Fish & Shellsh Immunology 59 (2016) 382e388

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Fish & Shellsh Immunology

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Full length article

Feeding pyridoxine prevents Saprolegnia parasitica infection in sh

Labeo rohita
Himadri Saha a, b, **, Asim Kumar Pal a, Narottam Prasad Sahu a, *, Ratan Kumar Saha b
a
Division of Fish Nutrition, Biochemistry and Physiology, Central Institute of Fisheries Education, Mumbai, Maharashtra, 400061, India
b
Department of Aquatic Health and Environment, College of Fisheries, CAU, Lembucherra, Agartala, Tripura, 799210, India

a r t i c l e i n f o a b s t r a c t

Article history: A 60-day experiment was carried out to delineate the role of dietary pyridoxine (DP) in Labeo rohita
Received 25 May 2016 ngerlings in modulating immunity and prevention of fungal infection. Two hundred and seventy n-
Received in revised form gerlings were randomly distributed into three treatments in triplicates. Three iso-caloric and iso-
14 September 2016
nitrogenous puried diets were prepared with graded levels of pyridoxine. Three experimental groups
Accepted 22 September 2016
Available online 23 September 2016
were C (0.0% DP), T1 (0.01% DP) and T2 (0.02% DP). The role of dietary pyridoxine in modulating im-
munity and prevention of fungal infection was assessed by haemato-immunological parameters like
erythrocyte counts (EC), leucocyte counts (LC), haemoglobulin (Hb), packed cell volume (PCV), mean
Keywords:
Dietary pyridoxine
corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin
Labeo rohita concentration (MCHC), nitro-blue tetrazolium (NBT), phagocytic activity, albumin, globulin, total plasma
Saprolegnia parasitica protein, albumin/globulin and by challenge study with Saprolegnia parasitica, where relative percentage
Hemato-immunological survival (RPS) were recorded. Hb, PCV, MCV, MCH, NBT, total plasma protein, albumin, globulin contents,
Fungus lysozyme and phagocytic activity was signicantly (P < 0.05) higher in DP fed group. Signicantly
(P < 0.05) higher RPS was recorded from T2 group fed with 0.02% DP for 45 days. Hence, DP has the
capacity to stimulate nonspecic immunity and increase resistance to S. parasitica infection in L. rohita
ngerlings.
2016 Elsevier Ltd. All rights reserved.

1. Introduction alternative medicines for treating and managing sh diseases [14].

Aquaculture has been expanded, intensied and diversied
globally [1] as fastest growing food producing sector to meet the
increased demand for quality protein. Asia is the leader in aqua-
culture production, where Indian major carps, rohu (Labeo rohita) is
the most preferred sh and share about 35% of the Indian major
carps production [2] However, the species is a host for many
pathogens for which many chemotherapeutants as well as antibi-
otics are used for their treatments. But the use of antibiotics and
cytotoxic drugs has been widely criticized for their negative im-
pacts like development of drug resistance, accumulation in the
tissues, and immunosuppression [3]. Unlike antibiotics and live
vaccines, most of the nutraceuticals do not show any adverse
impact on sh and environment [4e13]. Thus, they can be used as
* Corresponding author .
** Corresponding author. Division of Fish Nutrition, Biochemistry and Physiology,
Central Institute of Fisheries Education, Mumbai, Maharashtra, 400061, India.
E-mail addresses: sahacofcau@gmail.com (H. Saha), npsahu1@rediffmail.com,
npsahu@cife.edu.in (N.P. Sahu).
Saprolegnia is a most abundant oomycete of aquatic origin are
present in water and cause great losses to aquaculture and sheries
around the world [15e19]. Earlier, Saprolegnia was classied as
pathogenic fungi [19,36] and considered to be opportunist facul-
tative parasite [20] saprotrophic and necrotrophic [21], however,
recent ndings categorise them as oomycete [16]. Saprolegnia in-
vades epidermal tissues visible as white or grey patches of la-
mentous mycelium on sh [21,22], generally beginning on the head
or ns [20,23] and can spread over the entire surface of the body.
Saprolegnia infection has cotton-like appearance that radiates out
in a circular, crescent-shaped or whorled pattern. The zoospores of
Saprolegnia may be transmitted by sh eggs, wild sh, water
sources, and equipment [21].
A number of workers have reported Saprolegnia infection in
shes; salmonids [22,24], channel catsh [25], pike [26], elver and
suckers [27], roach, orfe, carp, tench, lamprey, sturgeon, barra-
mundi, tilapia, and mullet [21], Labeo rohita, Catla catla, Channa
striatus, C. punctatus, Clarias batrachus, Mystus cavasius, M. seen-
ghala, Tilapia mossambica [28], kissing gourami, guppy, swordsh
and platysh [23,27], Anabas testudineus [29]. However, among

