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Soybean lecithin–based extender preserves spermatozoa membrane integrity and fertilizing potential during goat semen cryopreservation

Authors:
Soybean lecithinbased extender preserves spermatozoa
membrane integrity and fertilizing potential during goat
semen cryopreservation
Sara Chelucci
a
, Valeria Pasciu
a
, Sara Succu
a
, Daniela Addis
a
,
Giovanni G. Leoni
a
,
b
, Maria E. Manca
a
, Salvatore Naitana
a
,
b
,
Fiammetta Berlinguer
a
,
b
,
*
a
Department of Veterinary Medicine, University of Sassari, Sassari, Italy
b
Centro di Competenza Biodiversità Animale, Sassari, Italy
article info
Article history:
Received 20 June 2014
Received in revised form 3 December 2014
Accepted 6 December 2014
Keywords:
Comet assay
Acrosin activity
In vitro fertilization
ATP
Malondialdehyde
abstract
Soybean lecithin may represent a suitable alternative to egg yolk for semen cryopreser-
vation in livestock species. However, additional studies are needed to elucidate its effects
on spermatozoa functional properties. Semen collected from ve Sarda bucks was cry-
opreserved in Tris-based extender and glycerol (4% v:v) with different supplementations.
In a preliminary experiment, different soybean lecithin concentrations were tested (1%6%
wt/vol) and results in terms of viability, percentages of progressive motile and rapid
spermatozoa, and DNA integrity after thawing showed that the most effective concen-
tration was 1%. In the second experiment, semen was frozen in a Tris-based extender with
no supplementation (EXT), with 1% lecithin (EXT LC), and 20% egg yolk (EXT EY). The
effectiveness of these extenders was also compared with a commercial extender. The EXT
EY led to the highest viability and motility parameters after freezing and thawing
(P <0.0001). No signicant differences were observed in intracellular ATP concentrations.
Additional molecular features revealed that sperm functionality was affected in EXT EY, as
demonstrated by lower DNA and acrosome integrity (P <0.05), and higher lipid peroxi-
dation compared with spermatozoa cryopreserved in EXT LC (P <0.0001). Results
obtained in the heterologous in vitro fertilization test showed that EXT LC better preserved
spermatozoa functionality, as demonstrated by the higher fertilization rates compared
with the other media (66.2 4.5% for EXT LC vs. 32.7 4.5%, 38.7 4.5%, 39.6 5.2% for
EXT, EXT EY, and commercial extender; P <0.01). The present study demonstrated that
lecithin can be considered as a suitable alternative to egg yolk in goat semen cryopres-
ervation, because it ensures higher fertilization rates and a better protection from mem-
brane damage by cold shock.
Ó2015 Elsevier Inc. All rights reserved.
1. Introduction
Long-term storage of spermatozoa is one the most
important tool to improve the reproductive technologies
in the eld of animal and human medicine [1]. However,
semen cryopreservation leads to biological and functional
changes in sperm cells, which impair their fertilizing
ability. Over the past few years, there has been little
progress in techniques to freeze sperm [2,3]. One of the
features that most directly affect spermatozoa function-
ality is membrane integrity. Sperm cells have three
membranes: plasma and mitochondrial membranes,
*Corresponding author. Tel.: þ39 (0)79 229904; fax þ39 (0)79 229429.
E-mail address: berling@uniss.it (F. Berlinguer).
Contents lists available at ScienceDirect
Theriogenology
journal homepage: www.theriojournal.com
0093-691X/$ see front matter Ó2015 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.theriogenology.2014.12.012
Theriogenology 83 (2015) 10641074
which are involved not only in sperm viability and
motility, respectively, but also in the process of capacita-
tion, and the acrosome membrane for their nal mission
of penetrating the oocyte [4]. These membranes contain a
high concentration of polyunsaturated fatty acids, and
therefore are susceptible to oxidative stress, especially
during the freezing and thawing procedures [5].
Extender solutions are normally supplemented with
avian egg yolk. The main disadvantage in having an animal-
derived material in the constitution of the cryopreservation
media is represented by the sanitary risk of diseases
transmission. This has raised boundaries in international
semen transport legislations of many countries because of
biosecurity issues [6]. Therefore, research efforts are
focusing in nding a well-dened egg yolk substitute of
nonanimal origin to be used in the constitution of semen
cryopreservation media.
Soybean lecithin may represent a suitable alternative to
egg yolk for semen cryopreservation in livestock species.
The precise mechanism by which lecithin exerts its effects
on spermatozoa plasma membrane during freezing/thaw-
ing procedures is not clear. It has been suggested that the
lecithin protection mechanism is because of the replace-
ment of sperm membrane phospholipids, with the reduc-
tion in the freezing point. Alternatively, it may form a
protective lm around the spermatozoon preventing the
formation of intracellular ice crystals and avoiding the
mechanical damage on the sperm membranes [7].
Soybean lecithin has been used successfully to supple-
ment sperm cryopreservation media in several species such
as human [8,9], bovine [10,11], equine [12,13], ovine
[1416], buffalo [17,18], canine [19,20], and goat [21,22].
However, its effects on spermatozoa functional properties
still need to be fully characterized.
This study was designed to determine the proper con-
centration of soybean lecithin to be added to a Tris-based
extender for buck semen cryopreservation, and to assess
its effects on the acrosome, mitochondria, and plasma
membrane functions on DNA integrity and IVF potential. To
carry out the present experiments, we used Sarda bucks as
a model, because semen freezing and thawing procedures
are well established for this specie in our laboratory.
