Theriogenology 172 (2021) 73e79

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Theriogenology
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Effect of glutathione on pre and post-freezing sperm quality of Indian

red jungle fowl (Gallus gallus murghi)
M.S. Ansari a, B.A. Rakha b, *, S. Akhter c, A. Akhter b, E. Blesbois d, J. Santiago-Moreno e
a
Department of Zoology, Division of Science and Technology, University of Education, College Road, Township, Lahore, 54000, Pakistan
b
Department of Wildlife Management, Pir Mehr Ali Shah Arid Agriculture University Rawalpindi, 46300, Pakistan
c
Department of Zoology, Pir Mehr Ali Shah Arid Agriculture University Rawalpindi, 46300, Pakistan
d
INRA, UMR85 Physiologie de La Reproduction et des Comportements, F-37380 Nouzilly, 37380, Nouzilly, France
e
Department of Animal Reproduction, INIA, Avda. Puerta de Hierro Km 5,9, 28040, Madrid, Spain

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

Article history: During cryopreservation sperm encounter oxidative stress due to higher production of ROS molecules
Received 12 August 2020 and insufficient natural antioxidant defence system. Therefore, present study was designed to identify
Received in revised form the effects of various glutathione (GSH) concentrations on Indian red jungle fowl (Gallus gallus murghi)
29 May 2021
sperm quality and fertility pre-freezing and post-thaw incubation hours. Semen was collected from eight
Accepted 6 June 2021
Available online 8 June 2021
cocks and qualified semen ejaculates having motility >65% were pooled after initial evaluation. Semen
was divided in four aliquots, diluted with red fowl extender (1:5) at 37
C having GSH 0 mM (control),
0.1 mM, 0.5 mM and 1.0 mM, cryopreserved and stored at (196
C) in liquid nitrogen. Semen quality
Keywords:
Indian red jungle fowl
was assessed at post dilution, cooling, equilibration, and freeze-thawing at 0, 2 and 4 h of incubation at
Antioxidant activity 37
C. Sperm motility, plasma membrane integrity, viability, acrosome integrity and mitochondrial
Mitochondrial activity function were recorded highest (P < 0.05) with 0.5 mM GSH in extender at post-dilution, cooling,
Lipid peroxidation equilibration, freeze-thawing and 0, 2 and 4 h of incubation. Lipid peroxidation in sperm and seminal
Acrosome plasma were recorded lowest (P < 0.05) with 0.5 mM GSH during cryopreservation stages and post-
Sperm viability thawing incubation. Moreover, antioxidant activities (total antioxidant potential and free radical scav-
enging capacity) were recorded highest (P < 0.05) in extender having 0.5 mM GSH. Fertility rates were
recorded higher (P < 0.05) with 0.5 mM GSH compared to control. It is concluded that 0.5 mM GSH in
extender improves sperm structural (sperm viability, plasma membrane integrity and acrosome integ-
rity), functional integrity (motility, mitochondrial function) and fertility parameters of Indian red jungle
fowl through enriching antioxidant potential and ameliorating the oxidative stress.
© 2021 Elsevier Inc. All rights reserved.

1. Introduction cryopreservation [4]. Though, natural antioxidant defence system is

Semen cryobanking provides opportunity to conserve threat-
ened species and restoration of the genetic diversity through arti-
ficial insemination [1]. The major challenge in the successful
application of cryobanking is development of cryopreservation
protocols of sufficient efficiency [2]. The success of cryopreserva-
tion protocols for a species depends upon the sperm response and
its ability to encounter thermal, chemical, physical and osmotic
shocks [3]. Furthermore, oxidative stress also becomes a limiting
factor due to overproduction of reactive oxygen species from per-
oxidation of sperm polyunsaturated phospholipids in the process of
* Corresponding author.
E-mail addresses: bushrauaar@gmail.com, arbushra@uaar.edu.pk (B.A. Rakha).
consisting of various antioxidants that encounter the excessive
production of the ROS molecules that cause lipid peroxidation of
the plasma membrane [5]. ROS molecules beyond the physiological
limits have highly deleterious effects on motility, viability, plasma
membrane integrity, acrosome integrity and fertility potential of
spermatozoa [6].
Indian red jungle fowl (Gallus gallus murghi) is native to South
Asia, facing population decline due to hunting, poaching, habitat
destruction and hybridization with the domestic chicken. Cry-
obanking of semen is believed to be an appropriate option for the
management/conservation of captive populations of this precious
sub-species and can be used to maintain pure germplasm for in-
definite period [7]. Alternative methodologies such as primordial
germ cells preservation, oocyte or embryo preservation

