Journal of Research in Biology

ISSN No: Print: 2231 6280; Online: 2231- 6299

An International Scientific Research Journal

ORIGINAL RESEARCH

Journal of Research in Biology

Anesthetic efficacy of clove oil and its impact on hematological and

biochemical changes in Channa striatus (Bloch, 1793)
Authors:
Jeyasheela P, Haniffa MA
and Kavitha K

ABSTRACT:
Channa striatus is one among the dominant group of air breathing
freshwater fishes in Southeast Asian countries. In the present study, fish anesthetic
clove oil was used to study the hematological and biochemical changes at different
concentration (400 ppm, 450 ppm and 500 ppm) and time interval (0 h, 1h and 24 h)
in C. striatus. The induction and recovery time was noted for each treatment groups.
Erythrocyte count (T/L), Hemoglobin and Hematocrit values showed elevated levels
Institution:
Centre for Aquaculture
when compared to control. RBC, Hb and Ht values significantly increased 1 h after
Research and Extension
anesthesia and returned to normal after 24 h. Anesthetic treated fishes exhibited
(CARE), St.Xaviers College marked decrease in WBCs when compared to control group. The rest of the indices
(Autonomous),
(MCV, MCH, MCHC, Lymphocytes, Monocytes, Neutrophils) were at comparable levels
Palayamkottai,
in all groups. The anesthetic treated fishes were found to show a significant increase
Tamil Nadu-627005, India
in the concentration of glucose. The rest of the indices (TP, ALB, GLOB, ALT, AST) were
at comparable levels in all groups. Results of the study suggested that the use of clove
oil at the concentrations of 400, 450 and 500 ppm does not cause irreversible damage
on the blood parameters as well as biochemical profile in C. striatus.
Corresponding author:
Haniffa MA

Keywords:
C. striatus, anesthesia, induction and recovery time

Email Id:

Web Address:
http://jresearchbiology.com/
documents/RA0497.pdf

Article Citation:
Jeyasheela P, Haniffa MA and Kavitha K
Anesthetic efficacy of clove oil and its impact on hematological and biochemical
changes in Channa striatus (Bloch, 1793)
Journal of Research in Biology (2014) 4(8): 1595-1603
Dates:
Received: 09 Oct 2014

Accepted: 11 Nov 2014

Published: 31 Dec 2014

This article is governed by the Creative Commons Attribution License (http://creativecommons.org/

licenses/by/4.0), which gives permission for unrestricted use, non-commercial, distribution and
reproduction in all medium, provided the original work is properly cited.
Journal of Research in Biology
An International
Scientific Research Journal

1595-1603 | JRB | 2014 | Vol 4 | No 8

www.jresearchbiology.com

Jeyasheela, 2014
induction and recovery periods and mortality are also

INTRODUCTION:
Murrels, commonly called snakeheads are

studied.

important air breathing freshwater fishes and are highly

regarded as food fish in the South and Southeast Asian

MATERIALS AND METHODS:

countries. They belong to the family Channidae

The striped murrel, C. striatus acclimatized in

(Ophiocephalidae) (Wee et al., 1982). Murrels can be

cement tanks (15mX3mX2m) for a period of two weeks

kept alive for several hours outside water under slight

at CARE Aquafarm were used for this study. During this

moist condition which facilitates transportation to distant

period, the fishes were fed twice a day with chicken

markets in good condition. Even then often fishermen

intestine. Forty fishes (29.7 1.69 cm and 232 14 g)

meet heavy loss due to death of murrels during transport.

were selected and were divided into four groups each

Hence, sedation or use of anesthetics can be beneficial

with 10 fishes based on the concentration of clove oil

to calm excitable fish during bulk transportation of fish

(group I: control [0ppm], group II: 400ppm, group III:

stocks, especially over long distances and high density.

