Proc. Natl. Acad. Sci., India, Sect. B Biol. Sci.
(Oct–Dec 2017) 87(4):1185–1197
DOI 10.1007/s40011-015-0692-9
RESEARCH ARTICLE
Sex Specific Seasonal Variation in Hematological and Serum
Biochemical Indices of Barilius bendelisis from Central Himalaya,
India
Neeraj Kumar Sharma1,2 • M. S. Akhtar1 • N. N. Pandey1 • Ravindra Singh2
A. K. Singh1
Received: 24 April 2015 / Revised: 4 October 2015 / Accepted: 18 November 2015 / Published online: 7 January 2016
 The National Academy of Sciences, India 2015
Abstract The study aimed to assess the effects of sex and
season on hematological and serum biochemical indices of
Barilius bendelisis. Hematological and biochemical results
showed significant differences (p \ 0.05) among seasons
as well as between sexes. In summer, hemoglobin, hema-
tocrit, erythrocyte and WBC number were highest and
significantly (p \ 0.05) different between sexes. Differen-
tial WBC count also showed marked seasonal variation but
do not show any significant difference (p \ 0.05) between
male and female. Mean corpuscular volume and mean
corpuscular hemoglobin was high in winter and low in
summer. For males and females, mean corpuscular hemo-
globin concentration was high in summer, rainy, and low in
winter season. Cholesterol level and low-density lipids
were high in autumn and low in spring. High-density lipids
(HDL) and very low-density lipids (VLDL) also fluctuated
in different seasons with maximum HDL in both sexes in
autumn. Maximum VLDL for male was observed in sum-
mer and in spring for female. Triglycerides were high in
autumn and summer and low in spring. Throughout winter
the total protein, albumin and globulin levels were highest.
Electronic supplementary material The online version of this
article (doi:10.1007/s40011-015-0692-9) contains supplementary
material, which is available to authorized users.
& M. S. Akhtar
mdshahbazakhtar@gmail.com
1
Fish Nutrition and Physiology Laboratory, ICAR-Directorate
of Coldwater Fisheries Research, Anusandhan Bhawan,
Bhimtal 263136, Uttarakhand, India
2 the internal environment of the aquatic organism including
Department of Zoology, Hemwati Nandan Bahuguna
Garhwal University, Tehri Campus Badshahithaul,
Tehri Garhwal 249199, Uttarakhand, India
•
Glucose level was higher in summer and autumn whereas,
lower in winter in both male and female respectively. This
study revealed that the variation in seasonal environmental
parameters have direct effect on the health status of B.
bendelisis. The baseline data generated in this study will
serve as a tool for fish physiologists and pathologists in
monitoring the stress, health and nutritional status of B.
bendelisis under aquaculture production.
Keywords Season  Hematology  Barilius bendelisis 
RBC  Cholesterol  Globulin  Glucose
Introduction
Aquatic environments display enormous spatial and tem-
poral fluctuations in physicochemical elements that can
directly influence the physiology of fishes [1]. In teleosts,
numerous physiological variables manifest seasonal varia-
tion in response to environmental changes of which
hematological and biochemical indices are the most critical
ones serving as indicators of fish health [2]. Variations in
hematological parameters of fishes are primarily caused by
numerous factors such as temperature, environmental
stress, malnutrition, sex, age, seasonal differences, nutri-
tional effects and water quality parameters including water
temperature, salinity, oxygen availability and pH, etc. [2–
6]. Hematological studies help in understanding the rela-
tionship of blood characteristics to their habitat and species
adaptability to the environment including adaptation
mechanisms and evolutionary processes [7]. The serum
biochemical profile can provide imperative information on
fish [8]. Nowadays, hematology and serum biochemistry
data have been employed effectively in monitoring the
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health of both captive and wild population of fish species,
especially in fishery management programs [9].
The literature on seasonal effects on hematological
parameters is still scarce for most of the teleost fishes [10].
Many researchers reported sex-specific seasonal quantita-
tive and qualitative variations in hematological parameters
including RBC and WBC number, differential leukocyte
count, the percentage of hemoglobin and the dimension of
RBC and WBC in different fish species and suggest that
sex of the fish must be taken into account while assessing
the health status in wild as well as in captivity [2, 11].
Assessment of the hemogram implicates the evaluation of
the total erythrocyte count (RBC), total leukocyte count
(WBC), hematocrit (PCV), hemoglobin percentage (Hb),
erythrocyte indices (MCV, MCH, MCHC), WBC differ-
ential count and the estimation of stained peripheral blood
films [2]. Erythrocytes are the most dominant blood cells in
fish and their number is dependent on external i.e. seasonal
water temperature, dissolved oxygen level and internal
cues such as age, sex, reproductive stage and activity level
[12]. Hematocrit and Hb are the most readily determined
hematological indices in the field and captive conditions
[13]. Amongst diverse hematological parameters, hemat-
ocrit percentage is not easily altered as per other parame-
ters and should be used in concurrence with erythrocyte
and leucocytes count, Hb, osmotic delicacy, and differen-
tial leucocytes count [14]. Leucocyte count (WBC) is often
used as indicator of health status in fish [13]. These cells
are vital components of the innate immune system and are
mainly involved in non-specific defense mechanism in the
aquatic organisms including fish [15].
In fishes, blood [serum] protein is a complex combination
of albumin, globulin, and fibrinogen. Serum proteins have a
wide range of functions and are especially important for the
regulation of water balance in fishes [16]. The seasonal
