Fisheries Science

https://doi.org/10.1007/s12562-020-01469-3

ORIGINAL ARTICLE

Biology

The influence of sex and season on some hematological

and biochemical parameters of snow trout Schizothorax labiatus
in the Indian Himalayan Region
Kousar Jan1 · Imtiaz Ahmed1 

Received: 29 February 2020 / Accepted: 21 September 2020

© Japanese Society of Fisheries Science 2020

Abstract
Snow trout Schizothorax labiatus constitute a significant part of the diet of large human population of colder regions of the
Indian Himalayan subcontinent, and are also important for local livelihoods there. In the present study, the hematological and
serum biochemical parameters of S. labiatus were examined with respect to sex and season. Hemoglobin (Hb) concentration,
red blood cell (RBC) count and hematocrit (Hct) were higher in males than females in each season, while white blood cell
(WBC) count were notably higher in females. A significant difference (P < 0.05) was noted in Hb concentration, RBC and
WBC counts, and Hct between males and females during the summer compared to the other seasons. There was no significant
difference (P > 0.05) between erythrocyte indices with respect to sex and season. Serum biochemical parameters (glucose,
cholesterol and urea) differed significantly (P < 0.05) between the sexes, as well as among the seasons. However, no signifi-
cant differences (P > 0.05) were observed in biochemical parameters (total protein, albumin and globulin) with respect to
sex, although the highest values of all serum biochemical parameters were reported during the summer and the lowest values
during the winter. The data reported here on the influence of sex and season on hematological and biochemical parameters of
S. labiatus inhabiting the Sindh River could provide a reliable basis for the future monitoring of the health status of this fish.

Keywords  Blood parameters · Serum biochemistry · Sex · Season · Schizothorax labiatus · Sindh River

Introduction environmental stressors, seasonal variations and physico-

The measurement of hematological and biochemical param-
eters is essential for monitoring the health status of wild and
farmed fish (Satheeshkumar et al. 2011; Fazio et al. 2018;
Fazio 2019). These parameters provide vital information on
the physiological and pathological alterations that occur in
fish in response to fluctuations in their environment. Infor-
mation on the hematology and serum biochemistry of fish
is used in fisheries for the management and conservation of
both wild and reared populations (Gul et al. 2011; Sharma
et al. 2017; Sheikh and Ahmed 2019). Alterations in hemato-
logical and serum biochemical parameters of fish depend on
a number of factors, such as sex, age, nutrition, temperature,
* Imtiaz Ahmed
imtiazamu1@yahoo.com
1
Fish Nutrition Research Laboratory, Department of Zoology,
University of Kashmir, Hazratbal, Srinagar 190006, India
chemical parameters of water (Pradhan et al. 2014a, b; Fazio
et al. 2016; Sharma et al. 2017; Ahmed et al. 2019, 2020).
Besides these factors, transportation and handling stress
also influence fish blood chemistry (Barton 2000; Kori-
Siakpere et al. 2005; Omeji et al. 2017). A considerable
body of work on the variation in hematological and serum
biochemical profiles of fish with respect to sex and season
has been reported for various species (Aras et al. 2010; Bani
and Vayghan 2011; Acar et al. 2013; Karimi et al. 2013;
Pradhan et al. 2014a, b; Farshad et al. 2015; Sharma et al.
2015,2017; Suleiman et al. 2016; Zhao et al. 2018; Ahmed
et al. 2019; Sheikh and Ahmed 2019). These studies showed
that hematological and serum biochemical profiles of fish
did not only vary with environmental factors, but also with
sex, and thus indicated that both sex and season should be
considered as determining factors when analyzing the health
status of fish (Ahmed et al. 2020).

