potential signicance with regards to seahorse husbandry in
terms of stress, survival and production efciency.
2010 Elsevier B.V. All rights reserved.

1. Introduction
The use of anaesthetic agents is common in modern aquaculture
and has practical relevance in diverse husbandry manipulations such
as selection of sh, their measurement, sampling, tagging, transportation, articial reproduction and surgery procedures (Roubach et al.,
2005; Weber et al., 2009). These routine operations often induce a
physiological stress response which may result in undesirable
outcomes such as immunosuppression and growth retardation
(Roubach et al., 2005; Heo and Shin, 2010). The use of anaesthetic
agents may reduce stress-induced damage to the sh, but may also
attenuate the physiological response to stress (Weber et al., 2009). A
number of chemicals have proved effective in the anaesthetisation of
sh each having its own merits and demerits (Velek et al., 2006). The
most commonly used anaesthetics in aquaculture are MS-222 and 2phenoxyethanol (PE) (Svoboda and Kolarova, 1999). Recent studies
have evaluated the anaesthetic efcacy of clove oil, the main active
component (70% to 90%) of which is eugenol [2-methoxy-4-(2propenyl) phenol], (Walsh and Pease, 2002; Iversen et al., 2003; King
et al., 2005; Mylonas et al., 2005; Roubach et al., 2005; Hajek et al.,
2006), as well as benzocaine (ethyl aminobenzoate) (Heo and Shin,

Corresponding author. Tel./fax: +91 832 2450 426/606.

E-mail address: [email protected] (R.A. Sreepada).
0044-8486/$ see front matter 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.aquaculture.2010.12.007

2010; Pramod et al., 2010) in teleost species. These authors report

signicant species-wise differences in response to different tested
anaesthetics.
Choosing an appropriate anaesthetic depends mainly on its
effectiveness in immobilizing sh with good recovery rates (Gilderhus
and Marking, 1987; Burka et al., 1997). An ideal anaesthetic should
possess several attributes such as non-toxic, inexpensive, simple to
administer and result in rapid induction and calm recovery (TrevesBrown, 2000). The efcacy is conditioned by environmental (temperature, pH and salinity) and biological factors (size, weight, lipid
content and sh species) (Burka et al., 1997; Ross and Ross, 1999). It is
often advisable to identify the lowest effective doses of different
anaesthetics in a specied species, as the responses to the same
anaesthetic may vary considerably among different species (King et al.,
2005).
Seahorses and other Syngnathid shes are in great demand
worldwide. They form an important ingredient in traditional Chinese
medicine, as well as popular ornamental sh and curios. Different
seahorse species are harvested on a large scale, and traded by at least
77 nations in high volumes and various product forms (McPherson
and Vincent, 2004). It has been reported that at least 25 million
seahorses (N70 metric tonnes, dry weight) are traded globally
(Project Seahorse, 2006; Salin and Nair, 2006). In addition, more
than one million live seahorses are caught for the aquarium trade,
mostly destined for sale in North America, Europe, Japan and Taiwan

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H.B. Pawar et al. / Aquaculture 311 (2011) 155161