http://dx.doi.org/10.1016/j.fsi.2016.09.045
1050-4648/ 2016 Elsevier Ltd. All rights reserved.
 H. Saha et al. / Fish & Shellsh Immunology 59 (2016) 382e388
several species of Saprolegnia, S. parasitica, in particular, appears to
be largely responsible for such infections, Saprolegniasis [30,31].
Saprolegnia generally invades sh that have been stressed or
otherwise have weakened immune systems [21]. A number of
chemicals such as malachite green [21,31], formalin [32], hydrogen
peroxide [33,34], sodium chloride at high concentrations [34,35],
etc. are available for treatment of Saprolegnia infection but a few are
approved for use in aquaculture. Formalin treatment [36e38] is
currently the most used method against saprolegniasis in sh eggs
in sh farms. Heikkinen et al. [39] showed that strong UV irradia-
tion of inlet water decreased Saprolegnia infection on eggs. Several
studies to nd replacements for malachite green (banned in Eu-
ropean Union in 2001) have been performed [40], but no better
substitute has yet been found.
Many researchers have reported many functions of pyridoxine
(vitamin B6) including pyridoxal and pyridoxamine for absorption
and metabolism of amino acids and development of red blood cells
[41], coenzymes for transaminases [42], control the biosynthesis of
neurotransmitters like GABA, dopamine and serotonin (5-HT),
development and function of the central nervous system (CNS) [43]
and anti-stress function [12,44,45]. Chen et al. [46] has reported
that dietary pyridoxine plays an important role in stimulating the
immune responses by enhancing phagocytic activity and respira-
tory burst activity of abalone, Haliotis discus hannai. However, this
vitamin has a protective effect against pathogenic Vibrio anguilla-
rum, and Aeromonas hydrophila in Chinook salmon, Oncorhynchus
tshawytscha (Walbaum) ngerlings [47] and Labeo rohita nger-
lings [12]. However, in one of our pilot study (data not shown here)
revealed that pyridoxine has the antifungal property (in-vitro)
against S. parasitica. Hence, with all these backdrop information the
present study was envisaged to elucidate the possibility of using
dietary pyridoxine for protecting L. rohita ngerlings against
pathogenic S. parasitica.
2. Materials and methods
2.1. Experimental animals
The experiment was carried at the laboratory of Department of
Aquatic Health and Environment, College of Fisheries, Lembu-
cherra, Tripura. Apparently healthy L. rohita ngerlings (average
length 12.67 1.5 cm and average weight 22.56 7.5 g) were
collected from the college sh farm. The sh were allowed to ac-
climatize in circular ber reinforced plastics (FRP) tanks (Plasto
Craft, Mumbai) of 500 L under the laboratory condition for 15 days
and then used for the experiments. Fishes were provided with
adequate aeration and fed with control diet. Satiation level feeding
was done twice a day and 30% of water was exchanged daily. Deep
tube well water was stocked in 1000 L FRP tanks with aeration and
was used for setting the experiment. FRP tanks of 500 L size were
used for the experiment. Round the clock aeration was provided.
2.2. Feed preparation and proximate composition
Three iso-nitrogenous(35.16e35.95% crude protein) and iso-
caloric (1766.85e1794.61 kJ 100 g
1) puried diets were prepared
with graded levels (0, 100 and 200 mg pyridoxine/kg) of pyridoxine
(Himedia Laboratories, Mumbai, India) (Table 1). Inclusion rate was
decided based on the earlier ndings of Akhtar et al. [12,13].
Proximate composition of the experimental diets were analyzed
(Table 1) following standard method [48].
2.3. Experimental design and feeding diet
The acclimatized rohu ngerlings (n 270) were randomly
383
