Chapter 4

Print ISBN: 978-93-5547-306-6, eBook ISBN: 978-93-5547-314-1

Mitigation of Zinc Sulphate Induced Haematological

Toxicity in the Fish Mystus montanus by Probiotic
Bacteria
T. Sakthika a*# and J. P. Meshanth b
DOI: 10.9734/bpi/rabs/v1/2045A

ABSTRACT

The present study was designed to evaluate the zinc sulphate intoxicated haematological parameters
of the fish Mystus montanus in two different sub lethal concentrations of ZnSO 4 and to analyse the
effect probiotic bacteria Lactobacillus rhamnosus on the haematological parameters of the Zinc
intoxicated fish for 60 days. Significant differences of altered blood parameters were observed
(P<0.050) in the Full blood count, Haematological indices and Differential Leucocytes counts of fish
treated with Zinc. However the fish group treated with probiotic bacteria showed significant tolerant
ability for zinc induced hematological parameters. This research demonstrates that dietary
supplementation of probiotic bacteria can cope up with zinc pollutants in the aquatic medium.

Keywords: Zinc sulphate; Mystus montanus; Lactobacillus rhamnosus; Haematology.

1. INTRODUCTION

Zinc is non-toxic and is an essential metal that is an important constituent of cells. In addition, several
enzymes depend on zinc as a cofactor. Although, small quantities of zinc are required for the normal
development and metabolism but if its level exceeds the physiological requirements, it can act as a
toxicant [1].

Blood parameters are increasingly used as indicators of the physiological condition or sub-lethal
stress response in fish to endogenous or exogenous changes. Red blood cell indices provide
information about the haemoglobin content and size of red blood cells, abnormal values indicates
presence of anemia and type of anemia [2].

Probiotics are generally described as mono- or mixed cultures of live microbes that have a beneficial
effect on the host's health when applied to animals and human beings. These beneficial effects
include disease treatment and prevention as well as improvement of digestion and absorption in the
host [3].
The total erythrocyte count [4], total leucocyte count fed with Saccharomyces cerevisiae [5],
haemoglobin, mean corpuscular volume, haemoglobin concentration and mean corpuscular
haemoglobin concentrations, total protein, albumin, globulin, albumin-globulin ratio, alkaline
phosphatase activity, alanine and aspartate aminotransferase activities, creatinine, sodium, cortisol,
insulin and glucose were reported to increase in Labeo rohita provided with Bacillus subtilis as
probiotics [6]. Considering these backgrounds the present study is aimed to find out the lethal
concentration of Zinc sulphate on the fish Mystus montanus, to find out its toxicity on hematologicl
parameters and role of probiotic bacteria Lactobacillus rhamnosus on the intoxicated fish.

_____________________________________________________________________________________________________
#
Assistant Professor;
a
Department of Zoology, A. P. C. Mahalaxmi College for Women, Thoothukudi, Tamil Nadu, India.
b
B.Tech Chemical Engineering, Sri Venkateswara College of Engineering, Chennai, India.
*Corresponding author: E-mail: sakthika@apcmcollege.ac.in;
 Research Aspects in Biological Science Vol. 1
Mitigation of Zinc Sulphate Induced Haematological Toxicity in the Fish Mystus montanus by Probiotic Bacteria

2. MATERIALS AND METHODS

2.1 Experimental Animal (Mystus montanus)

The present study was carried out in the Department of Zoology, A.P.C.Mahalaxmi College for
Women, Thoothukudi. Healthy fishes of Mystus montanus with 8.9±0.507cm in length and weighing
3.74±0.292 g were captured in Perunkulam pond, near Eral in Thoothukudi District. Approximately
150 fish were collected and 100 healthy fish of Mystus montanus were selected for LC50 experiments
and fifty fish were used for toxicity studies. They were acclimatized for 15 days in the laboratory
conditions before starting the experiment. Physicochemical properties of the holding water during
acclimation were determined by standard [7] methods (Table 1).

2.2 Toxicant

The toxicant Zinc sulphate was sponsored by DCW Ltd, Sahupuram. Zinc sulphate was used in the
present study to test its toxicity on the test fish Mystus montanus. Using the chemical ZnSo4 a stock
solution was prepared by dissolving 4.56 gram in one litre of distilled water to get 1ppt fluoride
solution. From this stock solution various concentrations of fluoride solution was prepared and used
for LC50 analysis and toxicity studies.