variations in serum proteins are caused by the combination of
exogenous factors i.e. water temperature, availability of
food, feeding intensity and endogenous processes i.e.
maturity stage and reproduction, etc. [17]. Blood glucose is
an extremely variable parameter that is strongly influenced
by multiple factors including handling stress, ecological
strain, seasonal disparity and thermal stress, nutritional sta-
tus and sexual maturity [8]. After proteins, fish utilize lipids
as the principal substrate in the energy metabolism rather
than carbohydrates for maintaining various physiological
processes like other poikilothermic organisms in their
aquatic environment [18]. Lipids are transported all the way
through the circulation bound to specific macromolecular
protein complexes called lipoproteins [19]. Triacylglycerols,
cholesterol and other lipids classified according to increasing
density are transported in the blood by lipoproteins such as
very low-density lipoproteins (VLDL), low-density
lipoproteins (LDL), high-density lipoproteins (HDL) and
123
N. K. Sharma et al.
chylomicrons, [20]. In fishes, cholesterol is also the precur-
sor of steroid hormones such as progesterone, testosterone,
estradiol and cortisol [21]. Many researchers have suggested
that lipids and lipoproteins in fish show sex-specific seasonal
fluctuations, especially when dealing with temperature
changes, diet, age and the annual reproductive cycle [21, 22].
Amongst measured parameters, plasma protein, lipids,
cholesterol, triglycerides and glucose have potential as pre-
dictive diagnostic tools to evaluate the nutritional status of
fishes in aquaculture.
Barilius bendelisis, Indian Hill trout under family
Cyprinidae, is a widely distributed and predominant species
in the Brahmaputra and Ganga basins along the Himalayan
foothills of India, Bangladesh and Nepal [23] including
Myanmar, Pakistan, Sri Lanka and Thailand [24, 25]. B.
bendelisis shows marked sexual dimorphism. Males are
brightly colored, bigger in size showing rough body due to
miniature tubercles on both lips including head region and
dorsolateral scales that are lacking in females [26]. B. ben-
delisis is an asynchronous/intermittent spawner; first mas-
sive spawning occurs in March–April and second in August–
September having GSI peaks in March and August respec-
tively [27]. B. bendelisis is potential food fish consumed by
Himalayan population. It has achieved a market price of Rs
160–200 per kg [28]. The fish also plays substantial role in
the capture fishery along Himalayan region of India,
dwelling shallow, clear and cold lotic and lentic water
bodies where Indian major carps i.e. Labeo rohita; Catla
catla and exotic carps cannot be raised efficiently due to
harsh climatic conditions [29]. Despite being a popular
ornamental species among aquarists, as well as a food fish
for populace of Himalayan region, it is poorly studied and as
the result this species has suffered a drastic reduction in its
availability in the recent past [29]. Moreover, Himalayan
ecosystem is fragile and sensitive to temperature and hence
the species including B. bendelisis is predisposed to climate
change effects. Further, there is no information available in
the current literature on its hematology and serum bio-
chemistry. Therefore, keeping in view the economic
importance of the species, the present study was conducted
to document the sex-specific variations in hematological and
biochemical indices during different seasons of the year,
which could be applied as a diagnostic tool to evaluate the
health status in its natural habitat.
Material and Methods
Experimental Fish
A total of 288 adult (144 male and 144 female) of Indian
hill trout, B. bendelisis (age 2?; average ± SE; male
weight 19.26 ± 1.56 g; average male length
Sex Specific Seasonal Variation in Hematological and Serum Biochemical Indices of Barilius…
12.51 ± 0.29 cm and average female weight
9.57 ± 0.75 g; average female length 10.17 ± 0.24 cm)
were caught seasonally from a tributary of the river Kosi at
Ratighat area located in Kumaun hills (central Himalaya)
of Uttarakhand, India between the latitude 2927.350 N and
2927.260 N and longitude 7928.430 E and 7928.480 E
(Fig. 1) using cast nets. Adults of B. bendelisis were
transferred to the wet laboratory of ICAR-Directorate of
Coldwater Fisheries Research, Bhimtal-263136, Nainital,
India using 250 l plastic tanks containing water from the
sampling site with continuous aeration. Particular attention
was paid to maintain a consistent capture protocol
throughout the study from the wild as well in laboratory
after collection in order to minimize stress effects to
minimal level. The studies were conducted over all the
seasons viz. winter (January), spring (March), summer
(May and July), rainy (September) and autumn (Novem-
ber) of the year 2014. The winter season lasted from mid
November to February, spring from March to April,
Fig. 1 Map showing sampling site of B. bendelisis from River Kosi at Ratighat, Uttarakhand, Central Himalaya India
1187
summer from May to mid-July, rainy from mid-July to
September and the autumn lasted from October to mid-
November.
Physico-Chemical Parameters of Water
Water quality parameters like dissolved oxygen, alkalinity,
pH, ammonia, nitrite and nitrate of the sampling site were
assessed during each sampling season (Table 1). Temper-
ature, dissolved oxygen and pH were measured with a
multi-parameter probe (Hanna, model 9828). Other water
quality parameters like ammonia, nitrite, nitrate and alka-
linity concentrations were estimated colorimetrically using
Spectroquant Test Kits (Spectroquant Multy colorimeter,
Merk Millipore) following standard manufactures protocol.
The equipment is pre-programmed for the use of over 130
Spectroquant cell and reagent tests and covers all the
essential parameters considered under present study. The
concentrations were expressed as ppm.
123
1188 N. K. Sharma et al.

