Animal Reproduction Science 166 (2016) 122127

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Animal Reproduction Science

journal homepage: www.elsevier.com/locate/anireprosci

Quantication of leptin in seminal plasma of buffalo bulls and

its correlation with antioxidant status, conventional and
computer-assisted sperm analysis (CASA) semen variables
Pradeep Kumar a, , Monika Saini a , Dharmendra Kumar a , M.H. Jan a , Dheer
Singh Swami a,b , R.K. Sharma a
a
b

ICAR-Central Institute for Research on Buffaloes, Hisar, Haryana, India

Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar, Haryana, India

a r t i c l e

i n f o

Article history:
Received 29 October 2015
Received in revised form 5 January 2016
Accepted 5 January 2016
Available online 11 January 2016
Keywords:
CASA
Leptin
Semen quality
Buffalo
Cryopreservation

a b s t r a c t
The present study is the rst to quantify leptin in seminal plasma of buffalo and investigate its relationship with seminal attributes. Ten ejaculates each from 10 Murrah buffalo
bulls were collected. Semen quality variables such as semen volume, sperm concentration,
sperm abnormalities, membrane integrity, antioxidant enzyme activities (superoxide dismutase, catalase and total antioxidant capacity), malondialdehyde (MDA) concentration,
as well as sperm kinetics and motility variables were evaluated. The leptin concentration
in serum and seminal plasma were estimated by the ELISA method. Bulls were classied in
two groups on the basis of sperm concentration with Group I having >800 million sperm/mL
and Group II <500 million sperm/mL. Greater (P<0.05) mean sperm abnormalities, seminal
leptin concentrations and MDA concentrations were recorded in Group II than Group I. The
seminal leptin was positively correlated with sperm abnormalities and MDA concentration
while being negatively correlated with sperm concentration, but there was no correlation
with sperm kinetic and motility variables, sperm membrane integrity and seminal plasma
antioxidant enzyme activity. Thus, the data suggest that seminal leptin has a role in spermatogenesis and can be used as a marker for spermatogenesis to predict the capacity of
buffalo bulls for semen production.
2016 Elsevier B.V. All rights reserved.

1. Introduction
The seminal plasma is a mixture of secretions from
the testis, epididymis and male accessory sex glands. A
number of proteins are found in seminal plasma which
are associated with male fertility, but most of these have
not been studied in detail (Kumar et al., 2012). Among
these proteins, leptin has been reported to both positively

Corresponding author at: Animal Physiology and Reproduction Division, ICAR-CIRB, Hisar-125001, India. Fax: +91 1662 275004.
E-mail address: drpradeepkrvet@gmail.com (P. Kumar).
http://dx.doi.org/10.1016/j.anireprosci.2016.01.011
0378-4320/ 2016 Elsevier B.V. All rights reserved.

(Ando and Aquila, 2005) and negatively (Glander et al.,

2002) inuence the sperm cell. Leptin, a 167-amino acid
protein with a molecular weight of approximately 16 kDa
(Zhang et al., 1994), is an adipocyte derived hormone that
suppresses food intake, stimulates energy expenditure,
increases metabolic rate, and ultimately causes loss of body
fat (Kamohara et al., 1997; Rossetti et al., 1997; Bouloumie
et al., 1998).
Besides these principal roles, leptin has been implicated in a large variety of neuroendocrine, paracrine, and
autocrine actions involved in the regulation of reproductive
function in humans. In men, several studies have linked