distributed into three distinct experimental groups in triplicates
following a completely randomized design (CRD). Randomly
selected thirty sh were stocked in circular FRP tanks of 500 L ca-
pacity. Five ngerlings were randomly sampled on 15, 30 and 45 d
of feeding from each tank without replacement. The control group
was fed without pyridoxine whereas the remaining groups were
fed with either 0.01% (T1) and 0.02% pyridoxine (T2) for 45 days. For
the challenge experiment, remaining 10 shes from each treatment
including control were innoculated with the spores of S. parasitica
after 45 days of feeding. The water used for the experiment were
analysed for various environmental parameters such as tempera-
ture, pH, dissolved oxygen (DO), total alkalinity and total ammonia-
N following APHA [49]. The water quality parameters such as pH,
temperature, dissolved oxygen (DO), total alkalinity, and total
ammonia-N were found in the range of 7.0e7.5, 26.5e28.0  C,
5.2e5.6 mg l
1, 103e110, and 0.01e0.03 mg l
1, respectively, during
the experiments, which are in the optimum range required for rohu
[50].
2.4. Collection of blood and plasma
At different sampling days (0, 15, 30 and 45 d), each sh was
anesthetized with clove oil (Merck, Germany) at 50 ml of clove oil
per litre of water before collecting blood samples. Five sh from
each tank were used for blood collection and pooled samples from
each tank were used for analysis. Blood was drawn from the caudal
vein of sh by using 1.0 ml hypo-dermal syringe and 24 gauge
needles [51]. The collected blood was immediately transferred into
two vials, where vials were coated with thin layer of EDTA, an
anticoagulant. One for using blood to measure cell counts, packed
cell volume, haemoglobin, and NBT and other for plasma collection.
Collection of plasma was done by centrifuging blood samples at
3500 rpm for 15 min at 4  C and stored at
20  C until use.
2.5. Culture of Saprolegnia parasitica
2.5.1. Isolation of fungal strains from diseased sh
Isolation of fungal strains were done following Lilley et al. [52].
Briey, diseased sh samples were brought to the laboratory; sh
were killed by decapitation and pinned, to a dissecting board with
the lesions uppermost. Then the sh along with the board were
brought under laminar air ow. Using a sterile ne pointed forceps
and scalpel blade, the muscles pieces were removed. Using aseptic
technique, the muscle pieces were placed into a petridish con-
taining GP (glucose-peptone) PenStrep broth (composition:
glucose: 3 g L
1, peptone: 1 g L
1, MgSO4$7H2O: 0.128 g L
1,
KH2PO4: 0.014 g L
1, CaCL2.2H2O: 0.029 g L
1, FeCl3$6H2O:
2.4 mg L
1, MnCl2$4H20: 1.8 mg L
1, CuSO4$5H2O: mg L
1,
ZnSO4$7H2O: 0.4 mg L
1 autoclaved at 121  C for 15 min, cooling to
50  C and added penicillin-K: 250 units ml
1 and streptomycin
sulphate: 200 mg ml
1. Inoculated media were incubated at
20 2  C and after 24 h the emerging hyphal tips were repeatedly
transferred to fresh plates of glucose peptone agar (Composition:
glucose peptone broth technical agar: 12 g L
1 autoclaved at
121  C for 15 min, cooling to 50  C and added penicillin-K: 250 units
ml
1 and streptomycin sulphate: 200 mg ml
1) until cultures were
free from bacterial contamination. Conrmation of Saprolegnia
parasitica was done following Soderhall et al. [53], Willoughby [23],
Lilley et al. [52] and Eissa et al. [54].
2.5.2. Inducing sporulation in fungal culture
Sporulation was induced following Lilley et al. [52]. Briey, an
agar block of actively growing mycelium was placed on a petri-dish
containing glucose peptone yeast (GPY) broth (Composition:
glucose peptone broth Yeast extract: 1.5 g L
1) and incubated for
384 H. Saha et al. / Fish & Shellsh Immunology 59 (2016) 382e388