(Vincent, 1996). Seahorse aquaculture has been expanding considerably in the number and size of aquaculture operations and number of
species cultured to sustain increasing trade in traditional medicine,
aquarium shes and curios (Koldewey and Martin-Smith, 2010).
Some researchers have cited the use of anaesthetics in different
seahorse species (Woods, 2002; Woods and Martin-Smith, 2004;
Koldewey, 2005; Morgan and Bull, 2005; Castro et al., 2008; Mattle
and Wilson, 2009; Otero-Ferrer et al., 2010), however practical details
on their efcacy were seldom outlined. Before recommending the use
of a particular anaesthetic, a range of induction and recovery times
must be established to assess its efcacy. Such information would
therefore be necessary whenever a culture technology for a potential
species is being developed.
Given the growing interest in the culture of seahorses and lack of
detailed practical information on the administration of anaesthetics,
the overall aim of the present study was to determine the lowest
effective concentrations (LEC) based on anaesthetic induction and
recovery times of four most common sh anaesthetic agents (MS-222,
benzocaine, clove oil and PE) that could be efciently used in the
yellow seahorse, Hippocampus kuda (Bleeker, 1852) under controlled
conditions. Information on stages of induction and recovery associated with exposure of H. kuda to a wide range of concentrations of
anaesthetic agents provided here should be of practical interest to
seahorse aquaculturists.
2. Materials and methods
2.1. Fish and experimental facilities
The study was carried out at the seahorse hatchery adjoining the
aquaculture laboratory complex of the National Institute of Oceanography, Goa (India), where techniques for the standardization of
captive breeding, rearing and culture of Indian seahorse species are
being established. New born juveniles (pelagic phase) released from a
single Hippocampus kuda spawner belonging to the F2 generation
were reared in rectangular light blue background breglass reinforced
plastic (FRP) tanks (capacity, 100 L) at a juvenile density of 2 sh L1
until they attained settlement phase. Thereafter, juveniles were
reared in large FRP tanks (capacity, 500 L) secured with different
types and sizes of holdfasts depending upon the growth of juveniles.
Various husbandry practices as described in Murugan et al. (2009)
were followed. The seawater used was treated by rapid sand ltration,
bio-ltration and then passed through ultraviolet radiation. Adequate
aeration was provided to the FRP tanks using air blowers and a
photoperiod of 12 h L (07001900 h):12 h D (19000700 h) was
maintained using a uorescent bulb (100 W Philips build) providing a
light intensity ~800 lx at the water surface. Juveniles were fed ad
libitum three times per day (0600, 1400 and 2200 h) with different
live prey organisms such as copepodites, Artemia nauplii and mysids
(Mesopodopsis orientalis). The tanks were cleaned daily, and important water quality parameters measured twice a week. The measured
physico-chemical parameters of seawater in the rearing tanks fell
within the optimum levels recommended for culture of seahorses
(Murugan et al., 2009): temperature (28.5 0.5 C), salinity (31
1.5 ppt), dissolved oxygen (6.1 0.6 mg L1), pH (7.9 0.3), NO2-N
(b0.02 mg L1) and NH3/NH4 (0 mg L1). Healthy adults measuring
155.1 9.8 mm Ht (as in Lourie et al., 1999) and weighing, 11.15
1.68 g wet weight were selected. Seahorses from two different FRP
tanks were used to avoid a possible tank effect and were starved 24 h
prior to the initiation of anaesthetic experiments.
2.2. Anaesthetic agents
The anaesthetic agents, MS-222 (tricaine methanesulphonate,
HiMedia, India), 2-phenoxyethanol (ethylene glycol monophenyl
ether, Sigma Aldrich Chemie, Germany) (PE), benzocaine (Ethyl-p-

aminobenzoate, HiMedia, India) and clove oil (Sigma Aldrich Co. USA)
were used for the present study. Doses of the anaesthetic agents were
prepared a few minutes prior to anaesthetic induction experiments.
Since clove oil does not readily dissolve in water (Woody et al., 2002),
it was initially diluted in ethanol (ratio of clove oil to ethanol 1:9) to
prepare a stock solution (100 mg ml1). Similarly, to increase the
solubility of benzocaine in water, a stock solution was prepared with
ethanol (ratio of benzocaine to ethanol 1:9) to obtain a concentration
of 100 mg ml1. Aliquots of the stock solutions were used to achieve
the desired experimental concentrations. MS-222 with its inherent
solubility in water was added directly to the tanks to obtain the
desired test concentrations. Preliminary trials indicated that the
highest concentration of ethanol used in each trial did not have any
noticeable analgesic effect on H. kuda for at least 1200 s. A known
volume of PE was initially mixed with a little seawater in a reagent
bottle (200 ml) and then stirred to disperse the chemical to form
small droplets before adding to anaesthetic induction tanks.
2.3. Induction and recovery stages of anaesthesia
Since the characteristics that dene different stages of anaesthesia
induction and recovery in seahorses have not been reported
previously, a pilot study was conducted for describing these stages
by carefully observing the behavioural responses in H. kuda. For the
pilot study, clove oil (50 mg L 1) and H. kuda (n = 6) were used. Adult
H. kuda were netted from the rearing tanks and transferred to the
anaesthetic induction tanks (37.5 22.5 22.5 cm) lled with fresh,
aerated seawater (capacity, 15 L) for studying the stages of anaesthesia. Water temperature, dissolved oxygen and pH values in the
tanks were 29 0.2 C, 6.1 0.5 mg L1 and 7.9 0.1, respectively.
Water in each tank was stirred with a glass rod and aerated to
facilitate complete mixing. Changes in the physiological status of the
anaesthetized sh were assessed in three consecutive stages as
described by Summerfelt and Smith (1990), with slight modications
based on the species-specic behavioural response of H. kuda.
Different anaesthesia induction stages (IS) and recovery stages (RS)
were dened by checking opercular movements, equilibrium and
absence of response to tactile stimulus.
For practical purposes, four IS (IS1sedation, IS2partial loss of
equilibrium, IS3cessation of swimming and loss of reex reactivity,
IS4modulary collapse) were considered (Table 1). When the
seahorses reached IS3, they were immediately netted from the
experimental tank, and placed on a wet surface for a period of 90 s.