P. Kumar et al. / Animal Reproduction Science 166 (2016) 122127

leptin with puberty, spermatogenesis, functional regulation of the male gonadal axis, sperm maturation, sperm
capacitation and sperm motility (El-Hefnawy et al., 2000;
Zorn et al., 2007; Nicopoulou et al., 2009; Haron et al.,
2010). Unlike female reproduction, the role of leptin in
male reproductive system is not yet fully elucidated. Most
of the literature available regarding the physiological role
of leptin in male reproduction comes from human and
rodent studies. However, the presence of leptin has been
reported in boar sperm (De Ambrogi et al., 2007), bull
testis (Abavisani et al., 2009) and bull sperm (Nikbakht
et al., 2009; Abavisani et al., 2011). These ndings suggest that in addition to its endocrine actions at the
hypothalamicpituitary axis, leptin may have an autocrine
and/or paracrine role in male reproductive system. There
are, however, no reports regarding the concentration of
leptin in seminal plasma and serum of buffalo bulls and
its relation to seminal quality. Therefore, the present study
was designed to test the hypothesis whether there is any
correlation between serum and seminal leptin concentration with semen quality of buffalo bulls.
2. Materials and methods
2.1. Semen and blood collection
Semen was collected from ten Murrah buffalo bulls (35
years age) maintained under a progeny testing program at
Central Institute for Research on Buffaloes, Hisar, Haryana,
India. A total of 10 ejaculates from each bull were collected
using an articial vagina. At each collection, two ejaculates were taken with 2030 min elapsing between the
two successive ejaculates. Both ejaculates were pooled and
measurements of other semen variables occurred in the
pooled sample. The volume of semen in the study was the
volume of both ejaculates of individual bulls. Blood samples
were also collected and serum was separated for quantitating leptin concentrations. A small portion of ejaculate
was utilized for semen analysis and the rest of ejaculate
was centrifuged to collect the seminal plasma. The seminal
plasma was used for quantitation of leptin and antioxidant
enzyme activity.
2.2. Sperm concentration
Sperm concentration of each ejaculate was measured by
Accucell bovine photometer (IMV, LAigla, France). On the
basis of sperm concentration, bulls were classied in two
groups with Group I (n = 6) having >800 million sperm/mL
and Group II (n = 4) <500 million sperm/mL.
2.3. Sperm abnormality
The percentage of sperm abnormalities in fresh semen
was estimated using Eosin-Nigrosin staining as previously
described (Singh et al., 2013).

123

(2015a). Briey, the assay was performed by mixing 10 L

of fresh semen with 1 mL hypo-osmotic solution (0.735 g
sodium citrate dihydrate and 1.351 g fructose in 100 mL
distilled water). After incubation for 60 min at 37 C, 15 L
of well-mixed sample was placed on a warm slide (37 C)
and sperm tail bending/coiling were assessed under light
microscopy at 400x magnications. At least 200 sperm
were observed per slide. Sperm with a coiled tail after
incubation were considered to have an intact plasma membrane.
2.5. Sperm kinetics and motility
Sperm kinetics and motility of fresh ejaculates were
assessed using a computer-assisted sperm analyzer (CASA)
system (IVOS12.1, Hamilton-Thorne Biosciences, Beverly,
MA, USA) as previously described (Kumar et al., 2015b).
Before analysis using CASA, the semen sample was diluted
with pre-warmed Tris buffer to give a sperm concentration
of about 40 106 sperm/mL. The prepared semen sample
(1 L) was loaded in a pre-warmed (38 C) eight chamber
Leja slide (depth 20 m) and analyzed for sperm kinetics
and motility characteristics.
The following motion characteristics were recorded:
total motility (TM, %), progressive motility (PM, %), rapid
motility (RM, %), straight linear velocity (VSL, m/s),
average path velocity (VAP, m/s), curvilinear velocity
(VCL, m/s), average lateral head displacement (ALH,
m), beat cross frequency (BCF, Hz), straightness (STR,
%) and linearity (LIN, %) of the sperm. The CASA software settings were as follows: temperature = 38 C, frame
rate = 60 Hz, frames acquired = 30, minimum contrast = 35,
minimum cell size = 5 pixels, cell size = 9 pixels, cell intensity = 110 pixels, progressive cells (VAP cut-off = 50 m/s,
STR cut-off = 70%), slow cells (VAP cut-off = 30 /s and VSL
cut-off = 15 /s).
2.6. Bovine leptin assay
The leptin concentration in serum and seminal plasma
was estimated using a leptin ELISA kit (CSB-E06771b, Cusabio) as per the manufacturers instructions. This assay
was specic for detection of bovine leptin and its detection range was 3.1250 ng/mL. The standards or samples
and biotin-conjugated leptin were added to a pre-coated
antibody specic to bovine leptin microtiter plate wells.
A competitive inhibition reaction occurred between leptin (standards or samples) and biotin-conjugated leptin
for binding with the antibody coated wells. After washing,
avidin conjugated horseradish peroxidase was added to the
wells. Thereafter, substrate solution was added to the wells
and the color development occurred which was negatively
correlated with the amount of leptin in the sample. The
color development was stopped and the intensity of the
color was measured in the microplate reader at 450 nm.
2.7. Superoxide dismutase