2. Materials and methods
2.1. Chemicals
All chemicals in this study were purchased from Sigma
Chemical Co. (St. Louis, MO, USA) unless stated otherwise.
2.2. Animals and semen collection
All experimental procedures were carried out during
goat nonbreeding season (May-June) at the experimental
facilities of the Department of Veterinary Medicine at
the University of Sassari, Italy (latitude 40
43
0
N). These
facilities meet the requirements of the European Union for
Scientic Procedure Establishments (EU Directive 2010/
63/EU). This study followed ethical guidelines for care and
use of agricultural animals for research. Ejaculates were
obtained by articial vagina from ve adult Sarda bucks
aged 4 to 5 years, maintained in an outdoor environment
and fed a live-weight maintenance ration. Bucks were
kept isolated in separated pens, but with visual contact
between each others. A total of 25 ejaculates (ve ejacu-
lates from each buck) were collected twice a week (two
ejaculates a week from all bucks and on same days) and
were used for this study. Semen was transported to the
laboratory at a controlled temperature of 25
C within
5 minutes after collection, and it was immediately
processed. Volume, concentration, and total sperm output
were recorded for each ejaculate collected. Ejaculates
meeting the following criteria: volume of 1.8 mL or
greater, wave motion of 3 or greater, and sperm concen-
tration of 1.0 10
9
spermatozoa/mL or greater, were
pooled and processed as described subsequently.
2.3. Semen cryopreservation
After being washed in a Tris-based extender (Tris
375 mM, citric acid 124 mM, glucose 41 mM, plus an
antibiotic solution of streptomycin-penicillin, 50
m
g/mL-
50 IU/mL) by two consecutive centrifugations (1500gfor
20 minutes ), semen was diluted up to 400 10
6
sperm/mL
in a Tris-based extender with different supplementations,
as described in the experimental design, and glycerol (4%).
It was cooled to 4
C over a period of 2 hours and equili-
brated for 20 minutes before freezing. Finally, semen was
frozen in pellet form (0.25 mL) on dry ice with a freezing
velocity of 0.7
C/s. In particular, semen drops passed
from a temperature of þ4
Cto80
C in 2 minutes, and
then were plunged into liquid nitrogen and stored in
colour-coded goblets. Thawing was carried out by
plunging a sterilized glass falcon tube containing the
pellet in a 39
C water bath for 20 seconds.
The content of the Falcon tube was then emptied into a
conical tube containing 3 mL of Tris-based extender (Tris
375 mM; citric acid 124 mM; glucose 41 mM, pH 7;
osmolality, 385 mOsm/kg). Semen was centrifuged at
900gfor 3 minutes maintaining a constant temperature
(35
C) to remove the freezing medium, the sperm pellet
was then resuspended in a fresh Tris-based extender, and
aliquots immediately used for all the experimental
procedures.
2.4. Experimental design
To study the effects of the supplementation of soybean
lecithin to the extender used for goat semen cryopreser-
vation we carried out two consecutive experiments.
In a preliminary experiment, we determined the best
concentration of soybean lecithin to be added to the
extender by comparing the following concentrations: 1%,
2%, 3%, 4%, 5%, and 6% (wt/vol). The parameters evaluated
included sperm viability, percentage of progressive motile
and rapid spermatozoa before and after thawing, and DNA
integrity after thawing using a Tris-based extender as
control.
In the second experiment, having found 1% as the best
soybean lecithin concentration, different supplementations
to a Tris-based extender were compared: no supplemen-
tation (EXT), 1% lecithin (EXT lecithin), and egg yolk 20%
S. Chelucci et al. / Theriogenology 83 (2015) 10641074 106 5
(EXT egg yolk). In addition, the effectiveness of these ex-
tenders in protecting buck spermatozoa during the freezing
and thawing procedures was also compared with a com-
mercial extender (EXT CM; OVIXcell IMV Technologies Italy,
Piacenza, Italy). Osmolality and pH did not change among
the tested media, being adjusted to 7.2 and 385 mOsm/kg.
The parameters assessed included viability, percentage of
progressive motile and rapid spermatozoa, ATP intracel-
lular concentration, DNA integrity, acrosin activity, acro-
some integrity, and lipid peroxidation (LPO). To better
determine the functionality of buck spermatozoa after
cryopreservation in the previously mentioned extenders,
we performed a heterologous IVF test using IVM prepu-
bertal ewe oocytes.
2.5. Semen evaluation
The parameters analyzed included different semen
molecular and cellular features. Viability and motility pa-
rameters were measured both before and after thawing.
Adenosine triphosphate intracellular levels, DNA integrity,
LPO, acrosin activity, acrosome integrity, and in vitro
fertilizing ability were measured only after thawing.
2.6. Viability and motility parameters assessment
The eosin-nigrosin solution was prepared as described
by Pintado et al. [23].Briey, 10 g nigrosin was dissolved in
distilled water by boiling and ltered into a cylinder
containing 0.7 g eosin, 7.5 mL of 50 mmol glucose per litre,
and 7.5 mL tartrate phosphate buffer (50 mM Na
2
HPO
4
per
liter, 25 mM KH
2
PO
4
per liter, 77 mM potassium sodium
tartrate per liter), and the volume made up to 100 mL. The
solution was kept at 5
C. Staining was carried out by
mixing an aliquot of spermatozoa suspended in saline
medium with eosin-nigrosin solution (1:3 dilution) for 30
seconds before preparing a smear and drying on a warm
plate at 37
C. At least 200 cells were counted for each
slide.