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The determination of sex in S. labiatus has mainly been
carried out through anatomical examination of the gonads
following dissection of the fish. Various morphological
characteristic features such as body shape and size, snout
shape, anal fin length, shape of the dorsal fin spines and
caudal fin lobes, and color of the fish have also been used
for the identification of sex in this species (Tilak 1987; Rai
et al. 2002; Sharma and Mehta 2010). During the breeding
season, the urinogenital papilla, located just behind the anus,
becomes enlarged in almost all species of fish, and can be
used as a primary means of identifying sex (Tilak 1987;
Har 2000). Sexual dimorphism is also seen in S. labiatus
with the development of prominent, sharp, whitish tubercles
(knob-like structures) on the head and other regions of the
body of the males during the breeding season (Regmi 2019).
Male and female S. labiatus can also be distinguished during
the breeding season by observation of oozing milt and eggs
when slight pressure is applied to the abdomen. Moreover,
the females are often observed to have a bulging belly due
to extreme enlargement of the ovaries when they are filled
with mature ova (Hussain et al. 2018).
Hematological parameters are also useful for assessing
the effects of habitat on a species, its adaptation to its envi-
ronment, and its evolution (Fazio et al. 2012c; Fazio 2019).
Hemoglobin (Hb) concentration and hematocrit (Hct) are
the hematological parameters most widely used to evalu-
ate the well-being of fish (Pradhan et al. 2012; Sheikh and
Ahmed 2019), although, Hct has to be used in association
with other parameters like Hb concentration, red blood cell
(RBC) and white blood cell (WBC) counts, the WBC dif-
ferential and osmotic fragility (Sharma et al. 2017). RBC
are the most abundant constituents of fish blood, and the
RBC count varies with both endogenous (stage of reproduc-
tion, age, sex, level of activity) and exogenous factors (water,
pH, temperature, dissolved oxygen concentration) (Witeska
2013; Burgos-Aceves et al. 2019).
A number of studies have revealed a decline in the Hb
concentration, RBC count and Hct of diseased fish (Rehulka
2002; Harikrishnan et al. 2003; Shah and Altidang 2004;
Silverira-Coffigny et al. 2004) and fish exposed to different
toxicants (Svobodova et al. 2003; Silverira-Coffigny et al.
2004; Mostakim et al. 2015). Leucocytes are a fundamen-
tal component of the innate immune system of fish; they
play a vital role in non-specific defense mechanisms (Prasad
and Charles 2010; Ahmed and Sheikh 2020), and thus are
also significant indicators of fish well-being (Pradhan et al.
2012; Sharma et al. 2017). An elevated total leucocyte count
is thought to occur mainly in infected fish, whereas a decline
in this parameter has been reported in fish exposed to toxi-
cants (Atamanalp and Yanik 2003; Harikrishnan et al. 2003;
Silverira-Coffigny et al. 2004; Movahed et al. 2016). There-
fore, any abnormalities in the hematological parameters of
fish are considered indicative of general ill-health, infection
13
Fisheries Science
or disease (Walencik and Witeska 2007; Ahmed and Sheikh
2020).
The serum biochemical profile (glucose, total cholesterol,
urea, total protein, albumin, globulin) of fish also gives
insight into changes in the internal environment of fish, as
they change with the seasons, and can therefore also be
used to determine the health status of fish (Terasawa et al.
2002; De Pedro et al. 2005; Kulkarni and Bedjargi 2016).
These parameters are considered pathological markers, as
any fluctuation in them is immediately reflected in the serum
before the appearance of clinical disease; hence they are
helpful subsidiary tools of diagnosis in aquaculture (Tripathi
2003; Sheikh and Ahmed 2019). In fish, blood glucose acts
as the main energy source and is also considered the most
variable parameter. Blood glucose levels exhibit variation in
response to different external and internal factors like sea-
sonal changes, handling stress, thermal stress, ecological
stress, sexual maturity and nutritional status (Wagner and
Congleton 2004; Prasad and Charles 2010; Sharma et al.
2017). Tavares-Dias and Moraes (2004) found that basal
glucose levels changed in ecologically distinct species in
response to various environmental and non-environmental
factors, especially feeding habits and mode of life. Choles-
terol is a precursor of different steroid hormones like testos-
terone, estradiol, progesterone and cortisol in fish (Karatas
et al. 2014; Hoga et al. 2018). Gonads at an increased stage
of maturation, increasing age and the transport of nutrients
to the gonads from different organs bring about significant
variations in cholesterol, triglyceride and protein levels in
fish (Chatzifotis et al. 2004). Total serum protein is often
used as an indicator of physiological condition in fish, as it
is one of the most stable component of blood, being affected
by few factors (Maita 2007). Serum protein is also a vital
diagnostic tool as it can be used as a marker to indicate the
health status of fish (Satheeshkumar et al. 2012). Serum pro-
tein, which comprises albumin and globulin, performs many
physiological functions in fish, the most important of which
is the maintenance of osmoregulation (Peyghan et al. 2014;
Nopianti et al. 2019).
The Sindh River, which is about 108 km long, is the
major tributary of the Jhelum River, and flows from the Gan-
derbal district of the state of Jammu and Kashmir in India.
The Machoi Glacier, located at an elevation of 4800 m east
of Amarnath Temple and south of the Zojila Pass, is regarded
the main source of this river. The river flows predominantly
westwards along National Highway 1D and is fed by many
glacial streams before reaching Ganderbal town. It joins
the Jhelum River at Shadipora, Srinagar. The Sindh River
also flows through a famous alpine hill station, Sonamarg. It
is the only river in Jammu and Kashmir where three hydro-
electric power plants are in use. A variety of fish inhabit
the Sindh River, predominantly those belonging to the fam-
ily Cyprinidae. The schizothoracines, commonly known as
Fisheries Science
Himalayan snow trout, inhabit the colder zones of the Indian
subcontinent, and are mainly restricted to snow-fed rivers
and streams of the region, including the Sindh River. Among
the schizothoracines, Schizothorax labiatus, which belongs
to the family Cyprinidae and is locally known as ‘chush,’
is a vital indigenous food fish of the region. It has a charac-
teristic elongated and streamlined body, rounded lower jaw,
D-shaped mouth with lips restricted to wide lateral flaps, and
two pairs of barbels on the face (Kullander et al. 1999). The
population of S. labiatus in the Sindh River, which was pre-
viously a major source of this species, is currently exhibiting
a significant decline because of habitat destruction, overfish-
ing, climatic changes, lack of food and the impact of various
physicochemical parameters.
Although considerable work has been done on the hema-
tology and serum biochemical profiles of various fish spe-
cies, and reliable information is available on the normal
range and best range for evaluating their health status, no
such information is available for S. labiatus from the Sindh
River in the Kashmir Himalayas. Therefore, the aim of the
present study was to determine the normal hematological
and serum biochemical values of S. labiatus with respect to
sex and season to provide useful and reliable information
on these as biomarkers of stress and as reliable tools for the
diagnosis and monitoring of abnormalities and disease in
this species.
Fig. 1  Map indicating the three sampling sites (green circles) in the Sindh River
Materials and methods
Study area and fish sampling
Regular sampling was carried out to collect live and healthy
specimens (i.e., no external signs of abnormalities, injuries
or infestations) of S. labiatus of different size from the Sindh
River. Sampling was udertaken on a monthly basis from
three sites: site I (Manigam), site II (Bamloora) and site III
(Rabitar) (Fig. 1) for a period of 2 years from April 2017
to March 2019 with the assistance of local fishermen. The
specimens were identified by using the standard taxonomic
keys of Day (1878) and Kullandar et al. (1999). A total of
432 fish samples comprising 216 males (31.13 ± 4.51 cm
total length, 302.63 ± 31.53 g total weight) and 216 females
(33.00 ± 4.35 cm total length, 372.69 ± 40.09 g total weight)
were collected by using different fishing gear, e.g., cast nets.
During the 2–year study, an average of 18 fish comprising
nine males and nine females were collected every month.
The fish were divided according to the season in which they
were caught: spring (March, April, May); summer (June,
July, August); autumn (September, October, November); and
winter (December, January, February). The total number of
fish per season was 54 (27 males and 27 females), thus over
the 2 years of the study, a total of 108 specimens comprising
54 males and 54 females were examined.
The fish were transported live from the collection sites in
open tanks containing 20 l of water to the wet laboratory at
the Department of Zoology, University of Kashmir. The fish
were treated with a prophylactic dip of potassium perman-
ganate and then stocked in well-aerated 70-l circular plastic
13