https://doi.org/10.1016/j.theriogenology.2021.06.008
0093-691X/© 2021 Elsevier Inc. All rights reserved.
M.S. Ansari, B.A. Rakha, S. Akhter et al.
methodologies are too invasive for at risk populations [1]. However,
negative impacts of semen cryopreservation have been observed on
motility, plasma membrane integrity, sperm viability, and acro-
some integrity in this sub-species [8,9]. Studies indicated that
natural antioxidant system of avian semen is insufficient to protect
the spermatozoa quality and fertility during cryo-induced oxidative
stress [8] due to intrinsic factors viz; less volume of cytoplasm and
seminal plasma [10]. The decline in motility, plasma membrane
integrity, acrosome integrity and mitochondrial membrane po-
tential after freeze-thawing process are associated with elevated
oxidative stress in fowl semen [11,12]. Due to this reason, oxidative
stress is considered one of the major reasons for decline in sperm
quality and fertility [13,14]. It is believed that an appropriate
effective scavenging system consisted of exogenous antioxidant is
required to enhance quality and fertility of avian sperm [15,16].
Glutathione (GSH) is naturally occurring antioxidant in semen
and protects the sperm from ROS-mediated damages by main-
taining balance through glutathione peroxidase/reductase pathway
where GSH serve as substrate [17,18]. The freeze-thawing cycle
reduces the levels of GSH in many species and decrease in GSH
contents is associated with decline in semen quality and fertility
[17,19e23]. It was hypothesized that glutathione in extender may
improve the antioxidant potential, semen quality and fertility of
Indian red jungle fowl sperm. Therefore, present study was planned
to determine the effects of glutathione on Indian red jungle fowl
sperm structural integrity (viability, plasma membrane integrity
and acrosome integrity), functional integrity (motility, mitochon-
drial function), lipid peroxidation (in sperm and seminal plasma),
antioxidant activities (total antioxidant potential and free radical
scavenging capacity) and fertility attributes.
2. Materials and methods
2.1. Experimental birds
Eight mature male Indian red jungle fowl were used in this
study. The animals were housed individually in pens of
106.68 cm  121.92 cm. The birds were offered commercially
available poultry cock breeder feed (100 g/day) and maintained
photoperiod (16 h L: 8 h D) at Avian Research Centre, Pir Mehr Ali
Shah Arid Agriculture University Rawalpindi, Pakistan. The birds
were offered commercially available Islamabad poultry cock
breeder feed and fresh water ad libitum [20].
2.2. Semen collection and evaluation
Semen was collected from individual birds (n ¼ 8) in a gradu-
ated plastic tube. Semen volume was measured using micropipette.
Initial sperm motility of each ejaculate was determined by mixing
10 ml semen samples in 500 ml of phosphate buffer saline (pH 7.2,
300 mOsm/kg). The percentage of motile spermatozoa was deter-
mined by putting a drop of semen sample on a pre-warmed glass
slide (37
C) under phase contrast microscope (x400, Olympus
BX20, Japan). Sperm concentration was measured by taking 1 mL of
semen and 200 ml of formal citrate solution (1 mL of 37% formal-
dehyde in 99 mL of 2.9% (w/v) sodium citrate) with Neubauer
haemocytometer (Marienfeld, Germany) under phase contrast
microscope (x400, Olympus BX20, Japan).
2.3. Extender preparation and cryopreservation
Qualifying semen ejaculates (>65% motility) were pooled, ali-
quoted and diluted (37
C) with glutamate-fructose based medium
(red fowl extender; pH ¼ 7.0; osmolarity 380 mOsm/kg) containing
GSH (w/v) 0.0 mM, 0.1 mM, 0.5 mM and 1.0 mM. The diluted semen
74
Theriogenology 172 (2021) 73e79
(final concentration at 1.72   109 mL) was cooled to 4
C at the
rate of 0.275
C/min, equilibrated for 10 min after the addition of
20% glycerol. Cooled semen was filled in 0.5 mL French straws (IMV,
L'Aigle, France) and kept over liquid nitrogen vapours (5 cm above
the level of LN2) for 10 min, plunged into liquid nitrogen (196
C)
and stored for two weeks. The straws were thawed individually at
37
C for 30 s in water bath and evaluated for semen quality (sperm
motility, plasma integrity, viability and acrosome integrity), mito-
chondrial function (MTT assay), free radical scavenging activity
(DPPH), total antioxidant potential (FRAP) and lipid peroxidation in
sperm and seminal plasma (MDA Assay) at pre-freezing (post
dilution, cooling and equilibration) and freeze-thawing at 0, 2 and