Table 1 Water quality parameters of sampling site in different seasons during the study period (values expressed as mean ± SE, n = 3)
Seasons Water temperature Dissolved oxygen Ammonia–N Nitrite–N Nitrate–N Alkalinity pH
(C) (mg l-1) (ppm) (ppm) (ppm) (ppm)

Winter 10.64 ± 0.017a 10.62 ± 0.06e 0.03 ± 0.003b 0.003 ± 0.0006a 0.60 ± 0.06b 60.70 ± 0.30a 8.81 ± 0.003e
b d a a b a
Spring 16.25 ± 0.012 9.75 ± 0.03 0.02 ± 0.000 0.002 ± 0.0007 0.53 ± 0.07 60.67 ± 0.33 8.75 ± 0.003d
e a a a a,b a
Summer 22.11 ± 0.003 8.28 ± 0.04 0.02 ± 0.002 0.004 ± 0.0003 0.38 ± 0.04 60.55 ± 0.00 7.78 ± 0.007a
d b a a a,b a
Rainy 20.87 ± 0.012 8.90 ± 0.06 0.02 ± 0.000 0.004 ± 0.0007 0.40 ± 0.12 60.93 ± 0.12 7.84 ± 0.003b
c c b a a a
Autumn 17.77 ± 0.012 9.55 ± 0.05 0.03 ± 0.000 0.002 ± 0.0006 0.20 ± 0.06 61.00 ± 0.06 8.66 ± 0.006c
Different superscript letters (a–e) in the same column indicate significant difference (ANOVA, p \ 0.05)

Hematological Parameters provided in the kit following the manufacturer protocol

Firstly, body surface of male and female B. bendelisis was
cleaned with blotting paper to avoid any contamination.
Blood was collected quickly from the caudal vein of alive
fish without giving any anesthesia treatment with a 26-
gauge syringe using anticoagulant (2.7 % w/v) ethylene
diamine tetraacetic acid (EDTA) for estimation of blood
glucose, Hb, total erythrocyte count and total leucocyte
count. The total erythrocyte count (RBC) and total leuco-
cytes count (WBC) were determined using an improved
Neubauer-type hemocytometer with Hayem’s solution as
the diluting fluid for RBC and Truck’s solution for WBC
(CDH chemicals, New Delhi, India) [2, 13]. The results
were expressed as the number of RBC or WBC per 1 mm3
of blood. The total RBC count per mm3 was calculated by
the following formula.

Nikon Eclipse E100 microscope (Nikon, Tokyo, Japan) for
Total RBC count per mm3 ¼ 200  50  N
¼ 10; 000  N
where, N = number of RBC counted, dilution
factor = 200.
The total WBC or leucocyte count per cubic millimeter
was obtained by the following formula.

37] and. the percentage of each blood type was calculated
Total WBC count per mm3 ¼ ½ðL  20Þ=½ð4Þ  ð1=10Þ
¼ L  50
where, L = number of WBC counted, dilution factor = 20
[2, 13, 30].
Thrombocyte count was also determined using an
improved Neubauer-type hemocytometer with thrombocyte
counting fluid (CDH chemicals, New Delhi, India. Total
thrombocyte count per mm3 was calculated by the fol-
lowing formula.


Total thrombocyte count per mm3 ¼ N  20  50
¼ N  1000
where, N = number of thrombocyte counted, dilution
factor = 20 [31].
The hemoglobin level of blood was analysed following
the cyanmethemoglobin method using Drabkin’s fluid
(Qualigens, Mumbai, India) [32]. Absorbance was read
using a multimode microplate spectrophotometer (Model-
Eon-C, Biotek, USA). The final concentration was calcu-
lated by comparing with standard cyanmethemoglobin
(Qualigens, Mumbai, India). Blood glucose was estimated
by the method of Nelson and Somogyi [33] and the amount
of glucose expressed as milligrams per deciliter (mg/dL).
Hematocrit (Hct-%) or packed cell volume (PCV) was
determined by the Wintrobe and Westergreen method as
described by Blaxhall and Daisley [34]. Blood smears, 2
for each fish, were prepared immediately using a pinpoint
amount of blood which was air-dried, fixed in absolute
acetone free methanol for 1 min then stained with Wright–
Giemsa (WG) modified stain as per provided protocol
(Sigma-Aldrich, LOT NO. SLBJ3110 V). The stained
smears were observed under light microscope using a
differential count. Two hundred cells for each slide were
counted, classifying them based on morphological char-
acteristics and WG staining as documented in previous fish
leucocytes studies [30, 34–37]. The relative numbers of the
types (lymphocytes, neutrophils, eosinophils and mono-
cytes) in the stained peripheral blood were recorded [34,
as the average blood cell type/sum of averages of all types
of blood cells 9 100 [38]. The secondary erythrocyte
indices [mean corpuscular volume (MCV-fL), mean cor-
puscular hemoglobin (MCH-pg), and mean corpuscular
hemoglobin concentration (MCHC-%)] were obtained
from the hematocrit, hemoglobin and erythrocyte count
data according to formula proposed by Dacie and Lewis
[39].
MCV ¼ ðHct/RBCÞ  10
MCH ¼ ðHemoglobin/RBCÞ  10
MCHC ¼ ðHemoglobin/HctÞ  100
Serum Immunological Parameters
The blood in the centrifuge tubes was allowed to clot at
room temperature for 30 min. Serum was removed from