450 ppm and group IV: 500 ppm). Test fishes were

In aquaculture practices and research activities,

starved for 24 hours prior to the experiment and

fish handling are a common source of stress. Hence, a

mortality rate (if any) was recorded regularly throughout

variety of anesthetics are used mainly in order to reduce

the course of the study. Stock solution of clove oil was

stress level and to prevent fish injury/ death during their

prepared by dissolving 1 ml of clove oil in 9 ml of tap

handling. The most commonly used fish anesthetics are

water. The test fishes were exposed to the respective

tricaine methanesulfonate (MS-222), benzocaine (ethyl

concentrations and they were anesthetized.

paraaminobenzoate)

(Kiessling

et

al.,

2009),

The induction and recovery period of fishes as a

2-phenoxyethanol, metomidate (Weber, 2009), and

function of concentration were recorded. Blood was

carbon dioxide (Pirhonen and Schreck, 2003).

sampled from caudal vein of the anesthetized C. striatus

Clove oil is a dark - brown liquid, a distillate of

with heparin coated syringe. To stabilize the blood

flowers, stalks and leaves of the clove tree Eugenia

samples, aqueous solution of heparin sodium salt (5000

aromatica (Soto and Burhanuddin, 1995)

having a

U/ml) was added to blood (Svobodova et al., 1991). The

mild anesthetic effect on human (Nagababu and

blood sample was divided into two aliquots; one part was

Lakshmaiah, 1992; Taylor and Roberts, 1999) and

transferred to a 2 ml heparinized tube and stored in

fish

and Ross, 2008). Keene et al. (1998)

refrigerator prior to hematological analyses. The other

showed that the clove oil i s much less e x p e n s i v e

part of aliquots was transferred to 1.5 ml microcentrifuge

than

tubes and centrifuged for 15 min. at 4C. The plasma

(Ross

other

chemicals

including

MS222

and

recommended the same for fish transport.

was removed and transferred to another 1.5 ml microtube

Anesthesia may affect blood parameters and

hemolyse

tissues

(McKnight,

1966).

Since,

and stored frozen at 70C for biochemical analyses

(Fast et al., 2008).

hemodynamic is closely related to response of animal to

Hemogram was established by estimation of total

external environment (Fernandes and Mason, 2003). The

erythrocyte count (RBC), total white blood cell count

goal of this study was to assess efficacy of clove oil as an

(WBC), hematocrit (Ht), hemoglobin concentration (Hb),

anesthetic through the measurement of multiple blood

erythrocyte indices (MCV, MCH, MCHC) and white

parameters of striped murrel Channa striatus. The

blood

effect of different concentrations of clove oil on

Biochemical indices of blood plasma included glucose

1596

cell

differential

count

(Campbell,

2004).

Journal of Research in Biology (2014) 4(8): 1595-1603

Jeyasheela, 2014

Time (min)

Concentration (ppm)

Figure 1: Effect of Clove oil on Induction and Recovery time of C. striatus

(GLU), Total Protein (TP), Albumin (ALB), Globulin

complete when the fish lost its response to external

(GLOB), Aspartate amino Transferase (AST), and

stimuli. As per the results obtained, the optimum

Alanine amino Transferase (ALT). Statistical differences

concentration to anesthetize C. striatus was found to be

between groups if any at each time point (0 h, 1 h

450 ppm. The fishes underwent anesthesia through six

and 24 h) were tested using SPSS software and data

stages as described by Bowser (2001). The first stage of

were presented a s m e a n SD.

anesthesia was light sedation which involved: Slight loss

of reactivity to external stimuli; opercular rate slightly

RESULT AND DISCUSSION:

decreased with normal equilibrium. This was followed

Water quality parameters were measured and

by the next stage i.e., deep sedation involving total loss

recorded as pH 7.0, chloride 200 ppm, total hardness

of reactivity to all but strong external stimuli; slight

525 ppm, fluoride 0.5 mg/l, iron 0.5 mg/l, residual

decrease in opercular rate with normal equilibrium. The

chlorine 0 mg/l and nitrate 0.45 mg/l.

signs of next stage included: Partial loss of muscle tone

All the fish exposed to different concentrations

and swimming was erratic; increased opercular rate;

of clove oil recovered well and returned to normal

reactivity only to strong tactile and vibration stimuli.