4 h of incubation at 37
C. The whole experiment was repeated/
replicated for five times independently. All chemicals used in this
study were from Sigma-Aldrich, Co., 3050 Spruce street, St Louis,
USA.
2.4. Semen quality assays
2.4.1. Motility
Sperm motility was assessed by placing a drop of semen sample,
previously diluted to 1:5 (v:v) in the red fowl extender on a pre-
warmed (37
C) glass slide under a phase contrast microscope
(400x). Percentage of motile sperm was subjectively evaluated on a
scale ranging from 0 to 100%.
2.4.2. Plasma membrane integrity
Plasma membrane integrity of Indian red jungle fowl sperma-
tozoa was assessed by using hypo-osmotic swelling test [21]. The
HOS solution was prepared by adding 1 g of sodium citrate to
100 mL of distilled water. Previously diluted 25 ml semen was mixed
with 500 ml of a HOS solution (100 mOsm/kg) and incubated at
37
C for 30 min. A drop of incubated solution was placed on a pre-
warmed (37
C) slide and fixed in buffered 2% glutaraldehyde. The
spermatozoa showing swollen heads, swollen and coiled tails were
classified as normal spermatozoa having intact plasma membrane.
A total of 200 spermatozoa were counted at four separate fields
under a phase-contrast microscope (1000x with oil immersion).
2.4.3. Sperm viability
The sperm viability (% live/total sperm) of Indian red jungle fowl
spermatozoa was examined by adding eosin-nigrosin to the lake
glutamate solution. Lake's glutamate solution, prepared by adding
sodium glutamate (17.35 mg), potassium citrate (1.28 mg), sodium
acetate (8.50 mg) and magnesium chloride (0.67 mg) in 100 mL
distilled water. Water soluble nigrosin (5 g) and water soluble
eosin-bluish (1 g) were added into Lake's glutamate solution.
Twelve drops of stain were mixed with 1 drop of semen. A smear
was made on a glass slide, fixed and air dried. A total of 200 sper-
matozoa were assessed per slide under a phase-contrast micro-
scope (1000x with oil immersion). The mixture provides a clear
background in the smear to enhance the contrast of white, un-
stained “live” sperm or the pinkish stained “dead” sperm [7].
2.4.4. Sperm acrosome integrity
The acrosome integrity of Indian red jungle fowl sperm was
evaluated through Giemsa stain. The stain was prepared by adding
Giemsa (3 g) and phosphate buffer saline at pH 7.0 (2 mL) into
35 mL water. Smear was prepared by placing 5 ml of semen sample
on a clean glass slide, air-dried and fixed in neutral formal-saline
(5% formaldehyde) for 30 min. Fixed slides were kept in Giemsa
stain for 1.5 h. Sperm with normal acrosome appeared to be evenly
stained; abnormal spermatozoa were unevenly stained while
spermatozoa having ruptured acrosome were remained unstained.
A total of 200 spermatozoa were observed in at least four separate
M.S. Ansari, B.A. Rakha, S. Akhter et al.
fields under a phase-contrast microscope (x400; Olympus BX20,
Japan) at magnitude1000x with oil immersion [7].
2.5. Total antioxidant activity
Total antioxidant activity was measured by total antioxidant
potential (FRAP assay) and free radical scavenging activity (DPPH
assay).
2.5.1. Total antioxidant potential
Total antioxidant potential of semen sample diluted with GSH
was measured by Ferric reducing power (FRAP) assay. Working so-
lution for FRAP was prepared by adding 2,3,5-Triphenyltetrazolium
chloride [0.031 g TPTZ/40 mL of HCl], acetate buffer [sodium ace-
tate trihydrate (3.1 g), 16 mL of glacial acetic acid/1 L of distilled
water] and ferric chloride [0.054 g/10 mL of distilled water] in ration
10:1:1 respectively. A 3 ml of FRAP reagent was taken as blank and
standard was prepared by mixing 100 mL of ascorbic acid with 3 ml of
FRAP reagent. Aliquotes of 50 mL of semen of control and each of
experimental extender were mixed with 1000 mL of FRAP reagent
and absorbance was taken at 593 nm. FRAP value for each sample
was calculated by the following formulae [22]. Free
radical scavenging activity (mM) ¼ Abs sample/Abs blank x FRAP
value of standard
2.5.2. Ferric radical scavenging activity of spermatozoa
Free radical scavenging activity was measured by DPPH assay as
described by Zhang et al. [23]. For this purpose, sperm pellet was
obtained after centrifugation at 1500 rpm for 10 min and washed
with phosphate buffer saline and again centrifuged at 1500 rpm for
10 min. Sperm pellet was diluted with phosphate buffer saline to
obtain final concentration of 0.058 billion sperm/ml. A 200 mL of
sperm suspension was mixed with 2800 mL of DPPH working so-