Table 1
Stages of anaesthetic induction and recovery in Hippocampus kuda (ISinduction stage;
RSrecovery stage).
Stage Descriptor

Behavioural response of the seahorse

IS1

Sedation

IS2

Partial loss of equilibrium

IS3

Cessation of swimming
and loss of reex reactivity

IS4

Modulary collapse

RS1

Regaining of operculum
and n movement
Inability to balance head,
tapping head on tank
bottom
Normal

Slight fall in opercular rate; slow swimming;

partial loss of reaction to external stimuli
Striking the water column; erratic
swimming; increased opercular rate;
reaction only to strong tactile and vibration
stimuli
Complete cessation of swimming and
standing on the tank bottom; slow decrease
in opercular rate; no reaction to stimuli
Cessation of opercular movement (over dose
and or longer immersion in anaesthetic
solution); Subsequent death
Restarts operculum movement, starts erratic
movement of body.
Unable to balance the head, often keeping
the head down and trying to get up with tail
force; mouth tapping on the bottom of tank
Ability to swim normally and regular
opercular rate; reactive to the external
stimuli.

RS2

RS3

H.B. Pawar et al. / Aquaculture 311 (2011) 155161

Our previous studies (Pers. obs.) indicated that the duration of this
time period was sufcient to take length and weight measurements or
xing of a tag. Then the sh was transferred to a recovery tank
(capacity, 100 L) lled with fresh, aerated sea water for recording
stages of anaesthesia recovery. Three RS (RS1start of the operculum
and n movement, RS2inability to balance head, tapping head on
tank bottom, RS3normal) were described. Experiments were
repeated in triplicate to verify ndings. An induction time of 180 s
or less with complete recovery within 300 s suggested by Marking
and Meyer (1985) was used to establish a new set of anaesthesia
induction (IS3) and recovery (RS3) times for different anaesthetics
presented in this study (Table 1).

157

3. Results
3.1. Stages of anaesthesia
Signicant differences (P b 0.05) in the induction and recovery
stages at different concentrations of the four anaesthetic agents were
identied (Table 2). At certain concentrations of MS-222 (25, 50 and
75 mg L1) and 2-phenoxyethanol (PE) (250 l L1), all stages of
induction (dened in Section, 2.3) could not be attained. This may be
due to the concentrations applied were too low to reach complete
anaesthetic induction (IS3).
3.2. Induction and recovery times of anaesthesia

2.4. Anaesthetisation procedure

The efcacy of four anaesthetic agents in adult H. kuda was
assessed by testing several doses of each anaesthetic. Choice of
minimum and maximum doses of each anaesthetic was based on
previously published information for teleosts (Gomes et al., 2001;
Weber et al., 2009). The following doses of each agent were evaluated:
MS-222 and clove oil (25, 50, 75, 100, 125 and 150 mg L1),
benzocaine (75, 100, 125, 150, 175 and 200 mg L1) and PE (250,
500, 750, 1000, 1250 and 1500 l L1). Three individuals were
exposed to six concentrations of each anaesthetic totalling 72
individuals. Anaesthetic experiments performed were similar to the
pilot study and sh behaviour at each concentration was monitored
individually throughout the induction and recovery stages. The
induction and recovery times for four anaesthetic agents were
measured under similar set of environmental conditions using an
electronic stopwatch. When a particular seahorse reached stage IS3, it
was immediately netted from the anaesthetic induction tank and kept
on a wet surface for 90 sa time deemed sufcient to carry out
routine aquaculture procedures (e.g. length and weight measurements or xing a tag). After this, it was transferred to recovery tanks
for recording recovery stages.