123
Sex Specific Seasonal Variation in Hematological and Serum Biochemical Indices of Barilius…
the clotted sample after centrifugation at 3000 rpm for
5 min and frozen at -20 C until further analysis. Sam-
ples were analyzed within 48 h of collection and thawed
immediately prior to analysis for serum total protein,
serum albumin and lipid profile. Serum protein and
albumin were estimated using total protein and albumin
kit (Span Diagnostics Ltd India) following Biuret and
BCG dye binding method. Globulin was calculated by
subtracting the albumin values from the total serum pro-
tein. Serum cholesterol, LDL, HDL, VLDL and triglyc-
erides were measured by the autoanalyser (Vitalab
Selectra, Merck, Darmstadt, Germany) using standard kits
from Autospan Reagents, Span Diagnostics (Gujarat,
India) following standard protocols supplied with kits.
Serum triglyceride concentrations were estimated
according to the glycerol 3-phosphate oxidase–peroxidase
(GPO-PAP) method, serum cholesterol concentration
were determined according to cholesterol oxidase–per-
oxidase (CHOD-PAP) method, serum HDL cholesterol
concentration according to accelerator selective detergent
method and serum LDL concentration according to
selective detergent method [40]. VLDL cholesterol was
calculated by the Friedewald’s formula and all values are
expressed as mg/dL [41, 42].
Statistical Analysis
Hematological and biochemical seasonal data from the
study were subjected to one-way analysis of variance
(ANOVA) using SPSS 19.0 software. Differences between
means were determined by Duncan’s multiple range test
(p \ 0.05). The correlation between hematological vari-
ables were analysed by the Pearson coefficient for linear
correlation at p \ 0.05 and t test was used for determining
differences in parameters between sexes. All data are
reported as mean ± SEM.
Results and Discussion
Physico-Chemical Parameters of Water
The values of physico-chemical parameters observed dur-
ing study period are summarized in Table 1. There was a
significant difference (p \ 0.05) in water temperature,
dissolved oxygen, ammonia, nitrate and pH between dif-
ferent seasons. Nitrite and alkalinity showed no significant
difference (p \ 0.05) between seasons. In all seasons,
dissolved oxygen, ammonia, nitrite, nitrate, alkalinity and
pH were within optimum limit and found to be related to
respective seasons [28].
1189
Correlation Analysis
Overall highest positive correlation in male was observed
for Hb-MCHC (r = 0.983, p \ 0.01), Hb-Hct (r = 0.982,
p \ 0.01) and RBC-Hb (r = 0.977, p \ 0.01). The highest
positive correlations for females were observed between
Hb-MCHC (r = 0.994, p \ 0.01), Hb-Hct (r = 0.991,
p \ 0.01) and Hb-RBC (r = 0.980, p \ 0.05). The analy-
sis among serum biochemical parameters showed a strong
and positive correlation of TP-GLO (r = 0.996 at
p \ 0.01) and CHOL-GLU (r = 0.957 at p \ 0.01) in male
fish. Similarly in female highest correlation was observed
between TP-GLO (r = 0.969, p \ 0.05).
Hematological Parameters
Seasonal hematological data with mean, standard devia-
tion, minimum and maximum values are given in Table S2
(see supplementary data). There were significant
(p \ 0.05) seasonal differences between sexes in hemato-
logical parameters of B. bendelisis (Figs. 2, 3). In males,
the Hb concentration varied from 3.7 to 7.32 gm dl-1 with
the highest value recorded in the summer and lowest in
winter (Fig. 2A). Similarly, in females, the values ranged
from 3.2 to 6.48 gm dl-1 with highest value recorded in
summer. Hb concentration in males had significantly
higher values in all the seasons as compared to females.
The total number of erythrocytes for males ranged from
1.10 to 2.58 9 106 and 0.80 to 2.22 9 106 mm3 in
females. Male fish showed significantly (p \ 0.05) higher
number of RBCs than the female in spring, rainy and
autumn seasons with highest values in both the sexes
registered in summer (Fig. 2B). Mean values of hematocrit
ranged from 21.40 to 31.05 % for males and 19.60 to
26.45 % in females during the study period. Seasonal
hematocrit percentage was highest in summer and lowest in
winter for both sexes with male having significantly
(p \ 0.05) higher percentage during all the seasons
(Fig. 2C). The seasonal total leukocyte count (WBC) value
was higher in summer and lower in winter for both sexes of
B. bendelisis with female having greater value during all
the seasons but no significant difference (p \ 0.05) was
observed between sexes (Fig. 2D). WBC count varied
between 2.55–4.52 9 103 and 2.70–4.73 9 103 mm3 of
blood in males and females, respectively. Thrombocytes
also showed seasonal variation with a mean value ranging
from 31 to 66.35 9 103 and 35.60 to 72.60 9 103 mm3.
Highest value was recorded in summer and lowest in spring
for both the sexes with female having greater value in all
seasons and were significant (p \ 0.05) in all seasons
except spring and rainy season (Fig. 2E). Mean value of
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1190 N. K. Sharma et al.