lution (3.2 mg DPPH/100 ml of 80% methanol) and shaken vigor-
ously and incubated for 1 h. Blank was prepared by mixing 2800 mL
of methanol in 200 ml diluted sample and 3000 ml of DPPH was
taken as control. Free radical scavenging activity was measured at
537 nm and calculated by following formulae.
Free Radical scavenging Activity ð%Þ ¼ 100
ðAbs sample  Abs blankÞ
 x 100
Abs of control
2.6. Mitochondrial function
Sperm mitochondrial function was assessed by MTT reduction
assay [8]. Semen sample was centrifuged at 1500 rpm for 10 min
and diluted with phosphate buffer saline (pH 7.2 at 25
C) and
sperm pellet was obtained. Stock solution for MTT was prepared by
adding 5 mg of Methylthiazolyldiphenyl-tetrazolium bromide per
mL of phosphate buffer saline. The MTT stock solution (10 mL) was
added to 100 mL sperm suspension (0.058 billion sperm) and MTT
reduction rate was recorded immediately and after 1 h of incuba-
tion at 37
C at 550 nm with spectrophotometer.
MTT reduction rate ¼ Initial optical density-optical density after
1 h of incubation at 37
C
2.7. Lipid peroxidation of sperm and seminal plasma
Lipid peroxidation in sperm and seminal plasma was assessed
by thiobarbituric acid assay (TBA) and malondialdehyde (MDA)
concentration were taken as indices of lipid peroxidation a stable
peroxidation product, in the seminal plasma and spermatozoa as
75
Theriogenology 172 (2021) 73e79
suggested by Partyka et al. [12]. The technique is based on reaction
of a chromogenic reagent, N-methyl-2-phenylindole (NMPI), with
MDA at 45
C. For this purpose, 200 mL of seminal plasma was
mixed with 10 mL of probucol and 640 mL of NMPI (Total Lipid
Liquid, Fardiag, Italy) and vortexed. After vortexing, 150 mL of HCl
was added, mixed, and incubated at 45
C for 1 h. Clear seminal
plasma was obtained by centrifugation at 10,000 g for 10 min. MDA
in the sample was determined by measuring the absorbance at
586 nm. The MDA concentration in the seminal plasma was
expressed as mM/mL and in the spermatozoa as mM/109
spermatozoa.
2.8. Fertility measurements
The fertility attributes of Indian red jungle fowl spermatozoa in
best evolved extender i.e. 0.5 mM GSH and 20% glycerol (control)
was measured by inseminating 50 hens. Glycerol was removed
from control before use in artificial insemination in hen following
procedure suggested by Purdy et al. [24]. Intravaginal artificial in-
seminations were performed at a depth of 4 cm with AI gun (IMV
Technologies, France) having fitted glass pipette with a dose con-
sisted of 270e300 million sperm. The eggs were collected day after
insemination for five days. The eggs were set in incubator at tem-
perature 37
C and relative humidity 86e90%. Eggs fertility was
evaluated by candling after 7 days of incubation. The fertility at-
tributes were calculated with the following formulas:
Total Number of fertile eggs
Fertilityð%Þ ¼ x100
Total Number of eggs laid
Total Number of chicks hatched
Hatchability of fertile eggs x100
Total Number ofFetile eggs
2.9. Statistical analysis
The data (mean ± SEM) on the effect of GSH in diluent on semen
quality (sperm motility, plasma membrane integrity, viability and
acrosome integrity), mitochondrial function, antioxidant activity
(free radical scavenging activity and total antioxidant potential)
and lipid peroxidation in sperm and seminal plasma on pre-
freezing stages (post-dilution, cooling and equilibration) was ana-
lysed by analysis of variance in two factor factorial completely
randomized design using MSTAT-C® and body weight of the birds
were used as covariate (version 1.42, Michigan State University,
East Lansing, MI, USA). The data on the effect of GSH on above
mentioned parameters and post-thaw incubation at 0, 2 and 4 h
was analysed by One-way repeated measures ANOVA. Prior to
analysis, all percentage data were normalized with an arcsine
transformation. Post-hoc comparison between the means was done
though Fisher's protected LSD test. The data on fertility rate was
analysed using t-test with MegaStat® Version 7.25 Mc-Graw-Hill
New Media, New York, for excel.
3. Results
3.1. Effect of GSH on motility, plasma membrane integrity, viability
and acrosome integrity of Indian red jungle fowl spermatozoa
The data on the effect of GSH on sperm motility, plasma mem-
brane integrity, viability and acrosome integrity) at pre-freezing
and post-thaw incubation hours are shown in Table 1e2.
M.S. Ansari, B.A. Rakha, S. Akhter et al. Theriogenology 172 (2021) 73e79