Induction times generally decreased signicantly with increasing

doses for all of the anaesthetic agents evaluated. Induction time (IS3)
ranged from 359 36 s (100 mg L1) to 92 9 s (150 mg L1) for
MS-222, from 1165 49 s (75 mg L1) to 150 16 s (200 mg L1) for
benzocaine, 457 89 s (25 mg L1) to 36 7 s (150 mg L1) for clove
oil and 1154 69 s (500 l L 1) to 61 13 s (1500 l L1) for 2phenoxyethanol (PE) (Table 2). Recovery times increased with
increasing concentrations of anaesthetic agents (P b 0.05). Recovery
Table 2
Induction and recovery times (s) for Hippocampus kuda anaesthetised with six
concentrations of four anaesthetic agents (a) MS-222 (b) Benzocaine (c) Clove oil
and (d) 2-phenoxyethanol. Data are presented as mean s.d. denotes nonattainment of stage.
(a)
Stages

IS1
IS2
IS3
RS1
RS2
RS3

Concentrations (mg L1)

25

50

75

100

125

150

266 73

94 16
227 26

59 12
186 12

32 8
125 19
359 36
18 3
54 8
180 15

17 5
34 6
115 16
32 11
94 23
246 36

62
21 4
92 9
44 13
98 21
358 24

125

150

175

200

28 5
142 44
629 1.04
15 5
30 8
122 14

25 6
69 12
354 15
45 7
119 12
236 27

19 4
46 8
175 19
69 13
181 13
354 55

11 4
35 12
150 16
216 10
235 26
678 44

75

100

125

150

21 4
30 7
81 15
108 21
251 63
485 66

12 2
15 2
76 17
229 30
210 39
563 49

83
12 3
45 8
305 59
420 66
734 96

61
13 2
36 7
369 39
545 55
927 50

2.5. Effective anaesthetic concentration

(b)

An effective concentration of an anaesthetic is the concentration

that produced general anaesthesia (IS3) within 180 s and allowed
recovery (RS3) within 300 s (as in Marking and Meyer, 1985) was
used. The lowest observed concentration met this efcacy criteria was
considered to be the lowest effective concentration (LEC). Accordingly, LECs of each anaesthetic was determined.

Stages

Concentrations (mg L1)

75

IS1
IS 2
IS 3
RS 1
RS2
RS3

41 10
253 16
1165 49
52
11 3
41 6

100
42 9
178 19
999 82
10 5
19 6
50 8

2.6. Post-treatment survival

(c)

After anaesthetic treatment, seahorses were transferred to posttreatment tanks (capacity, 60 L) for 7 days to assess behavioural
changes and mortality. Seahorses that were exposed to the same
concentration of anaesthetic were kept in the same post-treatment
tank and fed twice a day ad libitum with live mysids, and 50% of the
tank water was exchanged on a daily basis.

Stages

Concentrations (mg L1)

25

IS1
IS2
IS3
RS1
RS2
RS3

31 7
47 16
457 89
31 6
52 5
238 33

50
21 3
28 3
115 28
51 18
70 27
385 37

2.7. Statistical analysis

(d)

A KruskalWallis test was used to assess the difference in

induction and recovery times of different concentrations of the
same anaesthetic agent (Zar, 1999). Regression analyses were used to
establish the relationship between dosage and induction time, as well
as dosage and recovery time. Signicance difference was tested at a
5.00% level, represented as P b 0.05. All results were processed and
analysed with the Graph Pad Prism 5 statistical software (San Diego,
California, U.S.A).

Stages

IS1
IS2
IS3
RS1
RS2
RS3

Concentrations (l L1)
250

500

750

1000

1250

1500

62 12
324 52

22 4
86 6
1154 69
51
16 4
170 21

15 3
59 5
796 55
14 3
61 12
208 22

13 4
44 7
176 22
43 10
94 23
271 37

11 2
34 5
93 11
92 21
186 20
382 47

72
30 6
61 13
127 12
190 15
426 45

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H.B. Pawar et al. / Aquaculture 311 (2011) 155161

time ranged from 180 15 (100 mg L1) to 358 24 s (150 mg L1)