behaviour with respect to feeding, surfacing activity,

Total loss of muscle tone and equilibrium; slow but

swimming and respond to external stimuli after the

regular opercular rate; loss of spinal reflexes especially

anesthetic treatment. Furthermore, no mortality was

loss of equilibrium observed in the fourth stage. The

noticed within 48 h following recovery from anesthesia.

changes that were noticed in the fifth stage were total

The technique anesthesia by immersion wa s

loss of reactivity; opercular movements were slow and

ap p li ed to provide the active ingredient into the fish

irregular, heart rate was very slow and loss of all reflexes

gills through water flow to travel through bloodstream

was noticed. The final stage during anesthesia was

to the central nervous system. Thus, the fish goes

medullary collapse (stage

through several stages of anesthesia viz ; light sedation,

anesthetized fishes showed opercular movements to

deep sedation, partial loss of equilibrium, total loss of

cease; cardiac arrest followed quickly. The time duration

equilibrium, loss of reflex reactivity and ultimately

for each stage was shown in the figure 1.

medullary collapse as described by Bowser (2001).

Induction of anesthesia was assumed to be
Journal of Research in Biology (2014) 4(8): 1595-1603

of asphyxia) and the

Increase in clove oil concentration resulted in

decrease in induction time whereas the recovery time
1597

Jeyasheela, 2014
increased significantly. However, the recovery and

were put into induction tray containing clove oil, they

induction time was also concentration dependent. The

became excited and hypermotile followed by bubbling;

mean induction time for C. striatus 400, 450 and 500

the gill and fin movements progressively decreased, the

ppm clove oil was found to be 7.10 1.10 min., 2.36

fish lost equilibrium and started swimming laterally.

0.42 min. and 2.020.52 min. respectively. The mean

Finally, the fish became immobile with full loss of

recovery time for each concentration was 5.211.1 min.,

equilibrium and consciousness. After transfer to recovery

3.200.40 min. and 6.381.15 min. respectively. The

tray, reappearance of gill movement was noticed first.

induction time was longer at the lower dose whereas it is

This was followed by fin and then tail movement. The

quick and short at higher dose. In the same manner, the

fish started moving laterally. Gradually full equilibrium

recovery time was quick in lower dose whereas it elapsed

was regained and normal behaviour was restored at

for a longer duration at higher dose. For morphological

5.211.1 min., 3.200.40 min. and 6.381.15 min. as a

evaluations, biopsy and stripping, long handling periods

function of 400 ppm, 450 ppm and 500 ppm clove oil

are required and hence, anesthetizing with clove oil

respectively.

would be an added advantage which produces longer

induction and recovery as a function of anesthesia have

recovery time (Seol et al., 2007; Park et al., 2009).

been reported by McFarland (1960).

Similar,

behavioural

changes

during

It was observed that if the exposure was

Hematological and biochemical profiles of blood

prolonged until the fish become anesthetized; the

are necessary to provide vital information about internal

recovery was concentration-independent and lasted for

environment of organism. Erythrocyte count (T/L)

about four minutes. In the present study, the tested

showed increased values in anesthetic treated groups

concentrations met the efficacy criteria specified by

(1.18-1.21) when compared to control (1.14) (Table 1).

Marking and Meyer (1985) and hence, dose of 450 ppm

Similarly, Hemoglobin and Hematocrit also showed

of clove oil can be suggested for transport or grading of

elevated levels in anesthetic induced fishes (Hb: 59.93

C. striatus. This finding is also in accordance with the

64.826 g/dl and Ht: 0.179 0.194 l/l) as compared to

results reported by Woody et al. (2002) where higher

control (Hb: 59.58 63.24 g/dl and Ht: 0.178 0.189 l/l

dose produced a rapid and uniform response in all size

respectively). RBC, Hb and Ht values significantly

classes, suggesting that a dose of 400 ppm might be well

increased 1 h after anesthesia and returned to normal 24

over the effective concentration for sockeye salmon

h post anesthesia.