2.3 Determination of LC50 Value

LC50 value is defined as the lethal concentration of a toxicant to kill 50% of the test animals in 96
hours. To find out the LC50 for Zinc sulphate, a series of concentrations ranging from 20mg/L to
80mg/L were prepared separately from the stock toxicant. Well acclimatized 10 fishes of uniform size
were selected and introduced into each toxicant concentration for a period of 96 hours. Every day the
fresh concentrations were prepared from the stock, to make the concentrations constant through the
experimental study [8]. During the experimental period the animal was not fed. The mortality of test
individuals in each toxicant concentration was recorded. The experiment was repeated twice,
simultaneously to confirm the result. During the experiment, a control group of fish was also reared.

2.4 Probit / Log Regression Analysis

Probit analysis of log dose against response (mortality) was performed by adopting standard protocol
given by Finney [9]. The regression equation was calculated and 96 hours LC50 values were derived
from the equation (Table 3). The LC50 value of Zinc sulphate to the fish for 96 hours is 45.6 mg/l. Two
th th
experimental concentrations 4.56mg ZnSo4/L (1/10 of LC50) and 2.28mg ZnSo4/L (1/5 of LC50) were
used for the present study.

2.5 Experimental Design for Zinc Toxicity Analysis

The experimental design for the present study is shown in Table 2. Fish were reared in 25 liter
capacity glass tanks. For the experiment 50 numbers of fish were divided into five groups. Ten
numbers of fish served as control and were maintained under normal conditions, while the other four
as experimental groups with 10 fish in each group. Two experimental groups were treated with
4.56mg ZnSo4 (1/10 of LC50) and they were represented as T1 and T2 (Table 2). The other two
experimental groups were treated with 2.28 mg ZnSo4 (1/5 of LC50) of water and they were
represented as T3 and T4. The T2 group and T4 group of fish were fed with probiotic diet containing
6
10 cfu/g of Lactobacillus rhamnosus. The water in the aquaria was changed every alternate day, after
feeding. The fishes were fed with the diet prepared as per indicating in the Table 3. The dietary
constituents were procured in the local medical and grocery shop in and around Thoothukudi. The
experiment was conducted for 60 days, during the experimental period the fish were fed prepared diet
once each day at 5% body mass. The fish were reared in five glass tanks of 25 liters capacity
sponsored by DCW Ltd, Sahupuram.

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Mitigation of Zinc Sulphate Induced Haematological Toxicity in the Fish Mystus montanus by Probiotic Bacteria

2.6 Haematological Procedures

The fish were transported alive in plastic trough containing chlorine free water to the Laboratory. The
o
fish were allowed to acclimatize at water temperature range of 24 to 27 C for two weeks prior to the
commencement of the study. Blood was collected through caudal vein of the fish by hanging the fish
in upright position using a 2ml sterile plastic syringe. Determination of full blood count, including
haemoglobin (Hb), PCV, RBC and ESR, Hematological indices including Mean corpuscular volume
(MCV), Mean corpuscular haemoglobin (MCH) and Mean corpuscular haemoglobin concentration
(MCHC) and Differential leucocytes counts including Heterophil, Monocytes, Eosinophil, Basophil and
Lymphocyte were all carried out using SYSMAX XP 100 Auto Analyzer in the Laboratory of DCW Ltd,
Sahupuram.

2.7 Statistical Analysis

The values were expressed as mean ±SEM. The altered blood parameters of the experimental fish
from the control group of fish were expressed inn percentage. It was calculated by the formula,
Control – Experiment/control.
Table 1. Water parameters

No Parameters Units Value

o
1 Temperature T C 29
2 pH 7.2
3 Total suspended solids mg/l ppm 28
4 Total dissolved solids mg/l ppm 650
5 Total Hardness as CaCO3 mg/l ppm 280
6 Calcium as CaCO3 mg/l ppm 44
7 Magnesium as CaCO3 mg/l ppm 43
8 Sodium as Na mg/l ppm 75
9 Potassium as K mg/l ppm 33
10 Carbonates as CaCO3 mg/l ppm 24
11 Sulphates as SO4 mg/l ppm 75
12 Chlorides as Cl mg/l ppm 327
13 BOD5 mg/l ppm 4