2.4. Plasma membrane integrity

Plasma membrane integrity was evaluated using hypoosmotic swelling test (HOST) as described by Kumar et al.

Seminal plasma superoxide dismutase (SOD) activity

was assayed using SOD Assay Kit (K335, BioVision, Mountain View, CA, USA). This assay is based upon utilization of

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P. Kumar et al. / Animal Reproduction Science 166 (2016) 122127

Table 1
Seminal variables and leptin concentration of Group I (>800 million sperm/mL) and Group II (<500 million sperm/mL) buffalo bulls.
Variables

Group I

Group II

P-value

Semen volume (mL)

Sperm abnormalities (%)
Sperm concentration (million/mL)
Membrane integrity (%)
Seminal leptin (ng/mL)
Serum leptin (ng/mL)

4.87 0.52
15.50 0.79*
1053.53 48.58*
73.62 2.12
4.52 0.11*
6.30 0.26

5.55 0.44
20.50 1.33
386.32 49.81
74.82 2.14
5.64 0.28
5.87 0.25

0.368
0.0016
0.0001
0.7037
0.0003
0.2710

Mean values differ (P < 0.05).

WST-1, a tetrazolium salt, which produces formazan dye

upon reduction with a superoxide anion. The assay was
performed as per manufacturers instruction. Briey, 20 L
samples were added into microplates designated as sample
and two blank wells. Then, 20 L distilled water was added
to Blanks 1 and 3. The WST working solution (200 L) was
pipetted in each sample and each blank (13) well. Enzyme
working solution was added at the rate of 20 L to samples and Blank 1. Subsequently, 20 L of dilution buffer was
added to Blank 2 and 3. The mixture was incubated at 37 C
for 20 min. The SOD activity was measured after recording absorbance at 450 nm using a microplate reader. The
results were expressed as the inhibition rate (%).
2.8. Catalase
The catalase activity in seminal plasma was estimated
by using the catalase activity assay kit (K773, BioVision,
Mountain View, CA, USA) as per the manufacturers instruction. In the assay, catalase (CAT) rst reacts with H2 O2 to
produce water and oxygen, the unconverted H2 O2 reacts
with OxiRedTM probe to produce a product which measured at 570 nm and expressed as mU/mL. One unit of CAT
is the amount of CAT that disassociates 1.0 mol of H2 O2
per min at a pH 4.5 and at 25 C.
2.9. Total antioxidant capacity
Antioxidants in seminal plasma were estimated by total
antioxidant capacity (TAC) colorimetric assay kit (K274,
BioVision, Mountain View, CA, USA) according to instructions of the manufacturer. This assay was based on the
principle of use of trolox as an antioxidant standard.
The Cu2+ ion was converted to Cu+ by antioxidants. The
reduced Cu+ ion was chelated with a colorimetric probe
giving a broad absorbance peak around 570 nm, proportional to the total antioxidant capacity and expressed as
mmol/mL.
2.10. Malondialdehyde (MDA)
The concentration of MDA in seminal plasma was determined using the TABARS assay kit (Cayman Chemical
Company). Briey, to each tube 100 L of sample/standard,
100 L of SDS solution and 4 mL color reagent were added.
The mixture was boiled in a water bath for 1 h. After 1 h,
the samples and standards were removed and placed in
an ice bath for 10 min to stop the reaction. After cooling, the suspension was centrifuged for 10 min at 1600 g
and at 4 C. The 150 L suspensions were loaded into the