troughs with a continuous flow-through system (1.5–2.5
l min−1) and kept overnight to reduce any stress caused
by handling, netting and transportation. During the entire
period of acclimation, S. labiatus were maintained under a
12:12 h light:dark photoperiod; no mortality was recorded.
Blood collection
After overnight acclimation, the live fish were placed in a
tank and anaesthetized (MS-222 at 0.3 g l­−1 water) prior
to blood sampling (Topic Popovic et al. 2012; Iaria et al.
2019). Blood samples were obtained from the caudal vein
by venipuncture by means of a disposable 22-gauge sterile
plastic syringe. After collection, part of the collected blood
was transferred into a standard lithium heparin vial (25 inter-
national units ­ml−1 heparin per 2 ml blood) and immediately
used for the hematological analyses, while the remaining
portion of blood was stored in Eppendorf tubes without anti-
coagulant for serum biochemical analyses.
Hematological parameters
Hb concentration, total red blood cell (RBC) count, Hct,
total white blood cell (WBC) count, mean corpuscular Hb
(MCH), MCH concentration (MCHC) and mean corpuscular
volume (MCV) were determined. Hb concentration was esti-
mated by using the cyanmethemoglobin method (Lavanya
et al. 2011). Total RBC and WBC counts were estimated
by using an improved Neubauer hemocytometer with Natt-
Herrick’s stain solution (Natt and Herrick 1952). Blood cells
were calculated as per Pal et al. (2008) and Parida et al.
(2011). The following equation was used to calcutlate total
RBC count per cubic millimeter: 200 × 50 × N = 10,000 N
(where N = number of RBCs calculated; dilution fac-
tor = 200). The total WBC count per cubic millimeter was
evaluated by: 20 × 1 × L/0.4 cells = 50 × L (where L = num-
ber of leucocytes calculated, dilution factor = 50). The Hct
estimation followed Adebayo et al. (2007), whereby sam-
ples were inserted into microhematocrit capillary tubes and
centrifuged at 12,000 r.p.m. for 5 min in a microcentrifuge
(REMI RM-12C BL; REMI India); the final values are given
as percentages. The different RBC indices, MCH, MCHC
and MCV were assayed as per the formulas given by Dacie
and Lewis (1975).
Serum biochemical parameters
The blood samples in the Eppendorf tubes were centri-
fuged at 5000 g for 5 min and the supernatant (serum) was
obtained. The serum was then used for the estimation of
glucose, cholesterol, blood urea, total protein, albumin and
globulin by a VETSCAN VS2 (Abaxis, USA).
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Fisheries Science
Physicochemical analysis
Since the Sindh River has a number of different types of
catchment, the sites selected for the collection of fish spe-
cies have different physicochemical characteristics, which
ultimately affect the health status of the fish. Hence, random
water samples were collected from the three study sites on
monthly basis for physicochemical analysis. The physico-
chemical parameters of the water in the stocking tank in the
wet laboratory were also recorded. The parameters, pH,
water temperature, dissolved oxygen, free carbon dioxide
and total alkalinity were assessed by using standard methods
(APHA 1998).
Statistical analysis
The data are presented as mean ± SD of triplicates, and
were subjected to one-way ANOVA in SPSS (version 7.5
for Windows 2010). Variations between means were deter-
mined by using Duncan’s multiple range test (Duncan 1955).
Pearson coefficient for linear correlation (r) at P < 0.05 was
used to determine correlations of hematological and serum
variables.
Results
The biometric data of S. labiatus of different size are pre-
sented in Table 1, and the physicochemical parameters of the
stocking tank and water samples collected from the differ-
ent sampling sites of the Sindh River in Tables 2 and 3, 4,
respectively. The sex-specific seasonal hematological data
are presented in Table 5, and the correlations of hemato-
logical parameters of male and females in Tables 6 and 7,
respectively.
Males had the highest Hb concentrations, Hct and total
RBC count in all the seasons. Hb concentration was highest
in summer and lowest value in winter in both sexes. Over-
all, in all the seasons, the Hb concentration of males was
significantly higher (P < 0.05) than that of females (Fig. 2).
Similarly, a significantly higher (P < 0.05) RBC count was
noted in males compared to females, with maximum values
in summer and minimum values in the winter (Fig. 3). In
contrast, the WBC count was higher in females than in males
in all the seasons; the highest (P < 0.05) WBC count was
reported for both sexes during the summer and the lowest
noted in the winter (Fig. 4). Males had significantly higher
(P < 0.05) Hct than females. However, the Hct was high-
est in summer and lowest in winter in both sexes (Fig. 5).
MCH was highest in winter and lowest during summer for
both sexes (Fig. 6). A similar trend was noted in MCV val-
ues (Fig. 7). Conversely, the highest MCHC values were
recorded in summer for both the sexes, and the lowest values
Fisheries Science

Table 1  Biometric indices Season Sex Total length (cm) Total weight (g)
of Schizothorax labiatus
(mean ± SD) inhabiting the Range Mean ± SD Range Mean ± SD
Sindh River, Indian Himalayan
Region Spring Male 31.5–41.1 35.44 ± 5.03 311.2–627.0 356.38 ± 40.81
Female 26.5–37.1 30.58 ± 3.55 209.7–440.2 283.07 ± 33.28
Summer Male 25.6–36.4 29.68 ± 3.99 223.1–459.8 296.78 ± 26.21
Female 28.3–39.0 33.63 ± 5.23 246.6–509.8 300.17 ± 35.55
Autumn Male 24.0–36.1 28.97 ± 4.16 207.7–433.3 254.51 ± 37.44
Female 28.4–38.6 31.00 ± 4.23 243.3–501.3 298.83 ± 43.42
Winter Male 24.9–39.4 30.44 ± 4.88 220.3–518.3 302.63 ± 31.53
Female 33.5–41.5 36.81 ± 4.40 344.5–638.1 372.69 ± 40.09