Sperm motility parameters were assessed using a
computer-assisted sperm analysis system (Sperm Class
Analyzer, S.C.A. v 3.2.0; Microptic S.L., Barcelona, Spain)
with setting of 25 frames acquired to avoid sperm track
overlapping, minimum contrast 10, minimum velocity of
average path 30
m
m/s, progressive motility greater than
80% straightness. This system has a specicsetupforgoat
sperm evaluation. In particular, it was set up as follows:
phase contrast; frame rate, 60 Hz; minimum contrast, 70;
low and high static size gates, 0.6 to 4.32; low and high
intensity gates, 0.20 to 1.92; low and high elongation gates,
7 to 91; default cell size, 10 pixels; default cell intensity, 80.
For each sample, 5
m
L subsample of sperm suspension was
loaded into a prewarmed analysis chamber with a depth
of 10
m
m (Makler Counting chamber; Se-Medical
Instruments Ltd., Biosigma S.r.l., Italy), and a minimum of
500 spermatozoa per subsample were analyzed in at least
four different microscopic elds. Sperm motility was
assessed at 37
Cat40 magnication using a phase
contrast microscope. The percentage of progressive motile
(P motile) and average velocity values of spermatozoa
(% rapid) were evaluated.
2.7. Extraction and measurement of intracellular ATP
Determination of intracellular ATP concentration was
performed by the enzymatic assay as described by Tegge
and Bergmeyer [24]. Briey, 50
m
L of fresh semen and
250
m
L of frozen semen (approximately 1.5 10
9
cells/mL)
were washed twice with 0.1 mL of cold physiological
solution. For the extraction of nucleotides, 0.1 mL of ice-
cold 0.6 M perchloric acid was added to each Eppendorf
tube containing spermatozoa and kept for 15 minutes after
the suspension was centrifuged in an Eppendorf Microfuge
(3 minutes at 6000g) and the supernatant was neutral-
ized with 15
m
L of 3.5 M K
2
CO
3
[25]. After one successive
centrifugation in a Microfuge (3 minutes at 6000g), the
supernatant was analyzed spectrophotometrically with an
enzymatic assay.
Adenosine triphosphate levels were measured spectro-
photometrically at 340 nm using NADH-linked enzyme-
coupled assays. The enzymatic spectrophotometric ATP
assay was carried out at 37
C with a Beckman DU-7
spectrophotometer and performed using the coupling
enzymes, glucose-6-phosphate dehydrogenase and hexo-
kinase. Addition of excess hexokinase (2
m
L from 2 mg/mL)
and glucose-6-phosphate dehydrogenase (2
m
L from 1 mg/
mL) in the presence of excess glucose (8
m
L from 18 mg/mL)
and nicotinamide adenine dinucleotide phosphate
(NADP
þ
)(8
m
L from 20 mg/mL) to perchloric extract (25
m
L)
and to 400
m
L of triethanolammonium chloride buffer
(0.1 M, pH 7.6), the reaction begins and ATP was deter-
mined from the formation of nicotinamide adenine dinu-
cleotide phosphate.
2.8. Assessment of DNA integrity
The level of DNA damage was assessed by single-cell gel
electrophoresis (comet assay). Analysis of the shape and
length of comettail, just like the DNA content in the tail,
gives an assessment of DNA damage. The neutral comet
assay allows the detection of double-strand breaks by
subjecting lysed cell nuclei to an electrophoretic eld at
neutral pH [26], performed according to the method
described by Sakkas et al. [27] with slight modications.
Briey, sperm suspension (30
m
L) was diluted in low
melting point agarose at 37
C (80
m
L; 1% wt/vol). A 100-
m
L
mixture of sperm-agarose was immediately pipetted onto
1% wt/vol normal melting point agarosecoated slides.
Slides were immersed in ice-cold lysing solution (2.5 M
NaCl, 100 mM EDTA, 10 mM Tris, 1% Triton X, and 10 mM
dithiothreitol; pH ¼10) for 1 hour at 4
C. Slides were then
immersed in lysing solution supplemented with proteinase
K(10
m
g/mL). Incubation was performed for 1 hour at 37
C.
After this, slides were rinsed in PBS and then placed in a
horizontal electrophoresis tank lled with freshly prepared
electrophoresis neutral buffer (Tris-acetate-EDTA, pH 7.3).
Electrophoresis was performed at 10 V and 6 mA for
20 minutes. After electrophoresis, the slides were neutral-
ized with Tris-HCl buffer (pH 7.5) for 5 minutes and then
xed in methanol.
Slides were stained with propidium iodide (PI), moun-
ted with a coverslip, and analyzed under an epiuorescence
microscope. Digital comet images were captured with an
S. Chelucci et al. / Theriogenology 83 (2015) 10641074106 6
Olympus microscope equipped with a charge-coupled de-
vice digital camera and Olympus CellF software (Fig. 1).
Fifty comets were measured per replicate sample (i.e., slide
circle) using Comet Score software (TriTek Corp., Sumer-
duck, VA, USA). Scored parameters included percentages of
head and tail DNA (a measurement of the proportion of
total DNA that is present in the comet head and tail).