colorimetric plate and absorbance was measured at 535 nm

and concentration of MDA was expressed as nmol/mL.
2.11. Statistical analysis
All data were analyzed using the SPSS (Version 18) statistical software. The data were expressed as mean SE and
the differences were compared by using the unpaired t-test.
Relationships between values were analyzed by Spearman
correlation test. Spearmans correlation coefcient (r) with
a P-value was used to determine signicant differences
between variables. P<0.05 was considered signicant.
3. Results
Data for the seminal characteristics of two groups are
presented in Table 1. There were greater (P<0.05) sperm
abnormalities and seminal leptin concentrations observed
in Group II than Group I. However, there were no signicant
differences in the volume of ejaculates, sperm cell membrane integrity and serum leptin concentration between
these two groups. No signicant differences were observed
in seminal plasma antioxidant status (SOD, CAT and TAC)
between two groups except for a lesser (P<0.05) MDA concentration in Group I than Group II (Table 2). Furthermore,
there were no signicant differences in the sperm kinetics
and motility variables between the two groups (Table 3).
The data for correlation of seminal and serum leptin
concentrations, with each other, antioxidants status, conventional and CASA variables are presented in Table 4.
No signicant correlation was observed between seminal
and serum leptin concentrations. Further, serum leptin concentration were not correlated with antioxidant
enzyme activities or conventional and CASA attributes.
There were no correlations of seminal leptin with semen
volume, sperm cell membrane integrity, sperm kinetics and sperm motility. Interestingly, the seminal leptin
Table 2
Antioxidant status of seminal plasma of Group I (>800 million sperm/mL)
and Group II (<500 million sperm/mL) buffalo bulls.
Variables

Group I

Group II

P-value

SOD activity
(inhibition rate%)
CAT (mU/mL)
TAC (mmol/mL)
MDA (nmol/mL)

96.96 0.89

95.34 0.38

0.0620

4.82 0.25
19.94 0.14
16.34 0.34*

4.21 0.12
19.73 0.26
25.60 0.46

0.102
0.075
0.001

SOD: superoxide dismutase; CAT: catalase; TAC: Total antioxidant capacity; MDA: malondialdehyde.
*
Mean values differ (P < 0.05).

P. Kumar et al. / Animal Reproduction Science 166 (2016) 122127

Table 3
Sperm kinetic and motility variables (Mean SE) of Group I (>800 million
sperm/mL) and Group II (<500 million sperm/mL) buffalo bulls.
Variables

Group I

VAP (m/s)
VSL (m/s)
VCL(m/s)
ALH (m)
BCF (Hz)
STR (%)
LIN (%)
TM (%)
PM (%)
RM (%)

125.52
89.46
232.10
8.54
33.60
73.13
41.29
81.48
41.64
66.29

Group II

4.71
3.37
9.04
0.23
0.59
1.79
1.33
1.54
2.82
2.60

133.50
95.54
242.90
8.65
34.46
74.28
42.52
78.68
41.87
63.97

P-value
9.88
4.49
19.35
0.55
1.22
2.75
2.08
3.53
3.55
3.81

0.4467
0.3836
0.5930
0.8463
0.5088
0.7603
0.6627
0.4236
0.9687
0.6687

VAP: average path velocity; VSL: straight linear velocity; VCL: curvilinear velocity; ALH: average lateral head displacement; BCF: beat cross
frequency; STR: straightness; LIN: linearity; TM: total motility; PM: progressive motility; RM: rapid motility.

concentration were positively correlated with sperm

abnormalities and MDA concentration but negatively correlated with sperm concentration.
4. Discussion
To the best of our knowledge, this study was the rst to
determine the concentrations of leptin in seminal plasma
of buffalo bulls. Moreover, for the rst time, correlations
between the concentration of leptin in serum and seminal
plasma, along with the respective correlations with conventional and CASA sperm variables were also determined
in buffalo.
In the present study, concentrations of leptin were
quantied in seminal plasma of buffalo. Previously, leptin has also been identied in seminal plasma of men (Von
Sobbe et al., 2003; Jorsaraei et al., 2010), stallions (El-Maaty
Table 4
Correlation between seminal plasma and serum leptin concentration with
each other and with seminal attributes and sperm kinetic variables (r, P).
Variables