Table 1
Composition of experimental diets. Data expressed as Mean SE, n 3.

Ingredients (in g)/1000 g of feed Control diet Treatment diets

Diet T1 (0.01%) Diet T2 (0.02%)

Casein (vitamin free) 320 320 320

Gelatin 80 80 80
Dextrin 300 300 300
Cellulose powder 129.7 129.6 129.5
Carboxymethylcellulose 20 20 20
Sunower oil 45 45 45
Cod liver oil 45 45 45
Vitamin Mineral premixa 60 60 60
Betaine hydrochloride 0.2 0.2 0.2
Butylated hydroxyl toluene 0.1 0.1 0.1
Pyridoxine 0 0.1 0.2
Total 1000 1000 1000
Proximate composition of diets
Moisture 10.73 0.18 11.07 0.07 10.87 0.07
Crude protein (CP) 35.16 1.04 35.93 0.55 35.44 0.32
Ether extract (EE) 9.02 0.2 9.15 0.13 8.95 0.23
Ash 5.9 0.24 5.56 0.05 5.55 0.03
Total carbohydrate (TC) 49.9 0.95 49.36 0.65 50.08 0.37
Digestible energyb KJ 100g
1 1780.25 7.63 1788.51 3.09 1784.65 5.00
a
Composition of vitamin-mineral premix (PREEMIX PLUS) (quantity/2.5 kg): Vitamin A, 5500000 IU; Vitamin D3, 1100000 IU; Vitamin B2, 2000 mg; Vitamin E,
750 mg; Vitamin K, 1000 mg; Vitamin B6, 1000 mg; Vitamin B12, 6 mcg; Calcium Pantothenate, 2500 mg; Nicotinamide, 10 g; Choline Chloride, 150 g; Mn, 27,000 mg; I,
1000 mg; Fe, 7500 mg; Zn, 5000 mg; Cu, 2000 mg; Co, 450 mg; L-lysine, 10 g; DL- Methionine, 10 g; Selenium, 50 ppm.
b
Digestible energy (kJ 100 g
1) (% CPx17) (% EEx37) (% TCx17).

4 days at 20  C. The agar was washed out of the resulting mat by
sequential transfer through 5 petridishes containing ltered and
autoclaved pond water (APW) and kept overnight at 20  C in APW.
After about 24 h the release of secondary zoospores was observed
under microscope.
2.5.3. Saprolegnia parasitica infection
After 45 days of feeding, 10 sh from each group was taken for
challenge with S. parasitica following Firouzbakhsh et al. [55] with
slight modication. The challenge study was done with three rep-
licates for each treatments including control in 200 L capacity PVC
tanks. The method of ami-momi with slight modication [56] was
followed to create stress and reduce the protecting surface mucus
for the penetration of spores into the body of sh. This was carried
out by slowly shaking and rubbing groups of 10 sh in a net (6 mm
mesh size) for 2 min followed by washing mucus secretions and
placing the sh in the aquaria. Next, the zoospores were added
(3  105 L
1) to the treatments. The zoospores dispersed in the
water by suitable aeration. The water in the tanks was exchanged as
much as 50% every 3 days until 15 days with water containing
zoospores (3  105 L
1) [57], and daily mortalities as well as sh
contaminations were monitored. Direct microscopic examination
was employed to conrm fungal infections. The fungi at the internal
and external tissues were observed following Debnath et al. [58],
using haematoxylin and eosin (H & E) stains.
2.5.4. Relative percentage survival (RPS)
Mortality was observed for 15 days. Dead sh were collected
and tissues were removed aseptically for fungal culture to conrm
S. parasitica as the cause of death. Relative percentage survival was
calculated using following formulae: Relative percentage survival
(RPS) [1
(% mortality in treated group/% mortality in control
group)] x 100.
2.6. Haematological parameters
Red blood cells (RBC) and white blood cells (WBC) diluting uids
(Himedia) were used for counting total erythrocytes (EC) and total
leucocyte (LC) numbers, respectively. Briey it was carried out by
mixing 20 ml of blood with 3980 ml of respective diluting uid in a
clean test tube and was shaken well to suspend the cells uniformly
in the solution. The mixture was charged to Neubauer's counting
chamber of haematocytometer (Marienfeld, Germany) and count-
ing was carried out [50]. Both EC and LC were expressed in Nos.
mm
3.
Haemoglobin content in blood was quantied by using Sahli
Haemometer (Marienfeld, Germany). In this method, the
measuring tube was rst lled with 0.1N hydrochloric acid up to the
mark 2, and then 20 ml of blood was added [59]. The colour of the
sample was compared with the standard coloured rod (Marienfeld,
Germany) following dilution of the sample by adding distilled
water drop wise. Reading of the haemoglobin content was deter-
mined from the scale given in the measuring tube.
Packed cell volume (PCV) was measured following the method
of Schaperclaus [59] and expressed as the percentage fraction of
blood cells in the total volume (volume%). Mean corpuscular vol-
ume (MCV), mean corpuscular haemoglobin (MCH) and mean
corpuscular haemoglobin concentration (MCHC) was calculated by
following the formulae of Dacie and Lewis (2001) [60] and
expressed as 10
15 L, pg and g 100 ml
1, respectively.
2.7. Immunological parameters
Total plasma protein was measured using a diagnostic kit (Crest
Biosystems, India) based on Biuret method [61]. The sample was
measured by using micro-plate reader at 550 nm wavelength. Total
protein was calculated using the following formula: Total Protein
(in g dl
1) (Absorbance of Test/Absorbance of Standard) x 8
Albumin was measured spectro-photometrically by following
bromocresol green binding method [62] at 630 nm. Globulin was
calculated by subtracting albumin values from total plasma protein
[13].
Production of oxygen radicals from phagocytic cells was
measured using nitro-blue tetrazolium (NBT) dye as described by
Anderson and Siwicki [63] and absorbance was read at 540 nm
using a spectrophotometer.
Lysozyme activity of blood plasma was determined as described
by Anderson and Siwicki [63] with some modications. Plasma
 H. Saha et al. / Fish & Shellsh Immunology 59 (2016) 382e388 385