Table 1
Effect of different concentrations of glutathione (0 mM, 0.1 mM, 0.5 mM and 1.0 mM) on pre-freezing stages (Post-dilution, Post-Cooling and Post-equilibration) of cryo-
preservation of Indian red jungle fowl spermatozoa.

Semen Quality and Biochemical parameters Stage of cryopreservation GSH (mM)

(0 mM) 0.1 mM 0.5 mM 1.0 mM

a b a
Sperm Motility (%) Post-dilution 87.3 ± 1.4 82.0 ± 1.6 86.8 ± 2.4 84.3 ± 1.3ab
Post-cooling 76.0 ± 1.6c 72.8 ± 1.7c 75.5 ± 1.4c 73.0 ± 1.9c
Post-equilibration 63.8 ± 1.3d 65.0 ± 2.0d 72.5 ± 1.4c 62.2 ± 2.4d
Sperm Plasma Membrane Integrity (%) Post-dilution 91.3 ± 1.3a 91.3 ± 1.3a 92.5 ± 1.4a 91.3 ± 1.3a
Post-cooling 76.3 ± 2.4c 80.0 ± 2.0bc 83.8 ± 1.3b 81.3 ± 1.3b
Post-equilibration 67.8 ± 3.0d 70.0 ± 1.5d 67.8 ± 1.5d 62.0 ± 1.7e
Sperm Viability (%) Post-dilution 91.5 ± 1.7a 88.8 ± 2.2ab 85.5 ± 1.5bc 91.5 ± 1.6a
Post-cooling 83.8 ± 1.3c 82.5 ± 1.9cd 81.8 ± 1.5cd 84.8 ± 1.3bc
Post-equilibration 64.3 ± 1.5f 74.8 ± 1.9e 78.3 ± 1.2de 84.8 ± 1.3bc
Sperm Acrosome Integrity (%) Post-dilution 89.3 ± 2.0a 84.0 ± 0.9b 83.5 ± 1.0b 91.0 ± 1.5a
Post-cooling 77.8 ± 1.4cd 72.8 ± 1.3e 75.0 ± 1.9de 81.5 ± 1.0bc
Post-equilibration 59.3 ± 1.5f 62.5 ± 1.2f 71.0 ± 1.6e 58.8 ± 3.1f
Total Antioxidant Potential (mM/mL) Post-dilution 1.08 ± 0.03gh 1.14 ± 0.02fgh 1.34 ± 0.06bc 1.28 ± 0.08cde
Post-cooling 1.07 ± 0.03gh 1.17 ± 0.04efg 1.41 ± 0.05abc 1.28 ± 0.07gcde
Post-equilibration 1.06 ± 0.03gh 1.28 ± 0.05cde 1.46 ± 0.04ab 1.26 ± 0.05def
Free Radical Scavenging Activity (mg/mL) Post-dilution 0.040 ± 0.00a 0.030 ± 0.00b 0.025 ± 0.00c 0.029 ± 0.00b
Post-cooling 0.035 ± 0.00d 0.025 ± 0.00e 0.021 ± 0.00f 0.025 ± 0.00e
Post-equilibration 0.031 ± 0.00g 0.021 ± 0.00h 0.015 ± 0.00i 0.020 ± 0.00h
Mitochondrial Function (mM/min/106 Sperm) Post-dilution 0.014 ± 0.00 b 0.014 ± 0.00 b 0.017 ± 0.00a 0.015 ± 0.00 b
Post-cooling 0.011 ± 0.00c 0.012 ± 0.00c 0.015 ± 0.00 b 0.013 ± 0.00c
Post-equilibration 0.010 ± 0.00c 0.011 ± 0.00c 0.013 ± 0.00 b 0.012 ± 0.00c
LPO Sperm (mM/109 Sperm) Post-dilution 1.22 ± 0.03e 1.15 ± 0.02g 1.06 ± 0.01i 1.12 ± 0.01h
Post-cooling 1.24 ± 0.02d 1.26 ± 0.01c 1.12 ± 0.01h 1.13 ± 0.00g
Post-equilibration 1.35 ± 0.02b 1.43 ± 0.05a 1.11 ± 0.00hi 1.18 ± 0.01f
LPO Seminal Plasma (mM/mL) Post-dilution 1.25 ± 0.01e 1.20 ± 0.01f 1.12 ± 0.03h 1.19 ± 0.01g
Post-cooling 1.35 ± 0.02d 1.26 ± 0.01e 1.13 ± 0.01h 1.38 ± 0.03c
Post-equilibration 1.50 ± 0.02a 1.34 ± 0.02d 1.21 ± 0.01f 1.45 ± 0.02b

The values (mean ± SEM) having different superscript differ significantly (P < 0.05) for a given parameter.