Fig. 2 Sex specific seasonal

variation in hemoglobin,
hematocrit, RBC, WBC,
thrombocytes and lymphocytes
of B. bendelisis. Values in the
same series with different
superscripts (a, b, c, d) in each
panel differ significantly
(p \ 0.05, ANOVA). Values
expressed as mean ± SE;
n = 6. Asterisk in the series (if
any) in each panel implies sex
specific significant difference
(p \ 0.05, t test)

lymphocytes varied from 67 to 75 % for male and 67.60 to
78.0 % during study period with no significant difference
between sexes (Fig. 2F). Neutrophils also showed seasonal
variation with a mean value ranging from 15.33 to 23.17
and 16 to 24.33 % in male and female respectively. Neu-
trophil values were significantly (p \ 0.05) higher in
summer in both the sexes but no significant sex specific
difference (p [ 0.05) was observed (Fig. 3A). Mean eosi-
nophil percentage ranged from 2 to 6.68 % in males and
2.66 to 6.0 % in females. Eosinophils were found highest
in winter and lowest in autumn in both the sexes (Fig. 2B).
Difference between sexes was not significant (p [ 0.05) in
all seasons (Fig. 3B). Monocyte mean percentage varied
from 2.64 to 5.30 and 1.33 to 4.0 % in males and females
respectively. Significant sex specific difference (p [ 0.05)
was observed only during winter season. Higher values in
male and female fish were recorded in summer and rainy
seasons followed by lowest in winter and spring seasons
respectively (Fig. 3C). No basophils were observed in the
present study. The MCV value for males ranged between
122.07 and 196.42l and 122.24–249.29 fl for females with
no significant difference (p \ 0.05) between sexes
(Fig. 3D). The value of MCH throughout the study period
varied between 28.65 and 33.84 pg in males and
29.78–40.49 pg in female. MCH was significantly higher
in females as compared to males in all the seasons. In
males, higher value of MCH was observed in winter and
rainy seasons with lower values in summer and spring.
Similarly, females showed the higher value in winter and
spring with lower values in summer and autumn (Fig. 3E).
Significant difference between sexes was not observed. The
MCHC value ranged from 17.29 to 24.24 % in males and
between 16.32 and 24.48 % in females. In both sexes,
higher MCHC value was recorded in the summer and rainy
seasons and lower value in winter and spring seasons.
Difference between sexes was significant (p \ 0.05) during
summer season (Fig. 3F).
In fishes, hematological and serum biochemical param-
eters are considerably influenced by the wide range of
natural environmental factors i.e. age, fish size, stress,