Table 2  Physicochemical characteristics of the water in the wet labo- correlation of biochemical parameters of male and female
ratory tank during the acclimation of S. labiatus  in Tables 9 and 10, respectively. All serum biochemical
Parameters Mean ± SD parameters exhibited a significant difference (P < 0.05) with
respect to the sex except for total protein, albumin and globu-
pH 7.18 ± 0.45 lin (P > 0.05). The glucose concentration was notably higher
Temperature (℃) 16.96 ± 1.32 (P < 0.05) in both sexes during the summer and at its lowest
Dissolved oxygen (mg l−1) 6.48 ± 0.96
level in the winter (Fig. 9). A similar trend was observed
Free carbon dioxide (mg l−1) 13.52 ± 1.80
in the cholesterol concentration for both sexes through the
Total alkalinity (mg l−1) 143.04 ± 3.71
seasons, although it was significantly higher (P < 0.05) in
females than in males in every season (Fig. 10). The urea
concentration was significantly higher (P < 0.05) in males
in winter for males and autumn in females (Fig. 8). However, than in females in summer and autumn; the highest values
no significant differences (P > 0.05) were observed in MCH, were noted for both sexes during summer and the lowest
MCHC and MCV between the sexes or among the seasons. during winter (Fig. 11). Likewise, the total protein, albumin
The sex-specific and seasonal results of the serum bio- and globulin concentrations were highest during summer
chemistry of S. labiatus are presented in Table 8, and the and lowest during winter in both sexes (Figs. 12, 13 and

Table 3  Physicochemical characteristics of the Sindh River during spring and summer

Parameters Spring Summer
Site I (Manigam) Site II (Bamloora) Site III (Rabitar) Site I (Manigam) Site II (Bamloora) Site III (Rabitar)

pH 7.90 ± 0.16 8.00 ± 0.24 7.94 ± 0.20 7.80 ± 0.26 7.62 ± 0.12 7.54 ± 0.20

Temperature (℃) 5.20 ± 0.08 8.64 ± 0.18 9.00 ± 0.32 9.08 ± 0.45 12.02 ± 1.30 12.86 ± 1.56
−1
Dissolved oxygen (mg l ) 8.56 ± 0.24 8.16 ± 0.18 8.00 ± 0.40 7.92 ± 0.50 7.34 ± 0.34 7.00 ± 0.45
Free carbon dioxide (mg l−1) 5.28 ± 0.08 6.08 ± 0.16 7.32 ± 0.22 6.42 ± 0.33 7.21 ± 0.28 8.04 ± 0.24
Total alkalinity (mg l−1) 88.42 ± 3.82 103.57 ± 5.06 112.66 ± 6.38 83.50 ± 5.56 62.18 ± 2.15 105.08 ± 6.60

Table 4  Physicochemical characteristics of the Sindh River during autumn and winter

Parameters Autumn Winter
Site I (Manigam) Site II (Bamloora) Site III (Rabitar) Site I (Manigam) Site II (Bamloora) Site III (Rabitar)

pH 7.96 ± 0.76 7.80 ± 0.42 7.72 ± 0.58 8.10 ± 0.56 8.04 ± 0.32 8.20 ± 0.38

Temperature (℃) 8.24 ± 0.45 10.56 ± 1.38 11.04 ± 1.65 2.42 ± 0.06 5.26 ± 0.30 5.88 ± 0.52
Dissolved oxygen (mg l−1) 8.38 ± 0.40 8.02 ± 0.64 7.55 ± 0.36 9.14 ± 0.71 8.56 ± 0.64 8.24 ± 0.96
Free carbon dioxide (mg l−1) 5.96 ± 0.20 6.77 ± 0.51 7.78 ± 0.30 4.70 ± 0.15 5.36 ± 0.10 6.80 ± 0.38
Total alkalinity (mg l−1) 102.10 ± 4.84 131.94 ± 5.56 148.73 ± 7.62 128.40 ± 6.28 144.72 ± 6.44 161.58 ± 8.05

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Table 5  Sex-specific seasonal variation in hematological parameters of S. labiatus inhabiting the Sindh River, Indian Himalayan Region
(mean ± SD, n = 432)
Season Sex n Hb RBC WBC Hct (%) MCH (pg) MCHC (%) MCV (fl)
(g dl−1) (× 106 mm−3) (× 103 mm−3)

Spring Male 54 9.93 ± 0.86 d 1.78 ± 0.26 d 18.91 ± 1.34 c 34.46 ± 2.86 c 55.80 ± 7.04 ab 28.98 ± 2.13 ab 195.45 ± 24.69 a

Female 54 9.29 ± 1.02 e 1.69 ± 0.21 e 19.16 ± 1.19 c 33.08 ± 2.59 d 55.59 ± 8.99 ab 28.13 ± 2.52 b 197.80 ± 26.88 a
Summer Male 54 11.51 ± 1.23 a 2.12 ± 0.22 a 19.84 ± 1.30 b 38.84 ± 1.61 a 54.42 ± 5.54 b 29.62 ± 2.76 a 184.40 ± 17.57 b
Female 54 10.87 ± 1.01 b 2.01 ± 0.16 b 20.43 ± 1.08 a 37.40 ± 1.49 b 53.97 ± 3.46 b 29.08 ± 2.51 ab 186.35 ± 12.68 ab
Autumn Male 54 10.49 ± 0.83 c 1.89 ± 0.11 c 19.35 ± 1.21 bc 36.92 ± 1.77 b 55.29 ± 3.59 ab 28.45 ± 2.26 b 194.87 ± 12.15 a
Female 54 9.80 ± 0.82 d 1.81 ± 0.19 d 19.38 ± 1.30 bc 35.05 ± 2.50 c 54.32 ± 3.79 b 28.05 ± 2.55 b 194.98 ± 19.82 a
Winter Male 54 9.21 ± 0.93 e 1.66 ± 0.19 e 17.72 ± 1.29 d 32.68 ± 2.25 d 56.30 ± 5.20 ab 28.22 ± 2.40 b 198.49 ± 19.68 a
Female 54 8.83 ± 0.95 f 1.53 ± 0.20 f 18.08 ± 1.25 d 30.78 ± 2.33 e 57.61 ± 5.04 a 28.71 ± 2.40 ab 202.38 ± 22.13 a

Mean values in a column followed by different lowercase letters are significantly different (P < 0.05)
Hb Hemoglobin, RBC red blood cells, WBC white blood cells, Hct hematocrit, MCH mean corpuscular hemoglobin, MCHC mean corpuscular
hemoglobin concentration, MCV mean corpuscular volume