Percentages of sperm motility, plasma membrane integrity, and post-thaw incubation at 0, 2 and 4 h. Stage-specific negative
viability and acrosome integrity were recorded highest (P < 0.05) in effects (P < 0.05) were observed on aforementioned parameters
extender containing GSH 0.5 mM compared to 0.1, 1.0 GSH and irrespective of the experimental extenders.
control at pre-freezing (post dilution, cooling and equilibration)

Table 2
Effect of different concentrations of glutathione (0 mM, 0.1 mM, 0.5 mM and 1.0 mM) on post-thaw incubation of 0, 2 and 4 h of Indian red jungle fowl spermatozoa.

Semen Quality and Biochemical parameters Post-thaw incubation GSH (mM)

Control 0.1 mM 0.5 mM 1.0 mM

Sperm Motility (%) 0 h 50.0 ± 2.0de 56.3 ± 2.4bc 63.8 ± 1.3a 55.0 ± 2.0bcd
2 h 37.5 ± 1.4g 50.00 ± 2.0de 59.50 ± 1.7ab 47.50 ± 1.4ef
4 h 30.00 ± 2.0h 42.50 ± 1.4fg 52.00 ± 1.8cde 39.25 ± 0.8g
Sperm Plasma Membrane Integrity (%) 0 h 54.5 ± 0.6b 54.3 ± 1.5b 61.8 ± 1.2a 52.5 ± 1.9b
2 h 40.0 ± 2.0d 46.3 ± 2.4c 57.0 ± 1.2ab 45.8 ± 2.2c
4 h 32.5 ± 1.4e 37.5 ± 3.2de 52.0 ± 1.2b 37.5 ± 1.7de
Sperm Viability (%) 0 h 55.5 ± 1.0b 55.3 ± 1.9b 65.8 ± 1.8a 51.0 ± 2.6bcd
2 h 49.5 ± 0.5cd 48.8 ± 1.3cd 61.3 ± 2.4a 44.0 ± 2.9d
4 h 33.8 ± 2.4e 38.3 ± 1.2e 52.5 ± 1.4bc 36.8 ± 2.4e
Sperm Acrosome Integrity (%) 0 h 49.3 ± 1.8de 55.0 ± 1.7c 65.0 ± 2.2a 52.0 ± 0.9cd
2 h 42.3 ± 2.0f 49.5 ± 2.1de 60.0 ± 2.0b 47.0 ± 1.2e
4 h 31.3 ± 1.3g 41.3 ± 1.3f 52.5 ± 1.4cd 42.0 ± 1.2f
Total Antioxidant Potential (mM/mL) 0 h 1.03 ± 0.03g 1.32 ± 0.04g 1.53 ± 0.06ef 1.23 ± 0.05fg
2 h 1.33 ± 0.02e 1.58 ± 0.02c 1.90 ± 0.02b 1.53 ± 0.01cd
4 h 1.46 ± 0.01d 1.82 ± 0.03b 2.00 ± 0.03a 1.64 ± 0.03c
Free Radical Scavenging Activity (mg/mL) 0 h 0.027 ± 0.00b 0.019 ± 0.00b 0.017 ± 0.00a 0.021 ± 0.00ab
2 h 0.019 ± 0.00b 0.014 ± 0.00ab 0.013 ± 0.00a 0.019 ± 0.00b
4 h 0.012 ± 0.00b 0.012 ± 0.00b 0.011 ± 0.00a 0.012 ± 0.00b
Mitochondrial Function (mM/min/million Sperm) 0 h 0.008 ± 0.00a 0.009 ± 0.00ab 0.011 ± 0.00ab 0.007 ± 0.00ab
2 h 0.006 ± 0.00ab 0.008 ± 0.00bc 0.010 ± 0.00bc 0.006 ± 0.00bc
4 h 0.004 ± 0.00bc 0.007 ± 0.00bc 0.009 ± 0.00c 0.006 ± 0.00bc
LPO Sperm (mM/billion Sperm) 0 h 1.71 ± 0.03f 1.53 ± 0.02fg 1.23 ± 0.01h 1.33 ± 0.01gh
2 h 1.88 ± 0.00b 1.69 ± 0.03c 1.35 ± 0.01e 1.53 ± 0.05d
4 h 2.08 ± 0.05a 1.88 ± 0.00b 1.49 ± 0.03d 1.71 ± 0.03c
LPO Seminal Plasma (mM/mL) 0 h 1.85 ± 0.01e 1.63 ± 0.01e 1.39 ± 0.03d 1.53 ± 0.01e
2 h 1.99 ± 0.01bc 1.88 ± 0.00c 1.69 ± 0.05d 1.89 ± 0.01bc
4 h 2.33 ± 0.07a 2.06 ± 0.03b 1.76 ± 0.07d 2.00 ± 0.01bc

The values (mean ± SEM) having different superscript differ significantly (P < 0.05) for a given parameter.