for MS-222, from 41 6 (75 mg L1) to 678 44 s (200 mg L1) for
benzocaine, 238 33 (25 mg L1) to 927 50 s (150 mg L1) for
clove oil and 170 21 (500 l L1) to 426 45 s (1500 l L1) for PE
(Table 2).
3.3. Effective anaesthetic concentration
The determined lowest effective concentrations (LEC) of evaluated
anaesthetic agents were to be: 125 mg L1 for MS-222, 175 mg L1 for
benzocaine, 50 mg L1 for clove oil and 1000 l L 1 for PE. The LEC of
clove oil induced relatively quicker anaesthesia (115 28 s) and
longer recovery (385 37 s) in Hippocampus kuda. Induction and
recovery times exhibited by MS-222 and PE fell within the efcacy
criteria set out in this study (Section, 2.5). Induction times recorded
for benzocaine and clove oil were within efcacy criteria, while
recovery time limits exceeded by about 20 and 30%, respectively.
3.4. Induction and recovery in relation to concentration
A signicant correlation was observed between anaesthetic
concentration and induction time for all tested anaesthetic agents

(P b 0.05), whereas scatter plots yielded an inverse exponential

relationship (Fig. 1). The regression equations of times to reach IS3
and concentrations (C) of four anaesthetic agents in H. kuda were,
IS3 = 2748e 0.023C (R2 = 0.85) for MS-222; IS3 = 5419e 0.018C
(R2 = 0.96) for benzocaine; IS3 = 420e0.018C (R2 = 0.81) for clove
oil and IS3 = 6162e0.003C (R2 = 0.94) for PE. Similarly, a signicant
correlation (P b 0.05) was observed between anaesthetic concentration and times to reach RS3 for all anaesthetic agents, whereas scatter
plots showed exponential relationship (Fig. 1). The regression
equations established for recovery time and concentrations were,
RS3 = 68e0.010C (R2 = 0.78) for MS 222, RS3 = 6e0.023C (R2 = 0.97) for
benzocaine, RS3 = 206e 0.010C (R 2 = 0.93) for clove oil and
RS3 = 102e0.001C (R2 = 0.91) for PE.
3.5. Post-treatment survival
H. kuda reared in post-treatment tanks recovered well after the
anaesthetic experiment and showed no signs of stress or disease
symptoms and were feeding normally within 1 day. Furthermore, no
mortality was observed during the post-treatment period of 7 days
and no signicant decreases in mean body weight were observed
(data not shown).

Fig. 1. Induction (IS3) and recovery (RS3) times (s) in relation to anaesthetic concentrations for Hippocampus kuda (n = 3 for each trial).

H.B. Pawar et al. / Aquaculture 311 (2011) 155161

4. Discussion
Anti-stress agents form an integral component of modern day
aquaculture. Strong commercial demand coupled with concerns over
the long-term viability of exploiting natural populations have
stimulated a renewed interest in breeding and rearing of seahorses.
Because stress responses vary widely between species, it is often
necessary to screen dosages of different anaesthetic agents for each
cultured species (Ross and Ross, 1999). Although the use of
anaesthetic agents to reduce the stress associated with handling,
transport, connement, etc, is well established in sheries and
aquaculture research, their administration and efcacy in seahorse
husbandry has received scant attention. Some published papers have
cited the use of MS-222 (Boisseau, 1967; Bull, 2002; Jones and Lin,
2007; Olivotto et al., 2008), AQUI-S (Isoeugenol) (Woods, 2002;
Woods and Martin-Smith, 2004; Koldewey, 2005; Morgan and Bull,
2005; Mattle and Wilson, 2009), benzocaine (Wilson et al., 2006;
Martins et al., 2010) and clove oil (Castro et al., 2008; Otero-Ferrer
et al., 2010) in a few seahorse species. For recording length and weight
measurements, juveniles of H. reidi were lightly anaesthetized at MS222 concentration of 50 mg L1 (Olivotto et al., 2008). Wilson et al.
(2006) used benzocaine at 10 mg L1 for recording weight in
H. abdominalis juveniles, whereas adults of H. reidi were anaesthetized
in a benzocaine solution (50 mg L1) before sacricing them for
histological analysis by Martins et al. (2010). Castro et al. (2008)
anaesthetised adult H. reidi with clove oil (0.05%) for assessment of
diet composition. Also, 25 mg L1 clove oil was found effective and
safe in anaesthetizing H. hippocampus juveniles for taking biometric
measures (Otero-Ferrer et al., 2010).
Unfortunately, the aforementioned studies do not provide protocols of their administration and detailed information on the
anaesthetic induction and recovery times of the various anaesthetic
agents. Castro et al. (2008) measured induction and recovery times in
anaesthetized adult H. reidi with clove oil, but at a single concentration (0.05%). Our study differed from those conducted previously on
the use and efcacy of anaesthetic agents in seahorse species. Firstly,
different stages of anaesthetic induction and recovery in seahorse
species were dened and secondly, the lowest effective concentrations (LEC) of different anaesthetic agents based on the anaesthetic
induction and recovery times using a wide range of doses were
determined.
In the present study, the induction times decreased signicantly
with the increase in concentrations of MS-222, benzocaine, clove oil
and 2-phenoxyethanol (PE) (P b 0.05). The results are in good
agreement with previous studies that suggested the induction time
decreases inversely according to the concentration of anaesthetic
agent in teleost shes (Mattson and Riple, 1989; Hseu, et al., 1998;
Mylonas, et al., 2005; Gullian and Villanueva, 2009; Weber, et al.,
2009; Heo and Shin, 2010). On the other hand, recovery times
increased progressively with increasing concentration of anaesthetic
in adult Hippocampus kuda. Prolonged recovery with increased
anaesthetic dosage has been reported in seven species of tropical
reef teleosts (Cunha and Rosa, 2006), sockeye salmon (Woody et al.,
2002) and cobia, Rachycentron canadum (Gullian and Villanueva,
2009). However, decreasing recovery times with an increase in
concentration of clove oil and 2-phenoxyethanol (PE) for European
sea bass (Dicentrachus labrax) and gilthead sea bream (Sparus aurata)
been reported by Mylonas et al. (2005). The dynamics of recovery
times in anaesthetized sh seems to be more complex. For example, in
Senegalese sole (Solea senegalensis), recovery times decreased with
increasing doses of PE and metomidate, whereas those for clove oil
and MS-222 were dose-independent. The explanation put forward by
these authors for such an unusual trend is that with the highest doses,
the sh is not in contact with the anaesthetic for long, which would
allow faster recovery. Other factors such as differences in the
physiological responses of sh to the anaesthetic agents also inuence