Table 2. Study design

Fish group No of fish Treatment Duration Feeding %

Control 10 Basal diet 60 days 5% body mass
th
T1 10 1/10 LC50 dose of ZnSO4 and Basal diet 60 days 5% body mass
th
T2 10 1/10 LC50 dose of ZnSO4 and Probiotic 60 days 5% body mass
diet
th
T3 10 1/5 LC50 dose of ZnSO4 and Basal diet 60 days 5% body mass
th
T4 10 1/10 LC50 dose of ZnSO4 and Probiotic 60 days 5% body mass
diet

Table 3. Ingredients used for feed preparation

S. Ingredients Purpose of Inclusion Protein Lipid Carbohydra

No inclusion level (%) (%) (%) te (%)
1 Fish meal animal protein 16 62 5.8 3.7
2 Groundnut oil cake plant protein 16 45.6 40.9 8.7
3 Soya flour plant protein 16 71.6 10.7 9.8
4 Rice bran carbohydrate 18 13.5 1.8 75.5
5 Tapioca flour binder 18 5.8 12.5 76.3
6 Vitamins & Vitamins & minerals 0.5 - - -
minerals mix
6
7 PrePro KID Lactobacillus 10 cfu/g - - -
rhamnosus

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Mitigation of Zinc Sulphate Induced Haematological Toxicity in the Fish Mystus montanus by Probiotic Bacteria

Table 4. LC50 value calculation

2
Concentration mg/lit (X) X Probit Kill (Y) XY
10 100 00 000
20 400 1.72 34.40
30 900 3.48 104.4
40 1600 4.90 196
50 2500 5.25 262.5
60 3600 5.25 315
70 4900 5.52 386.4
80 6400 5.84 467.2
90 8100 6.28 565.2
100 10,000 6.28 628
2
∑X = 550 ∑X = 38,500 ∑Y = 51.34 ∑XY = 2959.1

Table 5. Haematological parameters of the fish

th th th th
Parameters Control T1 (1/10 of T2 (1/10 of T3 (1/5 of LC50) T4 (1/5 of
LC50) LC50 Zn LC50 Zn &
&Probiotics) Probiotics)
b c a b
Hb (g/dl) 8.45 ±0.43 5.34 ± 0.72 6.14 ± 0.32 4.66 ± 0.12 5.66 ± 0.14
a a a a
PCV (%) 13.22 ± 1.86 10.70 ± 2.1 11.10 ± 1.1 7.50 ± 1.18 9.50 ± 1.48
a a a a
RBC × 3.86 ± 0.17 2.20 ± 0.15 3.20 ± 0.45 1.95 ± 0.32 2.95 ± 0.12
6 3
10 /mm
a a b b
ESR (mm) 1.15 ± 0.26 1.65 ± 0.35 1.25 ± 0.65 1.87 ± 0.20 1.37 ± 0.40
b a b b
MCV (fl) 8.53 ± 0.61 6.53 ± 0.31 7.74 ± 0.95 5.74 ± 0.75 6.86 ± 0.16
a a a a
MCH (pg) 30.56 ± 3.13 27.56 ± 1.43 29.30 ± 2.28 23.28 ± 2.68 26.50 ± 1.48
a a a a
MCHC (%) 28.48 ± 2.32 23.48 ± 2.72 26.57 ± 2.06 22.67 ± 3.01 21.95 ± 0.14
a a b b
HET (%) 43.00 ± 4.56 32.00 ± 3.32 34.00 ± 2.32 23.00 ± 2.54 33.00 ± 1.54
a a a a
MON (%) 13 ± 3.67 10.45 ± 2.21 11.45 ± 1.21 7.87 ± 3.24 9.87 ± 32.24
b b b b
EOS (%) 11.57 ± 2.23 9.43 ± 1.77 10.43 ± 00.77 8.60 ± 1.71 8.20 ± 0.71
a a a a
BAS (%) 3.45 ± 1.76 2.70 ± 1.25 3.10 ± .25 1.63 ± 0.55 2.23 ± 0.67
a a a a
LYM (%) 30.46 ± 2.40 25.70 ± 2.60 27.70 ± 1.60 25.36 ± 3.10 28.36 ± 2.10
Where Hb: Haemoglobin; PCV: Packed Cell Volume; RBC: Red Blood Cells; WBC: White Blood Cells; ESR:
Erythrocyte Sedimentation Rate; MCV: Mean Corpuscular Volume; MCH: Mean Corpuscular Haemoglobin;
MCHC: Mean Corpuscular Haemoglobin Concentration; HET: Heterophil; MON: Monocyte; EOS: Eosinophil;
BAS: Basophil; and LYM: Lymphocyte In the table, a is Significant at 5%, b is Significant at 1% and c is
Insignificant

3. RESULTS
3.1 LC50 Analysis
The toxicity tests were conducted in constant temperature, light conditions and constant water quality
parameters. Fish mortality data at 96 hour duration and the results of it probit/regression analysis are
presented in Table 4. The observed LC50 value of Zinc sulphate to the fish for 96 hours is 45.6 mg/l.