2.9. Acrosome integrity
Acrosome integrity was evaluated by incubating sper-
matozoa with uorescein isothiocyanateconjugated Pisum
sativum agglutinin (FITC-PSA) [28]. The aliquots of sperm
suspension were incubated for 15 minutes at 39
Cwith
FITC-PSA (5
m
g/mL in PBS, pH 7.4) and PI (14
m
g/mL in PBS,
pH 7.4). To reduce background uorescence, unbound PSA
and PI were removed by adding 200
m
LofPBSandsper-
matozoa were washed by centrifugation in a micro-
centrifuge at 800gfor 2 minutes. The supernatant was
aspirated and the pellet resuspended in 100
m
L of PBS. After
washing, a 10
m
L sample was put on a slide and coverslipped.
The slide was immediately dried by leaving at 37
Cfor
10 minutes for immobilization of sperm cells. To evaluate the
stained sperm cells, at least 200 cells were counted in
duplicate for each sample, using a Diaphot (Nikon, Japan)
epiuorescence microscope. Spermatozoa with intact
plasma membrane and intact acrosome were PI and FITC-
PSA negative, those with intact plasma membrane and
damaged acrosome were PI negative and FITC-PSA positive,
those with damaged plasma membrane and intact acrosome
were PI positive and FITC-PSA negative, and nally those
with damaged plasma membrane and damaged acrosome
were PI and FITC-PSA positive (Fig. 2).
2.10. Acrosin activity
Working solutions for acrosin activity assay were pre-
pared as follows [29]: benzamidine: a peptidase inhibitor
was dissolved in water (0.5 mol/L); N-alpha-benzoyl-DL-
arginine p-nitroanilide (BAPNA): the solution was prepared
by dissolving N-
a
-benzoyl-DL-arginine para-nitroanilide-
HCL in dimethylsulfoxide to a nal concentration of
23 mmol/L. This solution was diluted nine times with BAPNA
extender; BAPNA extender: 0.01% Triton X-100 in 0.055 mol/
L HEPES, 0.055 mol/L NaCl at pH 8.0.
Briey, 4 10
6
frozen and thawed spermatozoa were
added to a tube with BAPNA extender containing BAPNA and
100
m
L of benzamidine solution; they were incubated at 37
C
for 90 minutes [30]. Two or three incubations were per-
formed (one for the control and one or two for the sample).
After incubation, 100
m
L of benzamidine solution was added
only to the sample tubes. All tubes were centrifuged at
1000gfor 15 minutes. Supernatants were collected and
total acrosin activity was measured spectrophotometrically.
N-alpha-benzoyl-DL-arginine p-nitroanilide, when hydro-
lyzed by acrosin, released the chromoforic product 4-nitro-
anilin that was detected (410 nm) on Thermo Electron
Corporation Genesys 10UV spectrophotometer (Thermo
Fisher Scientic, Rodano, Milano, Italy). Acrosin activity was
dened as the quantity of enzyme that hydrolyzes 1
m
mol
BAPNA/min at 23
Cto25
Candspecic acrosin activity was
calculated using the following formula:
Fig. 1. Percentage of viable, progressive motile, and rapid spermatozoa in goat buck semen fresh and cryopreserved in a Tris-based extender supplemented with
different concentrations of soybean lecithin (1%6% wt/vol; EXT, no supplementation). A total of 10 ejaculates collected from ve goat bucks were used. Data are
expressed as the mean SE. Different letters indicate statistical differences in viability and motility parameters among the tested media (ANOVA): P <0.0001.
Activity ð
m
IUÞacrosin106spermatozoa ¼mean Abssample Abscontrol106
½Eincubation time ðminutesÞ=total volumesnumber of sperm ;
S. Chelucci et al. / Theriogenology 83 (2015) 10641074 106 7
in which E¼9.9/mM/cm; incubation time ¼180 minutes;
total volumes 1.2 mL. Abs ¼absorbance.
2.11. Quantication of LPO end products: malondialdehyde
Malondialdehyde (MDA), one of the several low mo-
lecular weight end products of LPO, was evaluated by the
thiobarbituric acid reactive substances assay using thio-
barbituric acid and a spectrophotometric method accord-
ing to the thiobarbituric acid test described by Spanier and
Traylor [31], with some modications. After thawing,
semen was centrifuged for 7 minutes at 7000gand 100
m
L
of supernatant was added to 100
m
L glacial acetic acid 33%,
75
m
L SDS 10%, 100
m
L Tris-HCl 50 mM, pH 7.4, and 250
m
L
thiobarbituric acid 0.75%. The mixture was then incubated
for 1 hour at 100
C and immediately cooled on ice. After
10 minutes, 200
m
L of acetic acid 33% was added and
samples were centrifuged for 20 minutes at 7000g. The
supernatant absorbance was then read with Thermo Elec-
tron Corporation Genesys 10UV spectrophotometer
(Thermo Fisher Scientic), at 535 nm. The values of MDA in
the samples were expressed in micrometer units and
calculated using a standard curve.
2.12. Heterologous IVF test
Ovaries were recovered from prepubertal ewe lambs at
the local slaughterhouse and transported to the laboratory
within 1 hour in Dulbeccos PBS at a temperature between
25
C and 35
C. After washing in fresh medium, ovaries
were sliced using a microblade and the follicle content was
released in tissue culture medium 199 (with Earles salts
and bicarbonate) supplemented with 25 mM HEPES,
penicillin and streptomycin, and 0.1% (wt/vol) of polyvinyl
alcohol. The cumulus-oocyte complexes (COCs) comprised
4 to 10 layers of cumulus cells and oocytes with a uniform
cytoplasm, homogenous distribution of lipid droplets in the
cytoplasm, and with an outer diameter of about 90
m
m
(mean) were selected for the experimental procedure. The
selected COCs, after three washes in the same fresh
Fig. 2. Deoxyribonucleic acid integrity (comet assay) in goat buck spermatozoa after freezing and thawing in Tris-based extenders supplemented with different
concentration of soybean lecithin (1%6% wt/vol; EXT, no supplementation). A total of 10 ejaculates collected from ve goat bucks were used. Data are expressed
as the mean SE. Different letters indicate statistical differences among the tested media (ANOVA): P <0.0000.