(0.1 ml) was placed in test tubes and 0.9 ml of a 0.75 mg ml
1 Table 2
Micrococcus lysodeikticus (Sigma, St Louis, MO, USA) suspension in Haematological parameters (erythrocyte count, leukocyte count, packed cell vol-
ume, haemoglobin, mean corpuscular volume, mean corpuscular haemoglobin and
phosphate buffered saline, pH 6.2 was added and mixed well. The mean corpuscular haemoglobin concentration) of L. rohita ngerlings fed with di-
absorbance was measured at 450 nm by a spectrophotometer at etary pyridoxine supplemented diet (control diet, diet T1 and diet T2) for 0, 15, 30
1 min intervals for 10 min after mixing with bacteria and rate of and 45 d.
change of absorbance was calculated. Days T1 T2 C
Phagocytic cells were detected using Pseudomonas aeruginosa as
Erythrocyte count (x 106cells mm3)
described by Anderson and Siwicki [63]. Briey, 0.1 ml of blood
0d 3.54 0.03 3.52 0.02 3.54 0.03
sample was placed in a microtiter platewell and 0.1 ml of Pseudo- 15 d 3.53 0.03 3.55 0.01 3.5 0.02
monas aeruginosa (1  107 cells) suspended in phosphate buffered 30 d 3.53 0.02 3.56 0.03 3.5 0.02
saline (pH 7.2) was added and mixed. The mixed solution was 45 d 3.52 0.02 3.54 0.02 3.42 0.01
Leucocyte count (x 104 cells mm
3)
incubated for 20 min at room temperature. After incubation, a
0d 2.43 0.02 2.44 0.02 3.10 0.68
smear was prepared with 5 mL solution. The smear was air dried, 15 d 2.56 0.06 2.64 0.16 2.44 0.02
then xed with ethanol (95%) and stained with Giemsa stain. Total 30 d 2.77 0.02 2.92 0.01 2.43 0.02
of 100 neutrophils and monocytes from each smear were observed 45 d 2.96 0.06 3.02 0.03 2.42 0.01
using light microscope. The number of phagocytizing cells and the Packed cell volume (%)
0d 39.63 0.90 39.80 0.91 40.67 0.53
number of bacteria engulfed by the phagocyte were counted.
15 d 42.97 0.67 ab 47.33 0.88b 40.43 1.66a
Phagocytic activity was calculated as: Phagocytic activity (%) (The 30 d 43.83 0.88a 49.87 1.56b 40.47 1.30a
number of phagocytizing cells/the total number of phagocytes 45 d 44 0.58a 52.47 1.30b 41.40 1.33a