Table 6  Seasonal correlation Season Variables HB RBC WBC Hct MCH MCHC MCV
analysis of hematological
parameters of male S. labiatus Spring HB 1 0.594** 0.279* 0.625** −0.004 0.425** −0.290*
inhabiting the Sindh River,
RBC 0.594** 1 0.250 0.571** −0.780** 0.032 −0.806**
Indian Himalayan Region
WBC 0.279* 0.250 1 0.337* −0.103 −0.032 −0.095
Hct 0.625** 0.571** 0.337* 1 −0.244 −0.415** −0.019
MCH −0.004 −0.780** −0.103 −0.244 1 0.248 0.823**
MCHC 0.425** 0.032 −0.032 −0.415** 0.248 1 −0.316*
MCV −0.290* −0.806** −0.095 −0.019 0.823** −0.316* 1
Summer HB 1 0.551** 0.356** 0.502** 0.486** 0.923** −0.381**
RBC 0.551** 1 0.118 0.455** −0.458** 0.425** −0.901**
WBC 0.356** 0.118 1 0.078 0.254 0.380** −0.115
Hct 0.502** 0.455** 0.078 1 0.073 0.132 −0.035
MCH 0.486** −0.458** 0.254 0.073 1 0.530** 0.545**
MCHC 0.923** 0.425** 0.380** 0.132 0.530** 1 −0.417**
MCV −0.381** −0.901** −0.115 −0.035 0.545** −0.417** 1
Autumn HB 1 0.606** 0.314* 0.319* 0.658** 0.800** −0.332*
RBC 0.606** 1 0.257 0.377** −0.197 0.375** −0.683**
WBC 0.314* 0.257 1 0.085 0.152 0.261 −0.176
Hct 0.319* 0.377** 0.085 1 0.046 −0.312* 0.415**
MCH 0.658** −0.197 0.152 0.046 1 0.624** 0.244
MCHC 0.800** 0.375** 0.261 −0.312* 0.624** 1 −0.601**
MCV −0.332* −0.683** −0.176 0.415** 0.244 −0.601** 1
Winter HB 1 0.590** 0.318* 0.537** 0.274* 0.740** −0.361**
RBC 0.590** 1 0.345* 0.544** −0.612** 0.260 −0.817**
WBC 0.318* 0.345* 1 0.525** −0.107 −0.049 −0.058
Hct 0.537** 0.544** 0.525** 1 −0.124 −0.169 0.030
MCH 0.274* −0.612** −0.107 −0.124 1 0.412** 0.622**
MCHC 0.740** 0.260 −0.049 −0.169 0.412** 1 −0.453**
MCV −0.361** −0.817** −0.058 0.030 0.622** −0.453** 1

For abbreviations, see Table 5

*P = 0.05, **P = 0.01

13
Fisheries Science

Table 7  Seasonal correlation Season Variables HB RBC WBC Hct MCH MCHC MCV
analysis of hematological
parameters of female S. labiatus Spring HB 1 0.284* 0.453** 0.611** 0.534** 0.690** 0.173
inhabiting the Sindh River,
RBC 0.284* 1 0.304* 0.334* −0.647** 0.057 −0.772**
Indian Himalayan Region
WBC 0.453** 0.304* 1 0.488** 0.104 0.116 0.019
Hct 0.611** 0.334* 0.488** 1 0.206 −0.149 0.324*
MCH 0.534** −0.647** 0.104 0.206 1 0.470** 0.833**
MCHC 0.690** 0.057 0.116 −0.149 0.470** 1 −0.088
MCV 0.173 −0.772** 0.019 0.324* 0.833** −0.088 1
Summer HB 1 0.745** 0.337* 0.376** 0.484** 0.905** −0.695**
RBC 0.745** 1 0.097 0.573** −0.221 0.538** −0.882**
WBC 0.337* 0.097 1 −0.141 0.360** 0.433** −0.207
Hct 0.376** 0.573** −0.141 1 −0.217 −0.053 −0.130
MCH 0.484** −0.221 0.360** −0.217 1 0.623** 0.135
MCHC 0.905** 0.538** 0.433** −0.053 0.623** 1 −0.688**
MCV −0.695** −0.882** −0.207 −0.130 0.135 −0.688** 1
Autumn HB 1 0.792** 0.293* 0.344* −0.042 0.654** −0.594**
RBC 0.792** 1 0.149 0.422** −0.639** 0.400** −0.773**
WBC 0.293* 0.149 1 0.081 0.110 0.219 −0.095
Hct 0.344* 0.422** 0.081 1 −0.270* −0.482** 0.234
MCH −0.042 −0.639** 0.110 −0.270* 1 0.175 0.518**
MCHC 0.654** 0.400** 0.219 −0.482** 0.175 1 −0.740**
MCV −0.594** −0.773** −0.095 0.234 0.518** −0.740** 1
Winter HB 1 0.742** 0.329* 0.644** 0.152 0.710** −0.438**
RBC 0.742** 1 0.413** 0.561** −0.547** 0.453** −0.811**
WBC 0.329* 0.413** 1 0.367** −0.193 0.099 −0.220
Hct 0.644** 0.561** 0.367** 1 −0.046 −0.078 0.018
MCH 0.152 −0.547** −0.193 −0.046 1 0.240 0.651**
MCHC 0.710** 0.453** 0.099 −0.078 0.240 1 −0.590**
MCV −0.438** −0.811** −0.220 0.018 0.651** −0.590** 1

For abbreviations, see Table 5

*P = 0.05, **P = 0.01

Fig. 2  Hemoglobin (Hb) concentration of S. labiatus in the different Fig. 3  Total red blood cell (RBC) count of S. labiatus in the different
seasons seasons

14), although there was no significant difference (P > 0.05) inhabiting the Sindh River varied throughout the seasons as
between the sexes for these parameters. The results clearly well as between the sexes, whereas the cholesterol concen-
demonstrated that the serum biochemical parameters glu- tration was higher in females than in males in all the seasons
cose, urea, total protein, albumin and globulin of S. labiatus of the year.