S. Chelucci et al. / Theriogenology 83 (2015) 10641074106 8
medium were IVM in tissue culture medium 199 supple-
mented with 10% estrous goat serum, 1 IU/mL ovine FSH,
1 IU/mL ovine LH, and 100
m
M of cysteamine. Cumulus-
oocyte complexes were put in groups of 30 to 35 in
600
m
L of the maturation medium in a four-well Petri dish
(Nunclon; Nalgene Nunc International, Roskilde, Denmark)
layered with 300
m
L mineral oil and cultured for 24 hours in
5% CO
2
in air at 39
C.
After maturation, the COCs were partially stripped of the
cumulus cells and fertilized in vitro at 39
C and 5% CO
2
,5%
O
2
, and 90% N
2
atmosphere in four-well Petri dishes (Nun-
clon; Nalgene Nunc International). Synthetic oviduct uid
(SOF) containing 3% BSA-fraction V supplemented with
25 mm HEPES (sperm-SOF) was used for sperm preparation.
For IVF, SOF medium was supplemented with 10% estrous
goat serum, 20
m
g/mL heparin, and 1
m
g/mL hypotaurine
(IVF-SOF). Percoll gradients were prepared as described by
Rosenkrans et al. [32]. In brief, 100% Percoll solution was
mixed with a 10salt solution (NaCl 2.889 g; KCl 0.238 g;
KH
2
PO
4
0.116 g; CaCl
2
0.112 g; HEPES 0.163 g; 50 mL of Milli-
Q water) to form 90% Percoll solution. A 45% Percoll solution
was prepared from this by addition of an equal volume of
sperm-SOF. The gradient was formed by pipetting 1 mL of
90% Percoll solution into a 15 mL conical tube and then
overlaying it with 1 mL of 45% Percoll solution.
Frozen-thawed semen (500
m
L derived by thawing two
pellets) was placed onto the top of the 45% layer and then
centrifuged at 800gat room temperature for 15 minutes
through the gradient. After removal of supernatant, the
resulting pellet was transferred in a sterilized conical glass
tube below1 mL of warmed IVF-SOFand incubated at 39
Cin
a humidied atmosphere at 5% CO
2
in air for 15 minutes.
Swim-up derived motile spermatozoa were diluted in IVF-
SOF at a 1 10
6
spermatozoa/mL nal concentration and
incubated for 45 minutes at 39
Cunder5%CO
2
,5%O
2
,and
90% N
2
. For IVF, spermatozoa were coincubated under
mineral oil in four-well Petri dishes with a mean of 25
matured oocytes per well in the same atmosphere condition.
After 26 hours, presumptive zygotes were mechanically
denuded of their cumulus cells and stained with 1% lac-
moid in xing solution to evaluate chromatin conguration
[33]. Briey, oocytes were denuded and xed for at least
48 hours with acetic alcohol (1:3). On the day of the eval-
uation, the oocytes were placed on a slide, covered with a
coverslip and stained with 1% lacmoid in 45% glacial acetic
acid. Oocytes showing decondensing sperm chromatin or
pronuclei were classied as fertilized.
2.13. Statistical analyses
Statistical analyses were performed using the statisti-
cal software program Statgraphic Centurion XV (version
15.2.06 for Windows; StatPoint Technologies Inc.,
Warrenton, VA, USA) and a probability of P <0.05 was
considered to be the minimum level of signicance. Dif-
ferences in sperm parameters were studied by performing
an ANOVA analysis. Differences in fertilization rates per
extender were evaluated by performing a
c
2
test. All the
data are expressed as the mean SEM.
3. Results
The preliminary experiment demonstrated that the
soybean lecithin concentration most effective in preserving
buck spermatozoa viability and motility after freezing and
thawing was 1% (wt/vol). As depicted in Figure 1, buck
spermatozoa viability and motility signicantly decreased
after cryopreservation compared with fresh semen in all
the tested extenders (P <0.0001). This decrease was,
however, less marked when soybean lecithin was added at
1% concentration to the Tris-based extender. Deoxy-
ribonucleic acid integrity decreased as soybean lecithin
Fig. 3. Viability and motility parameters (percentage of progressive motile and rapid spermatozoa) in goat buck spermatozoa fresh and cryopreserved in a Tris-
based extender with different supplementation (EXT, no supplementation; EXT EY, egg yolk 20%; EXT LC, soybean lecithin 1%) and in a commercial extender (EXT
CM). A total of 15 ejaculates collected from ve goat bucks were used. Data are expressed as the mean SE. Different letters indicate statistical differences in
viability and motility parameters among the tested media (ANOVA): P <0.0001.
S. Chelucci et al. / Theriogenology 83 (2015) 10641074 106 9
concentration increased, the lowest concentration being
the most effective in protecting buck spermatozoa DNA
(P <0.0000; Fig. 2).