(Oncorhynchus nerka).

Anesthetic treated fishes exhibited marked

Similar observations were made by Inoue et al.

decrease in WBCs (0.876 1.109 g/L) when compared

(2003) on juveniles of matrinxa, Brycon cephalus,

to control group (0.981 1.138 g/L) (Table 1). The rest

where, the recovery in the experiment with a prolonged

of the indices (MCV, MCH, MCHC, Lymphocytes,

exposure was longer at all concentrations, but with 30

Monocytes, Neutrophils) were at comparable levels in all

and 40 mg /L, it was still below 10 min. Our results

groups. Results of the study suggested that the use of

showed that increasing the anesthetic dose significantly

clove oil at the concentrations of 400, 450 and 500 ppm

decreased induction time but prolonged recovery time.

does not cause irreversible damage on the blood

This was in agreement with reports of Salmo salar smolts

parameters in C. striatus.

(Iversen et al., 2003), Cyprinus carpio (Velisek et al.,

2005) and Silurus glanis (Velisek et al., 2006).
As reported by Matin et al. (2009), when the fish
1598

Tort et al. (2002) reported clove oil altering

hematocrit concentrations in rainbow trout. Velisek et al.
(2005) observed the same in rainbow trout and carp, both
Journal of Research in Biology (2014) 4(8): 1595-1603

151.918.98

50.582.98

332.960.53

Ht (l/l)

MCV (fl)

MCH (pg)

MCHC (g/L)

Leukocytes
(WBC) (g/L)
Lymphocytes
%
Neutrophils
%
Monocytes %

Journal of Research in Biology (2014) 4(8): 1595-1603

6.130.22

69.080.18

64.990.27

8.330.28

12.440.38

Glucose
(mmol/L)

Total Protein
(g/L)

Albumin
(g/L)

Globulin
(g/L)

AST (IU/L)

ALP (IU/L)

4.090.19

0h

Control

Biochemical
Indices

2.20.84

14.41.58

83.43.05

S.No

10

0.170.003

Hb (g/dl)

1.070.01

59.331.2

Erythrocyte
(RBC) (T/L)