159

this trend (Weber et al., 2009). This has been exemplied by Keene
et al. (1998) who documented lower recovery times with MS-222
compared to clove oil based on different effects on the cardiorespiratory system in rainbow trout. Further studies determining the
anaesthetic induction and recovery times in seahorse species exposed
to a wide range of anaesthetic concentrations are therefore necessary
for improved understanding of their interdependency.
The determined LEC of MS-222 (125 mg L1), benzocaine
(175 mg L1), clove oil (50 mg L1) and PE (1000 l L1) for H.
kuda were relatively higher than those obtained for teleost shes. A
concentration of 75 mg L1 of MS-222 was effective in anaesthetizing
Gadus morhua (Mattson and Riple, 1989) and S. senegalensis (Weber
et al., 2009), respectively. Gomes et al. (2001) documented that
application of benzocaine at doses of 100150 mg L1 resulted in
quick induction, total immobilization and fast recovery in juveniles of
tambaqui (Colossoma macropomum). Cunha and Rosa (2006)
recommended 20 mg L1 clove oil dosage for seven tropical reef
shes. Also, Weber et al. (2009) reported 30 mg L1 clove oil was
effective in anesthetizing S. senegalensis. PE at doses of 600 mg L1
and 0.12% produced optimal anaesthetic inductions in S. senegalensis
(Weber et al., 2009) and in goldsh (Carassius auratus) (Yasui et al.,
2009), respectively. One of the possible reasons for requirement of
higher doses of anaesthetics in seahorses could be related to modied
form of general teleost gill pattern (random tufted gills) and narrow
opercular opening (small cavity volume) which may entail lower
water volumes splashed onto the gills (inadequate irrigation) and
existence of slow rate of inhale and exhale water across the gills (Gert
Roos, personal communication). It has been reported that entry and
excretion of anaesthetics in sh takes place primarily via the gills,
whereas internal organs are only slightly involved (Ross and Ross,
1999; Hoskonen and Pirhonen, 2006). Further research on structure
of gills inuencing the anaesthetic dosage in different teleost species,
however is required.
Behavioural response in seahorse to anaesthetic agents is markedly
different from those in other teleosts. For example, it is difcult to
identify the state of death in seahorses, as many times they do not fall
or lie horizontally on the bottom of the tank. Instead, H. kuda stand
vertically on the bottom of tank and appear to be alive (pers. obs.). This
peculiar behaviour of seahorses is often misleading and might lead to
overdosing the sh as experienced during our pilot study. This calls for
careful observation when anaesthetising seahorses.
The onset of induction (IS3) and recovery (RS3) stages of
anaesthesia in H. kuda varied signicantly with the concentrations
of the anaesthetic agent (P b 0.05). A rapid anaesthetic induction time
of less than 180 s and recovery time not exceeding 300 s is considered
an ideal for sh (Marking and Meyer, 1985). All the anaesthetics
evaluated here met the rst criterion, while recovery times were
prolonged in H. kuda anaesthetized with clove oil and benzocaine
than with MS-222 and PE (Table 2). Relatively quicker induction in
H. kuda anaesthetized with clove oil may be due to its high lipophilic
nature which allows it to adhere easily and penetrate the gill
epithelium for adsorption in tissues (Mylonas et al., 2005). On the
other hand, delayed recovery might be due to its persistent nature on
the gill surface, which effectively increases the recovery time (Sladky
et al., 2001; King et al., 2005). Quicker induction (89 62 s) than
recovery (119 78 s) in adult H. reidi anaesthetised with a single
concentration of clove oil has been reported by Castro et al. (2008).
Although the induction time (115 28 s) obtained with clove oil in
adult H. kuda was comparable, the recovery times (375 22 s)
however were 3 times longer than those reported for H. reidi (Castro
et al., 2008). Such difference in the respective recovery times might be
related to species, size, physiological status and environmental
conditions (Burka et al., 1997; Ross and Ross, 1999).
According to Marking and Meyer (1985), the anaesthetic agent is
considered effective if it produces a complete induction within 180 s
and recovery within 300 s. In the yellow seahorse H. kuda, all four