Seminal leptin

Serum leptin

Serum leptin
Volume
Sperm abnormalities
Concentration
Membrane integrity
SOD
CAT
TAC
MDA
VAP
VSL
VCL
ALH
BCF
STR
LIN
TM
PM
RM

0.159 (0.455)
0.214 (0.168)
0.386* (0.011)
0.340* (0.025)
0.121 (0.437)
0.117 (0.421)
0.217 (0.165)
0.135 (0.385)
0.331* (0.035)
0.210 (0.174)
0.219 (0.156)
0.199 (0.200)
0.143 (0.357)
0.122 (0.433)
0.023 (0.882)
0.042 (0.788)
0.146 (0.347)
0.001 (0.993)
0.059 (0.704)

0.283 (0.179)
0.012 (0.954)
0.252 (0.234)
0.235 (0.268)
0.301 (0.151)
0.124 (0.415)
0.125 (0.401)
0.245 (0.238)
0.0249 (0.907)
0.197 (0.354)
0.108 (0.613)
0.113 (0.598)
0.326 (0.119)
0.282 (0.180)
0.347 (0.096)
0.054 (0.8004)
0.232 (0.274)
062 (0.772)

SOD: superoxide dismutase; CAT: catalase; TAC: total antioxidant capacity; MDA: malondialdehyde; VAP: average path velocity; VSL: straight
linear velocity; VCL: curvilinear velocity; ALH: average lateral head displacement; BCF: beat cross frequency; STR: straightness; LIN: linearity;
TM: total motility; PM, progressive motility; RM, rapid motility.
*
Mean values differ (P < 0.05).

125

et al., 2014), boars (Lackey et al., 2002) and zebu bulls

(Souza et al., 2012). The origin of leptin in seminal plasma
has not been clearly ascertained but some studies suggested that the presence of leptin in the seminal plasma is
due to the leakage of leptin through the testis-blood-barrier
(Banks et al., 1999; Von Sobbe et al., 2003). However, others
suggested that seminal leptin is also secreted by sperm cells
(Aquila et al., 2005). Recently, Ellithy et al. (2014) reported
that the leptin of seminal plasma may be secreted by the
genital tract and/or testicular tissue.
In the present study, the concentration of leptin in buffalo seminal plasma ranged from 4.00 to 5.61 ng/mL of
seminal plasma. The leptin in seminal plasma has been
reported to range from 0.75 to 4.72 ng/mL in normal fertile
men (Lackey et al., 2002; Jorsaraei et al., 2010; Guo et al.,
2014) and be 1.66 ng/mL in stallion (El-Maaty et al., 2014),
11 ng/mL in boar (Lackey et al., 2002) and range from 22
to 24 ng/mL in zebu bull semen (Souza et al., 2012). Thus,
among domestic animals, the leptin concentration in buffalo seminal plasma is greater than that of stallions but
lower than that of bulls and boars.
Sperm kinetics, motility, membrane integrity and
antioxidant status are commonly believed to be important characteristics for evaluating semen quality. The CASA
is a reproducible method and is used to detect different
sperm kinetics and motility variables. The accuracy and
precision of the CASA system allow for the detection of subtle changes in semen quality. In the present study, semen
samples from Group II bulls had a greater number of sperm
abnormalities and seminal leptin concentrations compared
to those of Group I. However, there was no signicant difference in sperm kinetics and motility variables between
two groups even though there was a difference in the seminal leptin concentration. Most of the available literature
indicates the leptin concentration is positively associated
with the azoospermic and ashthenozoospermic conditions
in men (Guo et al., 2014). The condition is rarely found
in bulls selected for articial insemination programs. The
results from the present study are in agreement with other
reports which suggested that leptin does not have a signicant effect on the motility of ejaculated mature sperm
in humans (Camina et al., 2002; Li et al., 2009). The activities of the antioxidant enzymes SOD, CAT and TAC were
similar irrespective of sperm concentration, whereas there
was a greater MDA concentration which is an indicator of
lipid peroxidation that was detected in the oligozoospermic (Group II) bulls. Interestingly, leptin has also been
suggested to elevate lipid peroxidation (Kutlu et al., 2005;
Ajala et al., 2009) similar to the present ndings.
No correlation was observed between serum and seminal leptin in the present study. This nding is in agreement
with ndings of Ellithy et al. (2014) where it was suggested
that the testis-blood barrier may prevent large molecules
like leptin from passing through the interstitial tissue and
the basal compartment of the tubule to adluminal compartment of the tubule and lumen. Others suggested that
leptin is not actively transported but rather leaks through
the testis-blood-barrier (Banks et al., 1999; Von Sobbe
et al., 2003). Further, there were no correlations observed
between serum leptin concentration and semen quality
variables in the present study. In non-human species there