3.2 Hemoglobin, PCV, RBC and ESR

Significant decrease of hematological parameters such as Hemoglobin, Packed Cell Volume, Red
Blood Corpuscle (p<0.05), and ESR (p<0.001) were observed in the zinc intoxicated fish in T3 from
the control group. However, all these parameters of the fish treated with probiotic diet in T4 showed
significant improvement (p<0.05) of PCV, RBC and Hb and ESR (p<0.001) from the antidote control
group of fish T3 (Table 4).

3.3 MCV, MCH and MCHC

The hematological indices such as MCH, MCHC (p<0.05), and MCV (p<0.001) (Table 4) decreased in
the T3 fish groups from the control group of fish. In T4 group the same hematological indices (MCH,
MCHC (p<0.05) and MCV (p<0.001) significantly increased from its antidote control T3.

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Mitigation of Zinc Sulphate Induced Haematological Toxicity in the Fish Mystus montanus by Probiotic Bacteria

3.4 Heterophil, Monocyte, Eosinophil, Basophil and Lymphocyte

The leukocytes such as HET, BAS, MON, LYM (p<0.05), and EOS (p<0.001) decreased significantly
in the T1 and T3 groups of fish (Table 4). All these counts (HET, BAS, MON, LYM (p<0.05) and EOS
(p<0.001) of the probiotic fed fish increased significantly in the T2 and T4 groups of fish.

4. DISCUSSION

During the course of experiment 100% mortality was not observed. There was no mortality at 45.6mg
ZnSo4/L and below this concentration. In higher concentrations of ZnSo 4, during the toxicant exposure
period the test fishes showed various behavioral responses like increased opercular movement,
mucous secretion and jerky movement, floating on the sides, hypersensitivity showing violent erratic
and fast swimming. Changes were also observed in the coloration and activeness of the fishes.
Clotting of blood on the gill surfaces also observed from the dead fishes.

Results of present studies clearly indicate that the rate of mortality for any fixed time increases with
increase in concentration and for a particular concentration with increase in exposure time and a
regular mode of toxicant, due to accumulation up to dangerous level leading to death as described by
Gomez et al. [10] in the fish Cnesterodon decemmaculatus with Zn concentration 44 mg/l. Das and
Sahu, [11] explained that the major cause of mortality of the Zn exposed fish might be due to
respiratory epithelium damage by oxygen accumulation during the formation of a mucus film over the
gills of fish. Observation of exposed gills in the present study during LC 50 analysis of the fish
confirmed the explanation of Das and Sahu, [11].

The hematological parameters such as Hemoglobin, Packed Cell Volume, Red Blood Corpuscle and
Erythrocyte Sedimentation Ratio of the Zinc intoxicated fish supplemented with probiotic diet showed
significant decrease (p<0.05) from the control group. However the fish treated with probiotic diet
showed significant recovery (p<0.05) from the antidote control group of fish.

The production of organic acids by lactobacilli is very important for the regulation of gut microflora
growth and composition. The inhibitive effect of organic acids is based on the reduction of gut content
pH to values where the growth of unfavorable microbiota is depressed [12]. In addition to lactic acid,
which is produced in highest concentrations, heterofermentative lactobacilli produce other types of
organic acids also in the gut, primarily acetic and acetoacetic acid. The L. plantarum CCM 7102
produces, in addition to lactic acid, relatively high concentrations of acetic and acetoacetic acids
which serve as a good prerequisite for the efficacy of a probiotic strain. Adams and Hall, [13] have
confirmed the synergistic effect of lactate and acetate in the inhibition of pathogens and favor the
growth of probiotics in the gut of fish. These evidences confirm the fact that sufficient probiotic
bacterial growth in the gut prevents the absorption of zinc sulphate, thus protect the blood parameters
of the fish.