1
counted) x 100. Haemoglobin (g dl )
0d 3.77 0.03 3.80 0.06 3.40 0.06
15 d 4.07 0.07 4.20 0.06 3.90 0.06
2.8. Statistical analysis 30 d 4.20 0.10a 4.67 0.07b 3.67 0.19c
45 d 4.10 0.06a 4.80 0.06b 3.60 0.15c
Statistical analysis of data was performed using SPSS-15.0 (SPSS Mean corpuscular volume (10
15 L)
Inc., Chicago, IL, USA). Results are presented as mean standard 0d 111.93 1.62 113.05 1.92 114.92 2.40
15 d 122.89 1.98 ab 133.44 1.97a 114.58 3.56b
error. Comparisons of mean values were determined by one way
30 d 125.40 3.34 ab 140.30 5.57a 114.61 3.12b
ANOVA and Tukey test. Probability levels of 0.05 were used to nd 45 d 128.53 1.43a 148.19 3.19b 117.46 3.01a
out the signicance. Mean corpuscular haemoglobin (pg)
0d 10.64 0.17 10.79 0.13 9.60 0.08
3. Results 15 d 11.63 0.25 11.84 0.21 11.06 0.27
30 d 12.02 0.35a 13.12 0.13a 10.39 0.56b
45 d 11.98 0.12a 13.56 0.20b 10.23 0.50c
3.1. Haematological responses Mean corpuscular haemoglobin concentration (g dl
1)
0d 0.10 0.00 0.10 0.00 0.08 0.00
There was no signicant (P > 0.05) effect of dietary pyridoxine 15 d 0.09 0.00 0.09 0.00 0.10 0.01
30 d 0.10 0.00 0.09 0.00 0.09 0.01
on both erythrocyte and leucocyte counts of L. rohita ngerlings
45 d 0.09 0.00 0.09 0.00 0.09 0.01
found at different sampling days (Table 2) in different experimental
groups. However, there was signicant (P < 0.05) effect of dietary Data (mean SE) at the same sampling days with different letters (a, b and c)
signicantly differ (P < 0.05) among different experimental groups (C, T1 and T2).
pyridoxine on blood haemoglobin content of different experi-
mental groups at different sampling time. The highest (P < 0.05)
haemoglobin content was observed in T2 group compared to T1 and respiratory burst activity reduction value was found in T2
control from 30th day of post-feeding (Table 2). Haemoglobin compared to other experimental groups from 15th day onwards.
content of T1 was signicantly (P < 0.05) different from control and However, T1 group showed signicantly higher value compared to
T2 from 30th day of feeding pyridoxine. Similarly, in comparison to the control on 15th day and onwards of feeding. In case of phago-
control MCH value was signicantly (P < 0.05) higher in T2 from cytic and lysozyme activity, both T1 and T2 values were signi-
30th day. However, both PCV and MCV were signicantly (P < 0.05) cantly higher (P < 0.05) than the control group from 15th day and
higher in T2 as compared to control on 15th day onwards. onwards of feeding (Table 3).

3.2. Total plasma protein, albumin and globulin

Highest albumin content was observed in T2 which was signif-
icantly (P < 0.05) higher than the control group between 15th and
30th days of post feeding (Table 3). However, globulin content of T1
and T2 were signicantly (P < 0.05) higher than the control group
on 30th day of post-feeding with pyridoxine supplemented feed.
Similarly, total plasma protein of T2 group was signicantly higher
compared to T1 and control on 15th day of post-feeding. However,
T1 was signicantly (P < 0.05) higher from control on 30th day of
feeding (Table 3).
3.3. Nitro blue tetrazolium (respiratory burst activity) and
phagocytic activity
The production of superoxide estimated by respiratory burst
activity reduction was signicantly (P < 0.05) inuenced by the
supplementation of dietary pyridoxine (Table 3). Highest (P < 0.05)
3.4. Challenge study with S. parasitica
All mortalities recorded during the trial were due to the chal-
lenge infection with S. parasitica. The mortalities were observed for
15 days and it revealed that the relative percentage survival of
group of sh fed with 0.02% pyridoxine was signicantly (P < 0.05)
higher than the other experimental groups (Fig. 1).
4. Discussion
The present study revealed that the supplementation of 200 mg
pyridoxine per kg diet has the immune-stimulatory effect on the
ngerlings from 15th day onwards of feeding and can greatly reduce
their susceptibility to fungal infection after 45 days of feeding.
Many researchers reported pyridoxine deciency causes
anaemia in chinook salmon, Oncorhynchus eshawytscha [64], and
rainbow trout, Salmo gairdneri [65]. Hoffman and Ross [66] have
386 H. Saha et al. / Fish & Shellsh Immunology 59 (2016) 382e388