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H.B. Pawar et al. / Aquaculture 311 (2011) 155161

anaesthetic agents evaluated were effective and presented a good

margin of safety when compared against the above efcacy criteria.
Amongst four anaesthetics, MS-222 and clove oil were found most
effective as least concentrations of these are required to achieve the
desired induction and recovery in H. kuda. However, a number of
other considerations such as availability, cost-effectiveness, nature of
the study, legislation and user safety determine the selection of
particular anaesthetic for aquaculture.
In many countries, the use of sh anaesthetics is a matter of
concern as there are no specic laws regulating their use. The
recommendations of the US Food and Drug Administration (FDA) are
usually followed. MS-222 is the only FDA-approved anaesthetic for
food sh in the USA. The drug is particularly effective on sh because
it is highly water and lipid-soluble and readily crosses the gill
membrane. It directly acts upon the central nervous system,
neuromuscular junctions and ganglion synapses preventing the
generation and conduction of nerve impulses (Treves-Brown, 2000).
But it is quite expensive and its procurement is also cumbersome as it
is not produced locally. On the other hand, clove oil has been
extensively used as an anaesthetic agent in aquaculture of freshwater
and marine shes as it is widely available, relatively inexpensive, ecofriendly and relatively safe to sh and humans (Hseu et al., 1998;
Keene et al., 1998; Woody et al., 2002; Iversen et al., 2003; Iversen
et al., 2009). Furthermore, clove oil was found to be effective at much
lower concentrations (2.5 times) than with MS-222 in achieving the
desired levels of anaesthetic induction and recovery in H. kuda. This
translates into larger safety margin and increasing cost-effectiveness
in favour of clove oil compared to MS-222.
5. Conclusion
In conclusion, MS-222 and clove oil were proven to be most
effective and the latter appears to be an ideal anaesthetic for the
yellow seahorse husbandry as it met many criteria used to dene sh
anaesthetic agents. The results of the study however, need to be
interpreted with caution as the conclusions drawn are solely based on
induction and recovery times. Further studies on different life stages,
gender, reproductive state and sizes, followed by assessments of the
effects of anaesthetics on haematological prole and respiration rate,
will considerably advance our understanding of anaesthesia in
husbandry of the yellow seahorse, H. kuda.
Acknowledgements
The authors are grateful to Dr. Satish R. Shetye, Director, National
Institute of Oceanography, Goa (India) for facilities and encouragement. Thanks are due to three anonymous reviewers for constructive
criticisms which improved the quality of the manuscript greatly.
Financial support came from the Department of Biotechnology
(Government of India). Permission granted by the Ministry of
Environment & Forests, Government of India for collection of
seahorses is gratefully acknowledged. This paper represents contribution No. 4871 of the National Institute of Oceanography (NIO), Goa
(India).
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