In the second experiment, the effectiveness of soybean
lecithin in protecting buck spermatozoa during the
freezing/thawing procedures was assessed by comparison
with egg yolk and with an EXT CM. The Tris-based extender
supplemented with egg yolk led to the highest viability and
motility parameters after freezing and thawing
(P <0.0001; Fig. 3). When soybean lecithin was used
instead of egg yolk, these parameters decreased signi-
cantly, but motility rates were higher than those recorded
in spermatozoa cryopreserved in an EXT CM.
When assessing spermatozoa molecular parameters after
freezing/thawing in the tested extenders, no signicant dif-
ferences were observed in intracellular ATP concentration,
even if absolute values were higher in spermatozoa cry-
opreserved in the Tris-based extender supplemented with
egg yolk or soybean lecithin (Fig. 4, panel A).
The quantication of the end products of LPO (i.e.,
malondialdehyde) showed that the highest concentration was
produced by spermatozoa cryopreserved in the Tris-based
extender supplemented with egg yolk, followed by those
cryopreserved in an EXT CM (P <0.0001; Fig. 4,panelB).
Deoxyribonucleic acid integrity, as evaluated by the per-
centage of DNA migrating after comet assay, was signicantly
affected when buck spermatozoa were cryopreserved in the
Tris-based extender supplemented with egg yolk (P <0.05;
Fig. 4,panelsCandD).Nosignicant differences were
observed among the other tested extenders.
In the same way, acrosome membrane integrity was bet-
ter preserved in spermatozoa cryopreserved in the Tris-based
extender supplemented with soybean lecithin, as demon-
strated by the higher percentage of spermatozoa showing an
intact acrosome after uorescent staining and by the lower
concentration of acrosin in the freezing media compared with
the other tested media (P <0.05; Fig. 4, panels E and F).
Results obtained in the heterologous IVF test showed
that the Tris-based extender supplemented with lecithin
better preserved spermatozoa functionality, as demon-
strated by the signicantly higher fertilization rates
compared with the other media (P <0.01; Fig. 4, panel G).
4. Discussion
The present study extends our knowledge on the effects
of the addition of soybean lecithin to semen freezing media
on spermatozoa functional properties and in vitro fertil-
izing capacity. Obtained results clearly demonstrate that
the cryopreservation of goat semen in the Tris-based
extender supplemented with soybean lecithin provides a
better protection to sperm cell membranes compared with
the supplementation with egg yolk and to an EXT CM.
Although cellular features, such as viability rates and
motility parameters, appeared to be higher in spermatozoa
cryopreserved in an egg yolk extender, molecular features
revealed that sperm functionality was affected, as
evidenced by lower DNA and acrosome integrity and
greater LPO compared with spermatozoa cryopreserved in
soybean lecithin extender. Moreover, our results demon-
strated that the EXT CM tested in this study was able to
preserve sperm DNA integrity and led to lower LPO
compared with the egg yolk extender, but motility pa-
rameters, and acrosome integrity suggested impaired
sperm functionality.
Finally, the signicantly higher fertilization rates ob-
tained with spermatozoa cryopreserved in a soybean leci-
thin extender compared with the other media conrmed
the more effective preservation of sperm cell function
exerted by soybean lecithin.
In this study, the best concentration of soybean lecithin
to be added to a Tris-based extender was 1%, and this result
conrms similar ndings in goat [21] and other species,
such as ram [34], human [8], and cat [35]. It has been re-
ported that high concentrations of lecithin increase vis-
cosity of extenders and suggested that particular debris in
the extenders could reduce fertility [36]. Therefore,
possibly the optimal amount of lecithin needed to protect
sperm membranes and increase tolerance to the freezing
process, without signicantly increasing extender viscosity,
can be obtained from 1% soybean lecithin.
The results of this study reveal that viability rates and
motility parameters decreased signicantly after cryopres-
ervation in all the tested extenders, but were higher in the
egg yolk extender compared with the others. The EXT CM
led to higher viability and motility rates compared with the
extender alone, but motility parameters were lower than
those recorded in a lecithin extender. Previous studies in
ram and goat semen cryopreservation reported no differ-
ences in viability and motility rates between egg yolk and
lecithinbased extenders [21,34], whereas other authors
found that lecithin-based extenders let to higher viability
rates compared with egg yolkbased extenders [14].Sperm
motility is essential for normal fertilization, and it is
currently the most common parameter of sperm quality,
acting as an indirect measure of metabolic activity and
sperm viability. However, motility parameters proved not to
be reliable factors in the prediction of sperm metabolic
status and IVF ability of cryopreserved goat semen [37].
In this study no signicant differences were found in
ATP intracellular concentration after freezing and thawing
in the tested media. Absolute values tended to be higher in
egg yolk and lecithinbased extenders compared with the
EXT CM and the extender alone, but differences did not
reach statistical signicance. Energy metabolism is a key
factor supporting sperm function. However, in a previous
study in goat we did not nd any predictive value of
spermatozoa energetic status, as evaluated by ATP intra-
cellular concentration before and after thawing, on IVF
outcome [37]. No previous studies reported ATP intracel-
lular concentration in sperm cell after cryopreservation
with lecithin-based extenders, but other authors exam-
ined mitochondrial activity in ram. Emamverdi et al. [14]
reported no signicant difference in the percentage of
live spermatozoa with active mitochondria, as evaluated
by ow cytometry analysis, between lecithin and egg
yolkbased extenders. Another study found that lecithin
induced serious mitochondrial damage that clearly
affected the inner mitochondrial membrane, and conse-
quently, sperm motility [38], but it used a higher lecithin
concentration (3.5%) and did not investigate sperm
fertilizing potential.