Table 3 count and Hb content etc. in L. rohita ngerlings (under endosulfan

Immunological parameters (total plasma protein, globulin, albumin, NBT, lysozyme stress and thermal stress) when fed with 100 mg pyridoxine kg
1
activity and phagocytic activity) of L. rohita ngerlings fed with dietary pyridoxine
supplemented diet (control diet, diet T1 and diet T2) for 0, 15, 30 and 45 d.
diet. However, in the present study, signicant (P < 0.05) increase in
the haemoglobin content, PCV and MCV without the signicant
Days T1 T2 C change in EC may due to the increase in the size and volume of
1
Total plasma protein (g dl ) erythrocytes contributed by the increase in the haemoglobin con-
0d 4.35 0.14 4.61 0.18 4.54 0.12 tent. Similarly, Lundby et al. [67] reported that a treatment with a
15 d 5.08 0.10a 5.37 0.09b 4.70 0.12a
glycoprotein growth factor, erythropoietin can elevate the hae-
30 d 5.25 0.05a 5.87 0.08b 4.00 0.06c
45 d 5.64 0.11a 5.92 0.16a 4.84 0.05b moglobin concentration by increasing the erythrocytes volume and
Globulin (g dl
1) depressing the plasma volume. Further like pyridoxine, erythro-
0d 2.58 0.20 2.55 0.22 2.81 0.12 poietin also has the role in potentiating the delta amino levulinic
15 d 2.88 0.14 3.05 0.082 2.88 0.17
acid synthetase for heme synthesis [68]. However, supplementa-
30 d 3.05 0.10a 3.66 0.29a 2.20 0.08b
45 d 3.21 0.06 3.47 0.14 2.73 0.06
tion of excess pyridoxine without any stress improved the eryth-
Albumin (g dl
1) rocyte count non-signicantly (P > 0.05) over time. This suggests
0d 1.77 0.06 2.06 0.03 1.73 0.05 that pyridoxine enhances erythropoiesis in L. rohita ngerlings [69].
15 d 2.21 0.05 ab 2.32 0.06a 1.82 0.06b However, further investigation need to be undertaken to elucidate
30 d 2.21 0.08 ab 2.21 0.22a 1.80 0.03b
the exact mechanism of these results.
45 d 2.43 0.06 2.45 0.03 2.11 0.02
Nitroblue tetrazolium (OD at 540 nm) On the other hand, leucocytes play an important role for
0d 0.10 0.00 0.09 0.00 0.09 0.00 enhancing non-specic or innate immunity, which is considered as
15 d 0.46 0.05a 0.90 0.00b 0.18 0.01c an indicator of the health status of sh [70]. In the present study
30 d 0.76 0.06a 1.24 0.06b 0.18 0.00c supplementation of dietary pyridoxine increased the leucocyte
45 d 2.14 0.06a 3.25 0.18b 0.29 0.03c
Lysozyme (U mg
1)
count non-signicantly (P > 0.05) as compared to the control group.
0d 0.12 0.00 0.12 0.00 0.12 0.00 Similar observation was also reported by Akhtar et al. [12,13],
15 d 0.88 0.11a 1.58 0.14b 0.19 0.07c however they have found signicant (P < 0.05) increase when
30 d 2.12 0.10a 2.75 0.10b 0.37 0.16c 100 mg pyridoxine kg
1 diet was supplemented. This suggests that
45 d 3.49 0.10a 4.28 0.19b 1.38 0.00c
supplementation of pyridoxine in diets helps in promoting leuco-
Phagocytic (%)
0d 36.67 1.67 33.33 1.67 35.00 2.89 cyte count.
15 d 48.67 1.86 ab 50.67 2.33a 37.00 2.08b Proteins are the most important compounds in the plasma or
30 d 61.00 3.79a 71.00 3.79a 44.33 1.86b serum, and its concentration is used as a basic index for the health
45 d 62.00 3.06a 74.33 0.67a 40.00 1.16b status of sh [71]. Among the plasma protein, albumin and globulin
Data (mean SE) at the same sampling days with different letters (a, b and c) are the major proteins, which play a signicant role in the immune
signicantly differ (P < 0.05) among different experimental groups (C, T1 and T2). response [72]. Plasma globulin consists of several components like
a-, b- and c-globulin. The c-globulin fraction is the source of almost
all the immunoglobulins of blood [73]. Increase in the total plasma
reported that delta amino levulinic acid synthetase is the rate-
protein, albumin and globulin levels is thought to be associated
limiting enzyme in the synthesis of haem and this enzyme re-
with a stronger innate immune response in shes [74]. Signicantly
quires pyridoxal 5-phosphate as a co-factor. Thus, in the present
(P < 0.05) higher content were observed in the groups fed with
study the increase in haemoglobin content may be due to the
0.02% pyridoxine supplemented diet as compared to control group
pyridoxine supplementation. T2 and T1 groups fed with 0.02% and
on 15 d, 30 d and 45 d of feeding. Aktar et al. [12,13] also reported
0.01% pyridoxine supplemented diets respectively showed signi-
that dietary supplementation of pyridoxine signicantly increases
cantly (P < 0.05) higher Hb content, respectively from 30 days of
albumin, globulin and plasma protein content in rohu. And it was
feeding as compared to the control. Similarly, other related hae-
suggested that the increased protein content may be due to