13

Fig. 4  Total white blood cell (WBC) count of S. labiatus in the differ-
ent seasons
Fig. 5  Hematocrit (Hct) of S. labiatus in the different seasons
Fig. 6  Mean corpuscular Hb (MCH) of S. labiatus in the different
seasons
Discussion
Fish are one of the largest and most diverse groups of
aquatic organisms, and respond with great sensitivity to
changes in their environment (Borkovic et al. 2008; Sahiti
et al. 2018). Physical and chemical alterations in a fish’s
environment can induce changes in its blood chemistry,
which can lead to physiological disturbances. Hence, it
13
Fisheries Science
Fig. 7  Mean corpuscular volume (MCV) of S. labiatus in the different
seasons
Fig. 8  Mean corpuscular Hb (MCHC) concentration of S. labiatus in
the different seasons
is important to take both the habitat and environment of
fish into consideration when studying their health. A large
body of research on different species of fish has shown
that water quality impacts their health status (Faggio
et al. 2012; Fazio et al. 2012c; Fazio et al. 2013a, b; Art-
hanari and Dhanapalan 2016; Demeke and Tassew 2016;
Sharma et al. 2017; Ahmed and Sheikh 2019; Kucukgul
et al. 2019). The analysis of blood is considered a vital
diagnostic tool for monitoring the health status of fish and
the impact of habitat on their biology (Gabriel et al. 2004;
Fazio et al. 2012a, b, 2013a, b, 2017a; b; Lorenz et al.
2018; Pula et al. 2018; Sheikh and Ahmed 2019). Diseases
in fish can be relatively easily diagnosed from authentic
data obtained from hematological analyses following non-
lethal sampling (Satheeshkumar et al. 2012). Standardized,
inexpensive and non-lethal methods are important tools for
the monitoring of the health status of fish, information on
which can ultimately be used to increase their production
(Hrubec et al. 2000).
The present study demonstrated that there were signifi-
cant alterations with respect to sex and season in both hema-
tological and serum biochemical parameters of S. labiatus.
Hb concentration, RBC count and Hct were higher during
Fisheries Science

Table 8  Sex-specific seasonal variation in serum biochemical parameters of S. labiatus inhabiting the Sindh River, Indian Himalayan Region
(mean ± SD, n = 432)
Season Sex n Glucose Cholesterol (mg dl−1) Urea Total protein (g dl−1) Albumin Globulin
(mg dl−1) (mg dl−1) (g dl−1) (g dl−1)

Spring Male 54 77.37 ± 11.84d e 143.57 ± 8.94 e 7.12 ± 0.78 f 5.71 ± 0.81 cd 1.26 ± 0.20 c 4.44 ± 0.77 bc

Female 54 82.31 ± 9.66 c 168.78 ± 16.11 d 7.83 ± 0.91 e 6.00 ± 0.90 bc 1.34 ± 0.30 bc 4.66 ± 0.83 b
Summer Male 54 110.88 ± 16.01 a 221.28 ± 12.61 b 10.78 ± 1.01 a 7.67 ± 1.14 a 1.70 ± 0.49 a 5.97 ± 1.10 a
Female 54 91.97 ± 12.14 b 245.58 ± 15.91 a 9.95 ± 0.64 b 7.32 ± 1.06 a 1.68 ± 0.26 a 5.64 ± 1.03 a
Autumn Male 54 94.46 ± 15.12 b 191.95 ± 10.49 c 9.21 ± 0.92 c 6.23 ± 1.20 b 1.46 ± 0.35 b 4.77 ± 1.11 b
Female 54 80.70 ± 10.69 cd 219.36 ± 14.63 b 8.58 ± 0.83 d 6.16 ± 1.08 b 1.43 ± 0.37 b 4.72 ± 0.95 b
Winter Male 54 73.99 ± 9.37 e 133.32 ± 10.09 f 7.07 ± 0.88 f 5.17 ± 1.07 e 1.20 ± 0.35 c 3.96 ± 0.99 d
Female 54 61.82 ± 11.55 f 145.33 ± 7.45 e 7.56 ± 1.07 e 5.42 ± 1.10 de 1.27 ± 0.42 c 4.15 ± 0.90 cd

Mean values in a column followed by different lowercase letters are significantly different (P < 0.05)

Table 9  Seasonal correlation Season Variables Glucose Cholesterol Urea Total protein Albumin Globulin
analysis of serum biochemical
parameters of male S. labiatus Spring Glucose 1 0.096 0.204 0.019 0.137 −0.017
inhabiting the Sindh River,
Cholesterol 0.096 1 0.436** 0.453** 0.340* 0.386**
Indian Himalayan Region
Urea 0.204 0.436** 1 0.339* 0.457** 0.235
Total protein 0.019 0.453** 0.339* 1 0.323* 0.967**
Albumin 0.137 0.340* 0.457** 0.323* 1 0.073
Globulin −0.017 0.386** 0.235 0.967** 0.073 1
Summer Glucose 1 0.160 0.164 0.287* −0.038 0.317*
Cholesterol 0.160 1 0.121 −0.093 0.214 −0.194
Urea 0.164 0.121 1 0.245 0.090 0.215
Total protein 0.287* −0.093 0.245 1 0.311* 0.903**
Albumin −0.038 0.214 0.090 0.311* 1 −0.128
Globulin 0.317* −0.194 0.215 0.903** −0.128 1
Autumn Glucose 1 0.257 −0.037 0.051 0.144 0.009
Cholesterol 0.257 1 0.176 0.259 0.258 0.198
Urea −0.037 0.176 1 0.316* 0.370** 0.225
Total protein 0.051 0.259 0.316* 1 0.393** 0.957**
Albumin 0.144 0.258 0.370** 0.393** 1 0.108
Globulin 0.009 0.198 0.225 0.957** 0.108 1
Winter Glucose 1 −0.200 −0.130 −0.189 −0.202 −0.134
Cholesterol −0.200 1 0.589** 0.336* 0.311* 0.254
Urea −0.130 0.589** 1 0.296* 0.502** 0.143
Total protein −0.189 0.336* 0.296* 1 0.396** 0.945**
Albumin −0.202 0.311* 0.502** 0.396** 1 0.074
Globulin −0.134 0.254 0.143 0.945** 0.074 1