S. Chelucci et al. / Theriogenology 83 (2015) 106410741070
The susceptibility of ruminant spermatozoa to
oxidative stress is a consequence of the abundance of
polyunsaturated fatty acids in sperm plasma mem-
brane, the presence of which gives the membrane
uidity and exibility needed to engage in membrane
fusion events associated with the fertilization.
Unfortunately,thepresenceofdoublebondsinthese
molecules makes them vulnerable to free radical attacks
and the initiation of LPO cascade. These attacks ulti-
mately lead to the impairment of sperm function
through oxidative stress and the production of cytotoxic
aldehydes, such as MDA [39].
Fig. 4. Adenosine triphosphate (ATP) intracellular concentration (A), malondialdehyde (MDA) concentration (B), DNA integrity (comet assay), as evaluated by the
percentage of DNA in the head (C) and in the tail (D), and acrosome integrity, as evaluated by the percentage of spermatozoa showing intact acrosome after
uorescent staining (E) and by the concentration of acrosin in the media (F) in buck spermatozoa after freezing and thawing in Tris-based extenders with
different supplementations (EXT, no supplementation; EXT EY, egg yolk 20%; EXT LC, soybean lecithin 1%) and in a commercial extender (EXT CM). Panel (G)
shows the percentages of fertilized oocytes after coincubation of IVM prepubertal ewe oocytes with buck spermatozoa cryopreserved in the previously described
extenders. A total of 15 ejaculates collected from ve goat bucks were used. Data are expressed as the mean SE. Different letters indicate statistical differences
among the tested media (ANOVA): panels (B) and (G): P <0.01; panels (CF): P <0.05. sptz, spermatozoa.
S. Chelucci et al. / Theriogenology 83 (2015) 10641074 1071
In the present study, the lowest concentration of MDA
was found in the lecithin-based extender and extender
alone, whereas the highest values were found in the egg
yolkbased extender. These results are in agreement with
recent studies in goat [21] and bull [40], which reported
that in egg yolkbased extenders the production of MDA
was signicantly increased compared with lecithin-based
extenders. Egg yolk contains high levels of unsaturated
fatty acid susceptible to LPO. Regardless of the source
(polyunsaturated fatty acids from sperm cell membranes,
from extenders, or both), LPO induced by reactive oxygen
species not only disrupts sperm motility, but also impairs
all the sperm functions, which are dependent on the
integrity of plasma membrane, including sperm-oocyte
fusion and ability to undergo acrosomal exocytose [41].
Lipid peroxides are also capable of inducing DNA damage
and decrease in fertility during storage of semen [42].
In the present study, the high levels of LPO end products
(MDA) found in the egg yolkbased extender were indeed
associated with DNA fragmentation. Among other sperm
tests, evaluation of DNA integrity has been considered
important as early embryo development depends on the
presence of normal DNA. After cryopreservation, sperma-
tozoa are particularly susceptible to DNA damage [43].
Therefore, the assessment of DNA integrity is of high value
in determining frozen/thawed semen quality. In a previous
study, we reported that DNA integrity, as evaluated by a
high sensible method such as the comet assay, has a high
predictive value on IVF outcome with frozen/thawed goat
semen [37]. In the present study, the better preservation of
DNA integrity in spermatozoa frozen in the soybean leci-
thinbased extender could be explained by a lower pro-
duction of lipid peroxides and by higher membrane
integrity. Lecithin is a naturally occurring mixture of
phosphatidylcholine with diverse fatty acid side chains
such as stearic, oleic, and palmitic acids. Such fatty acids,
the prevailing phospholipids in most of mammalian bio-
logical membranes, are known to confer structural stability
to cells [44]. Although the precise mechanism by which
lecithin exerts its effects on the plasma membrane of
spermatozoa during the freezing/thawing process is not
clear, it has been suggested that lecithin in soybean protects
sperm membrane phospholipids by occupying sites on the
plasma membrane and increases tolerance to the freezing
process [5]. The disruption of plasma membrane integrity
caused by disarrangement of lipids within the membrane
during cryopreservation may induce further cellular dam-
age and consequently lead to sperm death.
The better preservation of membrane integrity by
soybean lecithin compared with the egg yolkbased
extender is supported by higher rates of viable spermato-
zoa with intact acrosome and lower acrosin activity in
sperm cell extract. The acrosome is a Golgi-derived
organelle that stores enzymes necessary for sperm pene-
tration. Acrosin is one of these distinctive proteases that, as
a typical serine endoprotease with trypsin-like activity, is
stored in its inactive zymogen form (called proacrosin).
After the acrosome reaction, proacrosin is converted to
alpha-acrosin, which remains bound to the acrosome.
Alpha-acrosin is then autocatalytically converted into beta-
acrosin that is released to the extracellular milieu [45].
Proacrosin activation strongly relies on intra-acrosomal pH
[46]. Thus, it is possible that plasma membrane damage in
frozen/thawed spermatozoa allows an increase in the
intracellular pH, which in turn promotes proacrosin acti-
vation [47]. Thus, the higher acrosin activity in the egg
yolkbased extender and extender alone would suggest
membrane damage, as conrmed by the lower rates of
viable spermatozoa with intact acrosome. This result could
be explained by the presence of high-density lipoproteins
in egg yolk, which are regarded as one factor that decreases
the quality of semen by causing efux of cholesterol from
the sperm plasma membrane and resulted in the change in
uidity that increases the sensitivity to cold shock [48].
Results of the heterologous IVF test support this hypothesis.