matological parameters such as PCV, MCV and MCH were also
enhanced protein synthesis [75] which might be helping in miti-
improved signicantly (P < 0.05) in groups fed with 0.02% pyri-
gating stresses.
doxine supplementation. Akhtar et al. [12,13] also observed
Respiratory burst activity indicates the status of oxygen
improvement in the haematological parameters like erythrocyte
dependent defence mechanism in the phagocytic cells of verte-
brates, wherein there is generation of reactive oxygen in-
termediates with powerful microbial activity [76]. Thus, increased
respiratory burst activity can be correlated with increased bacterial
pathogen killing activity of phagocytic cells in blood [77] and hence,
a better immunity status of sh. In the present study, the respira-
tory burst activity was measured by reduction of nitro blue tetra-
zolium by intracellular superoxide radicals produced by leucocytes.
The respiratory burst activity was signicantly higher in pyridoxine
supplemented groups and best activity was shown by the group fed
with 0.02% pyridoxine diet. Aktar et al. [13] also showed supple-
mentation of 100 mg pyridoxine kg
1 diet enhances respiratory
burst activity. This suggests that higher respiratory burst activity
enhance the production of superoxide anion and hence, better
immunity status of L. rohita ngerlings. This is in agreement with
some previous studies [12,13,78].
Phagocytic activity in the present study was signicantly
Fig. 1. Relative percentage survival of L. rohita ngerlings challenged with S. parasitica
after 45 days of feeding with control diet, diet T1 and diet T2 to respective experi-
(P < 0.05) higher in 0.02% pyridoxine supplemented fed group from
mental groups C, T1 and T2. Data (mean SE) with different letters signicantly differ 15 days onwards and even 0.01% supplemented group had
(P < 0.05) among different experimental groups (C, T1 and T2). enhanced phagocytic activities from 30 days of feeding. Many
 H. Saha et al. / Fish & Shellsh Immunology 59 (2016) 382e388
studies revealed similar positive inuence of vitamins on macro-
phage activity [79, 80, 81, 82, 83].
Lysozyme act as primary defense system and it has the ability to
disrupt the cell walls of pathogens. Thus, its activity increases in
response to infection in sh [84]. In the present study lysozyme
activity was enhanced with dietary pyridoxine supplementation in
sh. Enhanced lysozyme activity in sh on supplementing their
diet with nutraceuticals were also observed by many researchers
such as Rao et al. [85] in L. rohita fed with the diet containing herb
Achyranthes aspera; Jian and Wu [86] in Jian carp with traditional
Chinese medicine; Yin et al. [87] in O. niloticus with Astragalus radix
root. However, Lygren et al. [88] and Verlhac et al. [89] reported
unaffected lysozyme activity in Atlantic salmon, S. salar, and
rainbow trout, O. mykiss respectively, fed with vitamin C.
Vitamin B6 (pyridoxine) and its derivatives are efcient in
quenching singlet oxygen and have potential antioxidant proper-
ties [90,91] and these properties might have inuenced the
macrophage activities in terms of NBT value, phagocytic activity
and lysozyme activity in the present study, as macrophages have
high demand for antioxidative substances, necessary for preventing
oxidative damage induced by free radicals produced [92]. This
clearly elucidates the immuno-modualtory property of dietary
pyridoxine and it can enhance non-specic immunity by 15 days of
feeding with 0.02% dietary pyridoxine in L. rohita ngerlings.
Saprolegnia is most abundant pathogenic sh fungi found in
freshwater ecosystems [19,36] and is considered to be opportunist
facultative parasite [20]. In the present study, feeding of L. rohita
ngerlings with 0.02% dietary pyridoxine for 45 days stimulated the
haemato-immunological parameter sufciently to provide protec-
tion from mortalities due to S. parasitica infection. Similarly, Das
et al. [74] reported increased protection to Anabas testudineus
(Bloch) from S. parasitica with b-glucan.
It is thus evident from the present study that supplementation
of 0.02% dietary pyridoxine in diet protect sh from fungal infection
of S. parasitica after 45 days of feeding. However, earlier co-workers
have reported 0.001% give better survivability against bacterial
infection of Aeromonas hydrophila [12]. Hence it can be concluded
that at dietary pyridoxine supplementation in the diet of L. rohita at
0.02% can provide protection to both fungal and bacterial
infections.
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