76
M.S. Ansari, B.A. Rakha, S. Akhter et al.
3.2. Effect of GSH on antioxidant activities (total antioxidant
potential and free radical scavenging capacity) of Indian red jungle
fowl semen
The data on the effect of GSH on antioxidant activities (total
antioxidant potential and free radical scavenging capacity) of In-
dian red jungle fowl semen are presented in Table 1e2 at pre-freeze
stages of cryopreservation and post-thaw incubation hours. The
antioxidant activity was recorded highest (P < 0.05) in extender
having 0.5 mM GSH compared to 0.1, 1.0 GSH and control at pre-
freezing (post dilution, cooling and equilibration) and post thaw
incubation at 0, 2 and 4 h.
3.3. Effect of GSH on mitochondrial function of Indian red jungle
fowl semen
The data on the effect of GSH on mitochondrial function of In-
dian red jungle fowl sperm at pre-freeze stages and post-thaw in-
cubation hours are presented in Table 1e2. The mitochondrial
activity was recorded highest (P < 0.05) in extender having 0.5 mM
GSH compared to 0.1, 1.0 GSH and control at pre-freezing (post
dilution, cooling and equilibration) and post thaw incubation at 0, 2
and 4 h.
3.4. Effect of GSH on lipid peroxidation of Indian red jungle fowl
semen
The data on the effect of GSH on lipid peroxidation in sperm and
seminal plasma of Indian red jungle fowl at pre-freeze stages of
cryopreservation and post-thaw incubation hours are presented in
Table 1e2. Lipid peroxidation levels in sperm and seminal plasma
were recorded lowest (P < 0.05) in extender with 0.5 mM compared
to 0.1 mM, 1.0 mM and control at pre-freezing (post dilution,
cooling and equilibration) and post-thaw incubation at 0, 2 and 4 h.
Lipid peroxidation was increased (P < 0.05) in stage-specific
manner irrespective of experimental extenders.
3.5. Effect of GSH on fertility attributes of Indian red jungle fowl
semen
The data on the effect of GSH on in vivo fertility rate of Indian red
jungle fowl semen is presented in Table 3. Fertility (%) and hatch-
ability of fertile eggs (%) were recorded highest (P < 0.05) with
extender having 0.5 mM GSH compared to control.
4. Discussion
During cryopreservation sperm encounter oxidative stress due
to cryo-induced alterations in sperm, lipid peroxidation and pro-
duction of higher levels of reactive oxygen species (ROS) molecules
[25e27]. In present study, an increase was recorded in total anti-
oxidant potential and free radical scavenging capacity of red jungle
fowl semen cryopreserved with 0.5 mM glutathione in extender.
Table 3
Effect of GSH (0.5 mM) vs Control (0.0 mM) on the fertility of spermatozoa and hatchability in Indian red jungle fowl (n ¼ 50).
Day after insemination
1 2
Fertility (%) Control 63.5 58.1
GSH 68.4 75.8
Hatchability of fertile eggs (%) Control 83.3 83.3
GSH 88.0 93.3
The values (mean ± SEM) having different superscript differ significantly (P < 0.05) for a given parameter.
77
Theriogenology 172 (2021) 73e79
Glutathione is major indigenous regulators of oxidation/reduction
activity in the cell that accords protection from oxidative stress
through various direct and indirect pathways [17,18,28]. It exists in
reduced and oxidized state; ratio of reduced glutathione to
oxidized glutathione is indicator of cellular oxidative stress [29]. In
the absence of oxidative stress, >90% glutathione in cell is found in
reduced form; however, under oxidative stress, oxidized gluta-
thione increases [30]. Higher accumulation of oxidized glutathione
is deleterious to cell, as it induces of apoptosis through activating
SAPK/MAPK pathway [31]. Glutathione can directly scavenge su-
peroxide anion, hydroxyl radical, nitric oxide, carbon radicals and
can catalytically detoxify hydroperoxides, peroxynitrites, and lipid
peroxides [32]. In addition, glutathione is involved in recycling of
the vitamin C and E to cope oxidative stress in the cell [33]. A
decline in glutathione level has been reported during freeze-
thawing process in various species [17,34e37]. A decrease in anti-
oxidant potential that was found insufficient in protecting the
semen quality after freeze-thawing process has also been reported
[6,22,38].
Sperm are rich in mitochondria that are the primary intracel-
lular sites of oxygen consumption and serve as major sources of
reactive oxygen species, most of them originating from the mito-
chondrial respiratory chain [39e41]. It has now been well estab-
lished that mitochondria are directly involved in the regulation of
cell death through ROS mediated oxidative stress [42e44]. Gluta-
thione is naturally synthesized in the cytoplasm, pumped into
mitochondria and have major role in cell antioxidant defence sys-
tem to detoxify oxidants. Mitochondrial glutathione is required in
high concentration to maintain mitochondrial redox balance to
avoid and/or repair oxidative modifications leading to mitochon-