0h

1.180.07

Hematological
Indices

S.
No.

63.780.49

4.510.28

68.290.42

14.520.36

6.60.45

24 h

2.41.52

13.51.14

84.12.71

0.980.01

333.550.69

52.212.99

156.538.90

0.160.004

59.501.28

1.140.05

0h

1.80.84

16.60.71

81.61.14

0.990.02

334.820.71

50.423.47

150.5910.23

0.180.004

59.601.32

1.180.08

2.80.84

16.01.48

81.21.14

0.920.02

334.600.63

52.797.08

157.7621.01

0.190.003

63.240.84

1.200.09

1h

400 ppm
24 h

2.01.0

14.91.52

83.11.58

0.930.02

334.630.52

51.092.34

152.676.98

0.180.30

60.230.30

1.180.06

0h

1.60.89

13.41.14

821.58

0.960.01

334.800.34

50.495.72

150.8017.26

0.180.003

59.930.89

1.190.09

3.01.22

15.60.89

80.41.14

0.900.02

333.050.44

53.437.20

160.4321.57

0.190.002

64.280.81

1.200.12

13.420.39

7.340.44

65.070.86

4.240.40

69.310.49

6.410.14

24 h

8.590.36

7.600.35

64.120.72

6.260.32

70.380.71

7.280.10

0h

400 ppm

12.630.29

5.040.20

65.850.36

5.320.50

71.300.42

8.420.21

1h

19.850.34

8.310.33

63.890.35

5.110.25

69.00.54

6.410.07

24 h

13.790.33

6.270.27

62.290.36

7.960.30

70.250.51

7.200.11

0h

450 ppm

1h

450 ppm

10.490.30

8.180.30

64.540.93

7.080.42

71.220.31

7.510.25

1h

24 h

13.390.31

14.140.32

63.770.67

5.380.23

69.150.58

6.250.18

24 h

2.20.84

131.58

81.80.84

1.110.02

333.630.3

49.482.10

148.316.37

0.180.004

58.391.21

1.180.05

Table 1: Effect of Clove Oil on Hematological Indices of C. striatus

Table 2: Effect of Clove Oil on Blood Plasma Indices of C. striatus

6.490.18

1h

2.41.14

14.02.08

83.62.41

1.140.02

333.440.52

52.084.09

156.2012.36

0.140.005

59.351.72

1.140.08

1h

Control
0h

16.520.33

12.330.33

56.291.09

13.110.61

69.40.53

7.320.10

0h

500 ppm

1.20.45

13.81.30

811.58

0.910.17

332.270.45

50.522.92

151.858.68

0.180.002

60.220.53

1.190.06

21.540.15

16.260.45

54.840.72

15.370.24

70.220.57

7.620.07

1h

2.60.89

14.80.84

80.61.14

0.880.26

334.150.57

53.4412.36

159.9337.22

0.190.002

64.830.60

1.210.07

1h

500 ppm
24 h

23.070.26

18.180.26

58.460.87

11.330.37

69.800.99

6.310.21

24 h

2.00.71

11.81.10

82.41.14

0.980.02

333.550.53

51.124.12

153.2612.58

0.180.001

60.370.42

1.180.08

Jeyasheela, 2014

1599

Jeyasheela, 2014
of them also exhibited significantly increased plasma

system was evaluated by the changes in White Blood

glucose concentrations after longer exposure periods (10

Cell (WBC). It showed a decline trend associated with

min.).

arresting in anesthetic. Increase in plasma cortisol

The increase in RBC level after anesthetic

concentration which is a glucocorticoid hormone, is also

treatment showed that fish's body is kept in touch with

acting as an immunosuppressive (Fast et al., 2008), so it

insufficient gas when anesthetized. When oxygen

could suppress humoral factors and lead in declining

becomes a limiting factor, RBCs increased for carrying

circulating WBC along with elevating cortisol.

more oxygen to the cells. Hemoglobin (Hb) is an

The anesthetic treated fishes were found to show

effective index of taking more oxygen in the blood and

a significant increase in the concentration of glucose i.e.,

hence Hb increased for obtaining more oxygen; the

7.196 7.318 mmol/l at 0 h and 7.514 8.422 mmol/l at

average volume value (MCV) with a red blood cell

1 h following anesthetic treatment. The glucose values of

increases after anesthetization and showed that dissolved

anesthetic treated fishes returned back to control values

oxygen amount decrease in fish's blood. So MCV is

after 24 h post treatment (6.134 6.486 mmol/l). The

strengthened by holding more oxygen, and keeping the

rest of the indices (TP, ALB, GLOB, ALT, AST) were at

basic physiological function to fish's body; the average

comparable levels in all groups (Table 2).

concentration of hemoglobin of red blood cells (MCHC)

These results revealed the significance of

increase after anesthetization and show that dissolved

exposure time and dosage on some physiological

oxygen amount is decreasing in fish's blood, so MCHC

indicators of anaesthetized fish. Post-exposure mortality

increase for combining more oxygen (Wu et al., 2000).

and lack of biochemical alteration in the present study

The respiratory actions lower resulting in

could be due to the short induction time after which

reduced O2 for circulation for breathing and survival

blood was collected. These results indicated the need to

creating

results

study possible physiological changes occurring in

physiological changes altering the blood factors like

different fish species exposed to different doses of clove

glucose and Hematocrit (Ht). Hyperglycemia (increased

essence.

hypoxic

environment

which

blood glucose), elevated HB and Ht were similar to other

Plasma cortisol as well as glucose is a

fishes anesthetized by Pirhonen and Schreck (2003);

physiological indicator of stress in fishes and their

Park (2009); Gomes et al. (2001). Reports by Velisek et

interactive effects on metabolism during recovery from

al. (2005) on Cyprinus carpio and Oncorhynchus mykiss

stress have recently become a subject of more intense

suggests

mg/l

study (Tytler and Hawkins, 1981; Woody, 2002; Weber

concentration and 10 min exposure did not produce any

et al., 2009). Clove oil was found to block the activity of

marked changes in the blood parameters after 24 h.