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P. Kumar et al. / Animal Reproduction Science 166 (2016) 122127

have been no previous reports, however, in men a number of studies have found positive correlations between the
concentration of leptin in serum and semen quality. Findings in the present study are in agreement with Zorn et al.
(2007) and Chen et al. (2009) where there was no difference
in serum leptin concentrations of normozoospermic and
azoospermic men. In contrast, a greater serum leptin concentration has been reported in azoospermic as compared
to normozoospermic men (Steinman et al., 2001). Findings
in the present study are in agreement with Guo et al. (2014)
where it was postulated that serum leptin increases only
in males with extremely poor spermatogenic function such
as the azoospermic condition. In the present study there
were no bulls having an extremely poor sperm production, therefore, no correlations of serum leptin and semen
quality could be established.
In the present study, seminal leptin is positively correlated with sperm abnormalities and negatively correlated
with sperm concentration. No report is available in domestic animals to compare the results; however, in men
there have been a number of reports studying this aspect.
Seminal leptin concentration was reportedly greater in
azoospermic men with low spermatogenesis capacity compared with that of the controls (Chen et al., 2009; Jorsaraei
et al., 2010). These ndings led Ma et al. (2011) to suggest that leptin may be a desirable marker for diagnosing
azoospermia in men. Similarly, Ishikawa et al. (2007) also
suggested that over-expression of the leptin gene may lead
to hypospermatogenesis, but the mechanism underlying
the effect of leptin on sperm production and morphology
is yet to be elucidated. The decreased sperm count and
increased fraction of abnormal sperm observed after leptin
administration has been suggested to be most likely due
to a direct effect of leptin, either on the sperm cell itself
or through its effect on the testicular tissues nurturing the
developing sperm cells (Haron et al., 2010).
In conclusion, the concentration of leptin in seminal
plasma of buffalo bulls was estimated for the rst time in
the present study and was found to be negatively correlated with sperm concentration and positively correlated
with sperm abnormalities and MDA concentration. These
data led us to propose that greater leptin concentrations
in seminal plasma are associated with hypospermatogenesis and sperm abnormalities. Further research is needed to
ascertain the exact mechanism of how leptin affects spermatogenesis.
Authors contributions
PK and DK involved in semen evaluation after semen
collection. PK, DK and DSS evaluated samples in CASA.
MS and PK estimated leptin, SOD, sperm abnormality and
membrane integrity. PK and MHJ performed statistical
analysis and prepared the manuscript. RKS conceived the
project, designed experiments and supervised the work.
Conict of interest
The authors declare that there is no conict of interest
that would prejudice the impartiality in this experiment.

Acknowledgments
The authors thank the Director, ICAR-CIRB and Coordinator, all India coordinated research project on nutritional
and physiological interventions for enhancing reproductive performance in animals for providing required
funding for conducting this study and technical staffs of
semen freezing laboratory for assistance in semen collection.
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