drial dysfunction and cell death [45]. It is relevant to mention that
mitochondrial function is positively associated with sperm motility,
plasma membrane integrity and DNA integrity while inversely
associated with lipid peroxidation in sperm and seminal plasma of
Indian red jungle fowl [6]. In present study, the sperm mitochon-
drial activity was recorded highest (P < 0.05) in extender having
0.5 mM GSH compared to control that is indicative of uptake of GSH
and maintaining a physiological balance combating ROS [46].
Polyunsaturated fatty acids (PUFA) of phospholipids play a
major role in membrane composition and function and are one of
the main targets of the lipo-peroxidative process. It is therefore the
saturation level of fatty acids that determine the ability of sper-
matozoa to maintain equilibrium in an oxidative environment [47].
Higher PUFAs content makes sperm membrane more sensitive to
lipid peroxidation in the presence of ROS [48,49] inevitably pro-
duced in excess during cryopreservation process. ROS are also
produced from abnormal, dead and/or damaged sperm that further
accelerate lipid peroxidation of membrane [2], a cascade of re-
actions that cannot be ceased without the inhibition of oxidation
process [50e52]. Lipid peroxidation in sperm and seminal plasma
negatively affected sperm motility, plasma membrane integrity,
DNA integrity and mitochondrial function in Indian red jungle fowl
[6,22]. In present study, lipid peroxidation in sperm and seminal
Mean ± SEM
3 4 5
48.0 43.9 46.7 52.0 ± 3.7b
75.3 73.5 61.3 70.8 ± 2.7a
85.1 83.3 83.8 83.8 ± 0.3b
92.9 91.7 91.4 91.4 ± 0.9a
M.S. Ansari, B.A. Rakha, S. Akhter et al.
plasma were recorded lowest in extender with 0.5 mM of gluta-
thione. Glutathione might have acted indirectly by partially
reversing the cell membrane damage through desaturation mech-
anism [47]. It is relevant to mention that fatty acid desaturation is
regulated by the presence of selenium, the prosthetic group of
glutathione peroxidase [53]. It is suggested that the administered
glutathione triggers the same enzymatic activities [47].
A physiological balance of ROS is required for normal cellular
functioning, while its overproduction, as encounters during cryo-
preservation process deteriorates sperm structure and function. In
present study, sperm motility, plasma membrane integrity,
viability, were highest in extender having 0.5 mM of glutathione.
This could be attributed to reduced lipid peroxidation levels in the
presence of glutathione that contributed to improved plasma
membrane integrity and viability [12,54,55]. Acrosome integrity of
red jungle fowl sperm with glutathione supplementation in
extender was also improved that supports the fact that lipid per-
oxidation promotes plasma membrane permeability alterations
and loss of control mechanisms required to avoid the premature
acrosome reaction [12,56]. Furthermore, our findings are in line
with the previous reports that demonstrated improvement in
semen quality and fertility by ameliorating antioxidant potential
through glutathione in various species [17,57e61]. In present study,
glutathione in extender improved the fertility outcome of Indian
red jungle fowl semen. It is pertinent to mention that results of
semen quality parameters are supported with fertility outcomes. It
is known that mitochondrial activity assessed through MTT
reduction is positively correlated with fowl sperm ATP content,
sperm mobility, sperm: perivitelline layer interaction and fertil-
izing ability [62]. It is believed that improvement in fertility out-
comes in red jungle fowl after semen supplemented with
glutathione is associated with reduced oxidative stress [32]. Pre-
sent study is an integrative approach in identifying best levels of
glutathione that can ameliorate cryodamages through reducing
oxidative damage, improving mitochondrial membrane potential,
reducing lipid peroxidation levels leading to improved post thaw
quality and fertility rates.
5. Conclusion
It is concluded that 0.5 mM GSH in extender improves sperm
structural (viability, plasma membrane integrity and acrosome
integrity), functional (motility, mitochondrial function) and fertility
parameters of Indian red jungle fowl through enriching antioxidant
potential and ameliorating the oxidative stress.
CRediT authorship contribution statement
M.S. Ansari: Formal analysis, and draft the manuscript. B.A.
Rakha: Experimental design, Sperm collection, sperm analysis,
Formal analysis, and manuscript writing. S. Akhter: Critical review
and proof read the article, Formal analysis. A. Akhter: Red jungle
fowl management, sperm collection, Formal analysis, and draft
manuscript. E. Blesbois: Experimental execution, proof read the
article. J. Santiago-Moreno: Discussion of results, draft the
manuscript.
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