cortisol, although not completely, in Brycon cephalus

However Sudagar et al. (2009), reported that a 7-min

(Inoue et al., 2005). Although the mechanism is not well

exposure to clove powder resulted in significant

known, Iverson et al. (2003) suggested that it blocks

reversible increase in Ht, Hb and RBC immediately after

transmission of impulses to the Hypothalamus-Pituitary

anesthesia in Roach Rutilus rutilus.

Interregnal axis (HPI).

that

clove

oil

anesthesia

at

30

Increase in the numbers of leukocyte (WBC)

In our experiments with murrel, an increase in

increase after anesthetization showed the unexpected

blood plasma glucose immediately after clove oil

changes of the living water environments or invasion of

anesthesia was observed. Increased glucose level

outside material (Wu et al., 2000). The fish immune

returned to normal 24 h after anesthesia. Increased blood

1600

Journal of Research in Biology (2014) 4(8): 1595-1603

Jeyasheela, 2014
plasma glucose level after anesthesia indicates that the

Advances in Veterinary Anesthesia and Analgesia:

procedure caused some stress in the experimental fish.

Companion

These findings are in accordance with results of

Information Service, Ithaca, NY.

Holloway et al. (2004) and Velisek et al. (2005) who

also detected increase of glucose concentration in
rainbow trout (Oncorhynchus mykiss) following clove oil
anesthesia. On the other hand, Iverzen et al. (2003)
found no change in the concentration of glucose in
Atlantic salmon (Salmo salar) following clove oil
anesthesia.

Animals,

International

Veterinary

Campbell TW. 2004. Hematology of lower vertebrates

In: Proceedings of the 55th Annual meeting of the
American College of Veterinary Pathologists (ACUPC).
www.ivis.org/proceedings/AVCP/2004/Campbell/
ivis.pdf
Fast MD, Hosoya S, Johnson SC and Afonso LOB.
2008. Cortisol response and immune- related effects of

CONCLUSION
According to these results, clove oil at a
concentration of 450 ppm could be an efficient and
relatively safe anesthetic agent, but further studies are
required to detect any possible toxicity effect on fish. In

Atlantic salmon (Salmo salar Linnaeus) subjected to

short-

and

long-term

stress.

Fish

and

Shellfish

Immunology 24(2):194-204.
d o i : 10.1016/j.fsi.2007.10.009.

conclusion, clove essence was found to be safe and can

Fernandes MN and Mason AF. 2003. Environmental

be effectively and easily applied in used dosages to

pollution and fish gill morphology. In: VAL, AL. and

anaesthetize various size groups of murrels with minimal

KAPOOR, BG., Ed. Fish adaptations. Enfield: Science

disruption in the physiological indicators studied and

Publishers. 203-231.

with zero mortality. Hence, considerations should be

given to the use of clove essence as a replacement for
synthetic forms of anesthetics.

acknowledge

R and Araujo-Lima CARM.

2001. Efficacy of

Benzocaine as an Anesthetic in Juvenile Tambaqui

Colossoma macropomum. Journal of World Aquaculture

ACKNOWLEDGEMENT
We

Gomes LC, Chippari-Gomes AR, Lopes NP, Roubach

the

financial

assistance

received from ICAR-NAIP (F.No. 1(5)/2007-NAIP dt.

Society 32(4):426-431.
doi: 10.1111/j.1749-7345.2001.tb00470.x

22 August 2008) to carry out this study. We are grateful

Holloway AC, Keene JL, Noakes DG and Moccia

to Dr.T.J.Pandian, Visiting Professor, CAS Marine

RD. 2004. Effects of clove oil and MS-222 on blood hor-

Biology, Annamalai University.

mone profiles in rainbow trout Oncorhynchus mykiss,

Walbaum. Aquaculture Research 35(11):10251030.

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