123
Sex Specific Seasonal Variation in Hematological and Serum Biochemical Indices of Barilius…
Fig. 3 Sex specific seasonal
variation in neutrophils,
eosinophils, monocytes, MCV,
MCH and MCHC of B.
bendelisis. Values in the same
series with different superscripts
(a, b, c, d) in each panel differ
significantly (p \ 0.05,
ANOVA). Values expressed as
mean ± SE; n = 6. Asterisk in
the series (if any) in each panel
implies sex specific significant
difference (p \ 0.05, t test)
nutritional state, season, spawning, sex, and genetic vari-
ations in the wild [2, 7, 36, 43]. Fish being an ectothermic
vertebrate accomplished of adapting to fluctuations in
ambient temperature and in view of that at elevated water
temperature there is an increase in metabolic activity and
that oxygen concentration in water reduces, fish is likely to
display an increase in erythrocyte number and hemoglobin
concentration because of hypoxia/lesser-dissolved oxygen
condition [12, 13]. In the present study, B. bendelisis shows
significant variation with respect to season and sex in
hematological parameters. In summer, due to high meta-
bolic rate and higher water temperature, most of the
hematological parameters (hemoglobin, erythrocytes, and
hematocrit) showed greater value in comparison to other
seasons whereas in winter the values are regressed due to
low water temperature and metabolic rate of fish species
(Fig. 2A–C). This seasonal response possibly compensates
for the effect of high water temperature reducing oxygen
availability [2, 44]. Hemoglobin, erythrocyte and
1191
hematocrit values in male fish were significantly higher
than that of female fish over all seasons that might be
potentially caused by greater physiological and metabolic
activity of male than female fish. These differences in
hematology about males and females may be related to
differential oxygen demand by sex, which in turn can be
linked to reproductive activity [43]. The decrease in
hemoglobin and hematocrit level in B. bendelisis in the
winter is probably due to the decline in metabolic activity
and reduction in feeding intensity at low water temperature
in the winter, which is supported by Pradhan et al. [13].
The relatively high number of RBCs seen in summer and
spring when compared to those observed in winter, this
study confirms that the water temperature plays a signifi-
cant role in the RBC counts in the fish. Similar results were
seen in Tinca tinca, C. catla, Cirrihinus mrigala and L.
rohita [2, 3, 10, 13]. Leukocytes are the second major cells
after erythrocytes in blood of fishes that are repeatedly used
as indicators of health status because white blood cells are
123
1192
essential constituents of innate immune defense and
involved in regulation of immunological function in the
aquatic organisms [45]. In this study, peak value of WBC
was observed in summer and least in winter season that
might be due to increase in water temperature which pos-
itively increases the number of circulating WBCs in the
blood (Fig. 2D). A similar rise in the white blood cell count
was found in C. mrigala [3], and Oncorhynchus mykiss
[46] at a higher temperature. Female fish have higher WBC
number throughout the different seasons probably due to
the differential genetic makeup of male and female fish.
Leukocyte levels in the blood may also fluctuate according
to environmental quality, nutritional status and presence of
infectious agents [36]. Immunosuppression described by
previous workers seemingly appears to be elicited by lower
water temperature during winter, therefore, is responsible
for the lower count of leucocytes. These findings showed
that immune cellular response could be affected by sex and
temperature alterations. Moreover, several authors reported
the effect of number, food density and age on hematolog-
ical parameters, which exert independent influence on these
parameters [36, 43, 46].
Differential leukocyte counts show the imperative
characteristics of health state of fish and are helpful in
evaluating the immune system. The differential leukocyte
count, like other hematological parameter, is dependent on
the sex, season and nutritional status [8, 14]. In teleost
fishes, thrombocytes are the most abundant blood cells
after erythrocytes, representing more than 50 % of circu-
lating leucocytes [47]. In the present study, the signifi-
cantly greater value was observed during summer that is
consistent with reports of Kfoury-Junior et al. [48]. Lym-
phocytes are involved in a diversity of immunological
functions such as immunoglobulin production and modu-
lation of immune resistance [49]. No significant difference
in lymphocytes between male and female was noticed, and
slightly higher value was obtained in spring and rainy
seasons and least in summer which is inconsistent with
findings of Silva et al. [49]. In the current study, temper-
ature-dependent change in neutrophil number was observed
with a maximum value in summer seasons. Neutrophils
being primary cells involved in phagocytosis increase
during the present study which might be due to enhanced
phagocytosis process at a higher water temperature [28].
Monocytes, which are long-lived phagocytic cells, are also
associated with defense of infections and bacteria [50].
Their number also increases in fish at high water temper-
ature during the summer and suppresses during the winter
in the current study. Increased number of monocytes at
higher water temperature is probably due to enhanced
immune response and phagocytic activity during warm
seasons [28, 51]. Seasonal changes in phagocytic activity
(neutrophil and monocyte) have also been previously
123
N. K. Sharma et al.
reported in several different fish species [52]. Eosinophils
play a primary role in the inflammation process and are
associated with defense against parasites [53]. The present
study shows higher value in winter that is consistent with
Mahananda et al. [54]. Vigliano et al. [44] reported that the
immune system of Odontesthes bonariensis was mainly
based on non-specific mechanisms at low temperature, but
at higher temperature, the activity of specific immune
response prevailed. As stated above, this is consistent with
the results obtained in present study on B. bendelisis that in
the cold season an increase in the number of cells
belonging to innate immune system such as eosinophils,
and a decrease in those cells involved in specific immunity
responses, i.e. lymphocytes and thrombocytes, were
observed (Figs. 2E, F, 3A–C). Secondary blood indices
(MCV, MCH, and MCHC) also showed slight fluctuation
during the study period. The maximum values of MCV and
MCH were obtained in winter and minimum in summer
whereas MCHC show higher value in summer and rainy
seasons and minimum during winter (Fig. 3D–F). Decrease
in MCV and MCH values during winter might be due to
increase in erythropoiesis process during the summer and
rainy months that may contribute to lower MCV values.
Gupta et al. [55] while studying seasonal variation in Tor
putitora noticed similar observations. Blaxhall and Daisley
[34] also observed lesser MCV values during that period of
the year when erythropoiesis is at peak. MCHC was higher
in summer and rainy seasons that might be related to
temperature increase during warm seasons. However,
Pradhan et al. [2] did not find any marked difference
between MCHC and MCH values between seasons and
sexes in L. rohita.
Serum Biochemical Parameters
Seasonal serum biochemical data with mean, standard
deviation, minimum and maximum values are given in
Table S2 (see supplementary data). The results of serum
biochemical parameters of male and female B. bendelisis
during different seasons of the year are shown in Figs. 4
and 5. Cholesterol ranged from 31.10 to 82.13 mg dl-1 in
males and 36.70 to 89.13 mg dl-1 in females respectively.
Values were significantly different (p \ 0.05) between
sexes during study period except in summer season. A
higher concentration of cholesterol was observed in warm
periods of the year (summer, rainy and autumn) in both
sexes (Fig. 4A). LDL concentration varied between 18.03
and 60.50 mg dl-1 for males and 26.13–70.40 mg dl-1 in
females. Seasonal LDL concentration was highest in
autumn and rainy seasons and lowest was found in spring
in both male and female but sex specific significant dif-
ference (p [ 0.05) was not observed during winter and
spring (Fig. 4B). HDL values fluctuated all over the period
Sex Specific Seasonal Variation in Hematological and Serum Biochemical Indices of Barilius… 1193