*P = 0.05, **P = 0.01

the summer and lower during the winter. These results can
be explained by the elevated water temperature during the
summer, which leads to an increase in metabolic rate. The
concentration of oxygen in water declines with an increase
in temperature, which causes an increase in Hb concentra-
tion, RBC count and Hct in fish due to hypoxia (Sharma
et al. 2017). Our results agree with those of other stud-
ies (Orun et al. 2003; Adebayo et al. 2007; Pradhan et al.
2012,2013; Yousefzadeh and Khara 2014; Sharma et al.
2015,2017; Sheikh and Ahmed 2019). Males had higher
Hb concentrations, RBC counts and Hct than females in all
the seasons, which could be linked to the higher metabolic
rate of the former (Orun et al. 2003; Svobodova et al. 2008;
Pradhan et al. 2012; Sharma et al. 2017; Ahmed et al. 2019).
The RBC count of an organism indicates the oxygen car-
rying capacity of the blood (Val 2000). It is regulated within

13
 Fisheries Science

Table 10  Seasonal correlation Season Variables Glucose Cholesterol Urea Total protein Albumin Globulin
analysis of serum biochemical
parameters of female S. labiatus Spring Glucose 1 0.255 0.055 0.142 0.143 0.101
inhabiting the Sindh River,
Cholesterol 0.255 1 0.258 0.390** 0.344* 0.296*
Indian Himalayan Region
Urea 0.055 0.258 1 0.025 −0.051 0.046
Total protein 0.142 0.390** 0.025 1 0.389** 0.941**
Albumin 0.143 0.344* −0.051 0.389** 1 0.054
Globulin 0.101 0.296* 0.046 0.941** 0.054 1
Summer Glucose 1 0.254 0.387** 0.065 0.346* −0.022
Cholesterol 0.254 1 0.084 0.044 −0.003 0.046
Urea 0.387** 0.084 1 0.138 0.202 0.090
Total protein 0.065 0.044 0.138 1 0.242 0.968**
Albumin 0.346* −0.003 0.202 0.242 1 −0.008
Globulin −0.022 0.046 0.090 0.968** −0.008 1
Autumn Glucose 1 0.034 0.097 0.240 0.299* 0.155
Cholesterol 0.034 1 0.109 −0.089 −0.049 −0.083
Urea 0.097 0.109 1 0.298* 0.272* 0.231
Total protein 0.240 −0.089 0.298* 1 0.499** 0.940**
Albumin 0.299* −0.049 0.272* 0.499** 1 0.172
Globulin 0.155 −0.083 0.231 0.940** 0.172 1
Winter Glucose 1 0.161 −0.102 0.081 0.039 0.081
Cholesterol 0.161 1 0.187 0.213 0.258 0.139
Urea −0.102 0.187 1 0.317* 0.413** 0.193
Total protein 0.081 0.213 0.317* 1 0.617** 0.930**
Albumin 0.039 0.258 0.413** 0.617** 1 0.284*
Globulin 0.081 0.139 0.193 0.930** 0.284* 1

*P = 0.05, **P = 0.01

Fig. 9  Glucose concentration of S. labiatus in the different seasons Fig. 10  Total cholesterol concentration of S. labiatus in the different
seasons

physiological limits through various mechanisms of physi-

ological compensation. In the present study, the RBC count
of S. labiatus was highest during the summer and lowest in
the winter. Similar results were reported for Capoeta trutta
(Orun and Erdeml 2002), Rutilus frisii (Nikoo et al. 2010),
Catla catla (Pradhan et al. 2012), Cirrhinus mrigala (Prad-
han et al. 2014a), Labeo rohita (Pradhan et al. 2014b), Esox
lucius (Adel et al. 2016), Barilius bendelisis (Sharma et al.
2017) and Schizothorax plagiostomus (Sheikh and Ahmed
2019), in which variation in the RBC count was attributed
mainly to changes in season, sex and age. Satheeshkumar
et al. (2011) concluded that a greater RBC count indicated
a higher demand for dissolved oxygen in wild marine fish in
water with low dissolved oxygen levels. Male S. labiatus in
the present study displayed a significantly greater RBC count
(P < 0.05) than females over all the seasons. Our results are
in agreement with those reported for C. trutta (Orun and
Erdeml 2002), R. frisii (Nikoo et al. 2010), C. catla (Prad-
han et al. 2012), Cyprinus carpio (Golemi et al. 2013), C.

13
Fisheries Science
Fig. 11  Urea concentration of S. labiatus in the different seasons
Fig. 12  Total protein concentration of S. labiatus in the different sea-
sons
Fig. 13  Albumin concentration of S. labiatus in the different seasons
mrigala (Pradhan et al. 2014a), L. rohita (Pradhan et al.
2014b), B. bendelisis (Sharma et al. 2015, 2017), E. lucius
(Adel et al. 2016), and L. rohita (Kumar et al. 2019).
Hct is also a useful indicator of the well-being and
physiological condition of fish. It is used in aquaculture
and fisheries management as an authentic index of anae-
mia and fish health in response to nutrition, stress and
disease (Brill et al. 2008). In the present study, the higher
Hct found in males compared to females in all the seasons
could be explained by the higher metabolic rate of the
Fig. 14  Globulin concentration of S. labiatus in the different seasons
former. Sheikh and Ahmed (2019) also observed higher
Hct in male compared to female S. plagiostomus inhabit-
ing different environments. WBC are the second major
constituent of fish blood after erythrocytes. They play a
vital role in the immune response of fish and constitute
an important part of their innate immune defense system
(Ballarin et al. 2004). The highest WBC count (P < 0.05)
was recorded in summer and the lowest in winter, simi-
lar to results recorded for Betta splendens (Motlagh et al.
2012), C. mrigala (Pradhan et al. 2014) and S. plagiosto-
mus (Sheikh and Ahmed 2019). Females exhibited greater
WBC counts than males in all the seasons of the year,
which indicates that the immune response of S. labiatus
is influenced by sex, and a higher water temperature also
lead to an increase in the number of WBCs in the blood
circulatory system; these results are in agreement with
those of Vazquez and Guerrero (2007), Yousefzadeh and
Khara (2014) and Sharma et al. (2017).
The erythrocyte indices MCH, MCHC and MCV also
varied with season. MCV, which indicates the size of RBCs,
reflects abnormal or normal cell division during erythro-
poiesis (Houston 1997). In spite of the low number of RBCs
recorded in the winter in our study, an increase in MCV was
noted, which might have indicated the enlargement of RBCs
(macrocytosis). The increase in MCV in winter could have
been due to the presence of larger mature RBCs in the blood
circulatory system, while the lowest MCV, in summer, may
have been due to a greater number of smaller RBC in the
circulatory system. A similar trend was seen in MCH, which
was higher during winter and lower in summer. The decrease
in MCH during summer was due to an increase in erythro-
poiesis. Our results are in agreement with those on R. frisii
(Bani and Vayghan 2011), Tor putitora (Gupta et al. 2013)
and B. bendelisis (Sharma et al. 2015). In contrast to MCH
and MCV, the maximum MCHC level was recorded in sum-
mer for both sexes; this might have been related to a rise in
temperature during this season, while the lowest MCHC was
noted in autumn for females. Similar results were reported
for B. bendelisis (Sharma et al. 2017).
13