The hematological indices such as MCV, MCH and MCHC of the experimental fish decreased
(p<0.001) from the control group and significant (p<0.005) decrease from the antidote control was
observed in the MCV. Kori-Siakpere, et al. [14] also observed decreased total white blood cell counts,
Plasma level of protein, glucose and haematological indices MCHC, MCH and MCV in the zinc (5.0
and 10.0 mg L-1) exposed fish, Heteroclarias sps.

Ologhobo, [15] reported that the most common blood variables consistently influenced by diet are
haematocrit (Ht) and haemoglobin (Hb). Bacillus subtilis and Saccharomyces cerevisiae significantly
(P < 0.05) increased Hematocrit level in the fish Oncorhynchus mykiss [16]. Concerning the effect of
the Lactobacillus rhamnosus on the haematological indices of Mystus montanus, the results indicated
a positive effect in the fish group supplemented with probiotic diet represented by increased MCV,
MCH and MCHC from the antidote control group. This could be attributed to the fact that, probiotic
diet increased the blood parameters than the antidote control group of fish, as a result of
hematopoietic stimulation. The result of the present study coincides with the results of Rajesh Kumar
et al. [17].

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Mitigation of Zinc Sulphate Induced Haematological Toxicity in the Fish Mystus montanus by Probiotic Bacteria

The leukocytes such as HET, BAS, MON, LYM (p<0.05), and EOS (p<0.001) decreased significantly
in the T1 and T3 groups of fish. All these counts (HET, BAS, MON, LYM (p<0.05) and EOS (p<0.001)
of the probiotic fed fish increased significantly in the T2 and T4 groups of fish [18]. The present study
confirms the role of probiotic bacteria Lactobacillus rhamnosus on for the improvement of differential
leukocyte counts in the Zinc toxicated fish. The application of a mixed probiotic species of B. subtilis
and S. cerevisiae in C. mrigala [19]. L. acidophilus and B. subtilis in Nile tilapia [18] have been
reported to increase the WBC levels. Munir et al. [20] reported that feeding fish with probiotic-
supplemented diets enhanced immune defense. This study confirms that Lactobacillus rhamnosus in
the gut competitively excludes other bacteria and stimulates the growth of probiotic bacteria in the gut
to protect the fish from the toxic effect of Zinc sulphate.

The present study confirms the role of probiotic bacteria Lactobacillus rhamnosus on the improvement
of hematological parameters in the Zinc intoxicated fish. Tahere et al. [21] observed high proportion of
Bacillus pumilus in the intestine of experimental fish fed with probiotic diet and they confirmed that
intestinal environment is suitable for the given probiotic bacteria to settle and grow and also lead into
harbor a great number of microbial cells of host intestine. This study confirms that improved
hematology of zinc intoxicated fish is associated with settlement of L.rhamnosus in the gut and their
protective effect from the toxic effect of Zinc.

5. CONCLUSION

The results of the study clearly indicated that application of Lactobacillus rhamnosus to the feed of
Mystus montanus gives good protection against Zinc toxicity. This study clearly demonstrated that
Probiotic bacteria can be effectively used as a therapeutic regimen for the Zinc intoxicated fish.
Further it is clearly evident that probiotic supplemented diet could be used to ameliorate any heavy
metal intoxication of fish.

COMPETING INTERESTS

Authors have declared that no competing interests exist.

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Biography of author(s)

T. Sakthika
Department of Zoology, A. P. C. Mahalaxmi College for Women, Thoothukudi, Tamil Nadu, India.

She is working in the Department of Zoology at A.P.C.Mahalaxmi College for Women, Thoothukudi. She has 24 years of
Academic experience, and 10 Years of research experience. She has published three books namely Nutritional concepts of
Diet, Nutraceuticals and Preparation of Probiotic Drinks and Detection of Adulterants. She has published 24 research articles in
National, International UGC care listed and Scopus indexed Journals and presented 5 research articles in National and
International conferences. She has been Question Paper Setter, Thesis Evaluator and Doctoral Committee Member in various
colleges and Review Board Member in the international journal of Acta Scientific Agriculture.
_________________________________________________________________________________
© Copyright (2022): Author(s). The licensee is the publisher (B P International).

DISCLAIMER
This chapter is an extended version of the article published by the same author(s) in the following journal.
Annals of Biological Sciences, 8 (2):34-37, 2020.

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