Fig. 4 Sex specific seasonal

variation in cholesterol, HDL,
LDL, VLDL, triglyceride and
total protein of B. bendelisis.
Values in the same series with
different superscripts (a, b, c,
d) in each panel differ
significantly (p \ 0.05,
ANOVA). Values expressed as
mean ± SE; n = 6. Asterisk in
the series (if any) in each panel
implies sex specific significant
difference (p \ 0.05, t test)

Fig. 5 Sex specific seasonal

variation in albumin, globulin
and glucose of B. bendelisis.
Values in the same series with
different superscripts (a, b, c,
d) in each panel differ
significantly (p \ 0.05,
ANOVA). Values expressed as
mean ± SE; n = 6. Asterisk in
the series (if any) in each panel
implies sex specific significant
difference (p \ 0.05, t test)

123
1194
ranging from 9.40 to 24.87 mg dl-1 in males and 12.60 to
22.47 mg dl-1 in females. Significant difference
(p \ 0.05) was observed between sexes during rainy sea-
son (Fig. 4C). VLDL also fluctuated during different sea-
sons under the present study and ranged from 7.03 to
16.93 mg dl-1 for male and 6.00 to 14.00 mg dl-1 for
females. Difference between sexes was significant
(p \ 0.05) during summer, rainy and autumn seasons
(Fig. 4D). Triglyceride mean value varied from 37.83 to
101.47 and 36.07 to 112.90 mg dl-1 for male and female
respectively (Fig. 4E). Higher value in autumn and lower
in spring for both sexes was observed and found signifi-
cantly different (p \ 0.05) during summer, rainy and
autumn between male and female. Total serum protein
content of fish showed seasonal variation, ranging from
1.35 to 2.89 g dl-1 in males and 1.38 to 2.66 g dl-1 in
females. The highest total protein value was found in
winter followed by autumn for both sexes and the lowest
value was observed in rainy season for males and in spring
for females. Significant difference (p \ 0.05) between
sexes was not recorded (Fig. 4F). Albumin concentrate
varied from 0.46 to 0.81 and 0.57 to 1.08 g dl-1 in male
and female respectively. Male and female showed signifi-
cant difference only during winter and rainy seasons
(Fig. 5A). Globulin follows same trend like that of albumin
with concentrate varied from 0.89 to 2.08 in male and 0.81
to 1.58 in female. No significant sex specific difference
(p [ 0.05) was observed (Fig. 5B). The concentration of
glucose varied from 24.40 to 50.90 mg dl-1 for male and
22.50 to 48.97 mg dl-1 in females. Highest values of
glucose in males were observed in autumn and summer
seasons in female. Lowest values were noticed in winter for
both sexes in B. bendelisis. There were significant differ-
ences (p \ 0.05) in glucose concentration in male and
female B. bendelisis in spring and autumn seasons
(Fig. 5C).
Lipid stores signify the primary energy reserves in
fishes. Seasonal fluctuation of the plasma/serum lipid levels
has been previously recorded for many fishes [20, 56].
Lipid fluctuations in fishes were also correlated with
reproduction and sexual maturation, as they are found to be
transported from stored tissues to gonads for gonadal
development [56, 57]. All the lipid class (except VLDL)
values in present study reached the maximum level in
summer, rainy and autumn seasons which could be asso-
ciated with increase in feeding intensity which was higher
because of high photoperiod and water temperature. White
et al. [56] observed fluctuations in lipids in plaice, Pleu-
ronectes platessa and correlated with the reproductive
cycle, which is also another reason that could not be
ignored. VLDL shows different pattern than other serum
lipids whose values were higher in spring and rainy season
that is attributed to reproductive activity of the fish, which
123
N. K. Sharma et al.
is supported by the finding of Aras et al. [22]. Cholesterol
is a precursor for the synthesis of sex steroids, and its levels
can fluctuate seasonally in teleost fishes [21]. Cholesterol
level was higher in warm seasons (summer, rainy and
autumn) and lower in winter in the present study. The
increase in cholesterol concentration during warm periods
of the year is due to higher feeding regime and metabolic
rate of fish [2, 3]. Cholesterol level is also found to be
fluctuated during the breeding cycle in many fishes [56,
58]. Higher levels of cholesterol during warm periods
could be due to its prerequisite for gonadal and adrenal
steroidogenesis and production of the basal steroid, which
is supported by previous works on different fishes [59].
Similarly, serum triglycerides concentrations were con-
siderably lower in winter and spring than those of summer,
rainy and autumn. This low level can be described by
starvation, which considerably affected the serum triglyc-
erides levels. Svoboda et al. [58] suggested that triglyceride
and cholesterol concentrations in blood serum of T. tinca
were higher in summer (22 C) than in winter (10.3 C)
due to different metabolic rates as it is seen that metabolic
rate and feeding intensity in fishes increase with the rise in
temperature. Subsequently, present study has been sup-
ported by previous studies and higher serum lipid and
lipoprotein concentrations in warm seasons (Fig. 4A–E)
were probably associated with high metabolic rate, feeding
regime and reproductive activity in these seasons [58].
Serum protein levels are often associated with fish
nutritional and physiological status and are affected by
starvation, stress and increased concentrations which can
be triggered by structural liver alterations reducing
aminotransferase activity, with concomitant reduction of