Alterations in the values of serum biochemical parame-
ters in fish are generally due to a number of factors, e.g., age,
season, environmental conditions, stress, diet and disease
(Zarejabad et al. 2010; Yeganeh 2012; Kumar et al., 2019).
However, it has also been reported that enhanced matura-
tion of gonads, and the transfer of nutrients to the gonads
from various organs, bring about significant variations in
cholesterol, triglyceride and protein levels (Chatzifotis et al.
2004). Serum glucose is the major energy source for fish
cells, and an abrupt increase in the level of glucose reflects
acute stress in fish (Barton 2002). In the present study, S.
labiatus showed significantly higher (P < 0.05) glucose val-
ues in summer and lower values in winter, in accordance
with findings on Tinca tinca (Guirjaro et al. 2003), C. mri-
gala (Pradhan et al. 2012), L. rohita (Pradhan et al. 2013), B.
bendelisis (Sharma et al. 2015), and S. plagiostomus (Sheikh
and Ahmed 2019). Glucose levels were lower during the
winter than in the spring and summer; this might be related
to lower feed intake and a decline in tissue nutrient uptake,
as the latter is generally facilitated by hormones produced
by the pancreas at higher temperatures than those occurring
in the winter (Pradhan et al. 2014a, b; Sharma et al. 2017).
Males had higher glucose levels in summer and winter,
which could be attributed to their activity and greater feed
intake (Giberson and Litvak 2003); in contrast, the glucose
levels of females were highest in spring and autumn. The
high levels of glucose, protein and triglycerides observed in
males were mainly due to an enhanced decline of liver gly-
cogen levels (Ojolick et al. 1995). Similar observations on
farmed Dicentrarchus labrax were reported by Coz-Rakovac
et al. (2005).
Seasonal fluctuations in cholesterol level were also noted
between the sexes during the current study. The highest lev-
els of cholesterol were recorded during summer in females,
which might have been due to greater energetic demand,
higher feed intake, higher metabolic rate and higher temper-
ature (Pradhan et al. 2014a, b). The lowest cholesterol levels
were recorded during winter, which might have been linked
to reproductive activity, as the breeding cycle of fish plays a
major role in cholesterol level (Svobodova et al. 2001). The
urea concentration remained high during the summer, which
might have been due to the availability of food, that leads to
an increase in metabolic activity, and thus higher levels of
urea in the blood. Similar results were reported for Wallago
attu (Chandra 2009).
Serum protein is a diagnostic marker used to assess the
nutritional and health status of fish (Friedrich and Stepanow-
ska 2001; Riche 2007; Satheeshkumar et al. 2011, 2012). In
S. labiatus, the total protein, albumin and globulin concen-
trations were higher during summer, followed by autumn,
while the lowest values were recorded in winter and spring.
Possible explanations for these results are augmented prote-
olysis and the probable utilization of protein in metabolism
13
Fisheries Science
or for the growth of the gonads, as winter and spring are con-
sidered to be the respective pre-spawning and peak-spawning
periods of this fish. Our results for serum protein agree with
those of Hrubec et al. (2001), Pradhan et al. (2014a, b) and
Sharma et al. (2015). Total protein concentration decreases
during reproduction, as fish utilize protein as an ultimate
energy source or fuel for energy-demanding activities like
swimming, as well as for gonad growth (Bani and Vayghan
2011). Female S. labiatus exhibited greater protein values
than males in spring and winter, while males showed higher
values in summer and autumn. Significant difference in the
levels of blood protein have also been reported between the
sexes in T. tinca and Thymallus thymallus (Svobodova et al.
2001). Serum albumin is also known to increase as a result
of sexual maturation (Di Marco et al. 2011), and similar
differences in the values of albumin to those of total protein
were also reported between the sexes in the present study.
As precursors of immunoglobulins, globulins are linked to
the innate immune response of fish (Palti et al. 1999). In the
present study, the highest levels of globulin were recorded
in summer in S. labiatus, followed by autumn.
The results of the current study show that most of the
hematological and serum biochemical parameters of S.
labiatus examined exhibit significant variations among the
seasons as well as between the sexes. The results clearly
show that season has a pronounced effect on the well-being,
as well as the health and nutritional status of S. labiatus. We
therefore recommend that both sex and season should be
taken into consideration when monitoring the health status
of this fish. This study provides the first baseline data for
rapid assays that can be used to assess the physiological and
pathological status of S. labiatus.
Acknowledgments  The authors are very grateful to the Head of the
Department of Zoology, University of Kashmir, for providing the nec-
essary laboratory facilities, and would like to thank the Department of
Science and Technology, Government of India, New Delhi for provid-
ing financial assistance through a DST-INSPIRE fellowship to K. J.
Compliance with ethical standard 
Conflict of interest  The authors declare that they do not have any con-
flict of interest.
Ethical approval  All applicable international, national, and/or insti-
tutional guidelines for the care and use of animals were followed by
the authors.
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