deamination capacity and impaired control of fluid balance
[60]. An increase or decrease of total blood proteins,
globulins and albumin has clinical relevance in fish. Total
protein content in the current study varied throughout the
seasons in both males and females, with maximum values
in winter followed by autumn (higher in males except rainy
season, Fig. 4F). The decrease in protein content in the
spring and rainy seasons is possibly due to increased pro-
teolysis and promising utilization of the product for
metabolic purposes and/or utilization in the growth of
gonads as both seasons are peak spawning periods of fish
[2, 17, 27, 58]. The depletion of protein in the blood during
spawning periods can be correlated with the increase of
protein in the ovary [16]. Albumin and globulin content of
B. bendelisis follows the same pattern as that of total
protein (Fig. 5A, B). Albumins in fish blood performs the
transportation of lipids and rise in albumin concentration in
animals due to loss through urine or faeces or through
break down may result in impaired synthesis [61]. In the
present study, higher albumin level was observed during
winter and summer i.e. pre-spawning period [27] as
Sex Specific Seasonal Variation in Hematological and Serum Biochemical Indices of Barilius…
preparatory phase storage for reproductive activity [egg
development]. Low level during spring and rainy seasons
(spawning period) might be due to the utilization during the
reproduction process. Marco et al. [59] studied blood
chemistry profiles of cultured sturgeon hybrids and
observed differences in albumin level in male and female
at the time of reproduction and sexual maturation stages. A
higher level of globulin in serum is thought to be associated
with stronger innate response in fish [62]. The present
study observes higher value of globulin in winter followed
by autumn that is consistent with findings of Bayir et al.
[63]. During post-spawning months/season (summer and
autumn), the amount of total protein, albumin and globulin
increased signifying the regaining of the fish from the
exhaustion of spawning activity. Male fish have higher
total protein and globulin during all seasons except rainy
season. Results of the present study are consistent with the
reports on Clarias batrachus [17] and three Indian major
carps [2, 3, 13].
Blood glucose in freshwater teleosts is an extremely
variable parameter that is strongly influenced by multiple
factors i.e. environmental and handling stress, seasonal
variation, nutritional status, sexual maturity, feeding habits
including locomotion and fish species [4]. An increase in the
blood glucose during summer and autumn in present study is
possibly due to increase in feeding intensity and the increase
in tissue uptake mediated by pancreatic hormones [3] at
higher temperature in contrast to winter when ambient tem-
perature is considerably low (Fig. 5C). Male fish had a rel-
atively higher value of glucose during autumn, rainy and
winter seasons that might be attributed to physical activeness
and higher food conversion efficiency of male than female
[64]. Previous studies on ecologically different species by
several workers substance the current findings [3, 4, 13].
Conclusion
In conclusion, this study indicates that hematological and
biochemical parameters in B. bendelisis are evidently
related to endogenous and exogenous factors such as sex,
reproductive state and seasonal variations. Among these
factors, exogenous ones (e.g. temperature, dissolved oxy-
gen and feeding regime) have a greater impact on hema-
tological parameters, whereas changes in biochemical
parameters are more related with endogenous factors (e.g.
reproduction) but effect of other abiotic factors cannot be
ignored. Therefore, seasons must be considered as key
factors when blood parameters are used as biomarkers for
environmental alterations. Since the data is collected over
all the seasons and gender wise with sufficient number of
samples, it is believed that the data would provide an ini-
tial/first baseline for blood parameter that can be
1195
effectively used as a health-monitoring tool in B. bendelisis
considering the concerns of climate change. This investi-
gation might also be useful to any advance study con-
cerning the acclimation mechanism that B. bendelisis has
adapted to survive the fragile and sensitive Himalayan
environment. Finally, the authors hope that the outcome of
the current study will be of help to environmental and
aquaculture officials responsible for future decisions in the
managing and rearing of fish used for human consumption.
Acknowledgments The authors are grateful to the Director, ICAR-
Directorate of Coldwater Fisheries Research, Bhimtal-263136,
Nainital, Uttarakhand and Head, Department of Zoology, HNB
Garhwal Central University, Uttarakhand for providing necessary
facilities and support to successfully carry out this study. This paper is
a part of thesis research of the first author registered for PhD at HNB
Garhwal University, Uttarakhand, India. The financial support pro-
vided by Hemwati Nandan Bahuguna Garhwal University, Srinagar
(under University Grant Commission, India) as student fellowship to
first author is duly acknowledged. Authors would like to thank the
anonymous reviewers for their critical evaluation and improvising the
manuscript.
Compliance with Ethical Standard
Conflict of interest Authors do not have any conflict of interest.
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