Spermatozoa cryopreserved in the lecithin-based extender
lead to signicantly higher fertilization rates compared with
the other tested media. It has been reported that lecithin in-
creases sperm binding to the zona pellucida, probably by the
reduction of the cholesterol to phospholipid molar ratio
[49,50] that results in increased uidity of the membrane,
whichinturnallowstransmembraneorsurfaceproteinsand
antigens to relocate over the plasma membrane [51].In vitro
fertilization has been reported to be one of the most adequate
parameters for semen fertility prediction because it evaluates
the spermatozoa-oocyte interactions occurring during in vivo
fertilization [52].
4.1. Conclusions
The present study demonstrated that soybean lecithin
not only can be considered as a suitable alternative to egg
yolk in goat semen cryopreservation, but also ensures a
better protection from membrane damage by cold shock, as
demonstrated by the higher DNA and acrosome integrity,
and higher fertilization potential. The lower semen quality
of spermatozoa frozen in the egg yolkbased extender may
be explained by the presence of high-density lipoproteins,
which are regarded as one factor that increases the
sensitivity to cold shock.
Acknowledgments
This study was supported by Autonomous Region of
Sardiniadspecial project Biodiversity (MIGLIOVIGENSAR).
S.S. was supported by a grant from Fondazione Banco
di Sardegna (Decreto n. 400, prot. 3943, Università degli
Studi di Sassari).
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/
j.theriogenology.2014.12.012.
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... It is worth mentioning that in the current study, there was also a conflicting result between two different assays evaluating a similar parameter. It is well known that many studies use the parameter 'viability' for the result of eosin-nigrosin staining (Chelucci et al. 2015;Salmani et al. 2014). In addition, the output of phosphatidylserine (PS) translocation assay is four sperm category which one of them is live spermatozoa. ...
... It is worth noting that a restriction suggested for traditional micelles is that they have just only monolayer determining its solubility, although breaking these structures down to nanoparticles can improve their biodistribution. However, structures having additional amphiphilic polymers, such as liposomes, results in better stability and distribution (Chen et al. 2016 (Chelucci et al. 2015). Semen cryopreservation can cause structural alterations in sperm DNA and induce DNA fragmentation (Fraser et al. 2011). ...
... The destruction of lipids in the membrane during cryopreservation causes the destruction of the plasma membrane integrity, which may lead to further cell damage, resulting in sperm death. (Chelucci et al. 2015). ...
Article
This study was to evaluate the effects of two different ultrastructures of lecithin including nanoparticles (NPE mostly nanomicelles) and lecithin nanoliposome (NLE) with egg yolk extender (EYE) on goat sperm cryopreservation. Semen samples were collected from 6 goats, then pooled, diluted and then frozen. Motility and motion parameters, plasma membrane integrity and functionality, morphology, apoptosis status (Annexin V-PI), acrosome integrity, DNA fragmentation and in vitro fertilisation were assessed. Total motility and most motion parameters were higher in EYE (p < .05) compared with the two lecithin extenders, while there were no significant differences between NLE and NPE. NLE and NPE had higher values for viable spermatozoa (Annexin V-PI) (p < .05) compared with EYE. The highest value for dead spermatozoa was observed in EYE (p = .08). A higher percentage of DNA fragmentation (p < .05) was detected in EYE compared with NPE. Plasma membrane integrity and functionality, morphology, acrosome integrity and fertility of spermatozoa indicated no significant differences between extenders. Data suggested that ultrastructural changes of lecithin (micelles versus. liposome) could not improve the sperm cryosurvival of goat spermatozoa. Moreover, we cannot also claim that lecithin-based diluent supplies better protection compared with the egg yolk in goat.
... This concentration of lecithin was higher than our soybean lecithin concentration. Chelucci et al. (2015) reported that the best concentration of soybean lecithin was 1%. Salmani et al. (2014) reported that optimal soybean lecithin concentration in tris extender was 1.5% for motility during cryopreservation of caprine semen. ...
... These concentrations are compatible with our study. Previous studies suggested that soybean lecithin extender provided better protection in goats (Jiménez-Rabadán et al., 2012;Vidal et al., 2013;Salmani et al., 2014;Chelucci et al., 2015;Lekshmi Bhai et al., 2015). Soybean lecithin contains antioxidant composites as glutathione, which helps to the reduction of lipid peroxidation and inhibition of MDA generation during semen processing (Salmani et al., 2013). ...
Article
The objective of this study was to evaluate the effect of supplementation of different concentrations of royal jelly (RJ) in Tris-egg yolk (TEY) and Tris-soybean (TSL) extenders on sperm quality of Damascus buck chilled semen. The ejaculates were collected from five bucks using an electroejaculator twice a week during the reproductive season. Semen samples were pooled and diluted in extenders supplemented with RJ (1%, 0,75%, 0,50%, 0,25%) with a final concentration of 150x10 6 spermatozoa/mL and stored for 72 hours. The samples were evaluated for sperm quality parameters, including motility, viability, abnormality, membrane integrity, pH, osmolarity. When the RJ doses were compared among each other, 1% RJ concentration caused an increase in the percentage of abnormal and dead spermatozoa in the TSL extender. In the TEY extender, the control and 0.25% RJ concentration groups had the lowest motility and viability values. Generally, the addition of medium and high doses of RJ in the TEY extender group was found to be beneficial to sperm motility and plasma membrane integrity. Although motility was terminated at the 48th hour in the TEY control and 0.25% RJ groups, other trial groups continued. In conclusion, it was determined that the TSL extender was superior to the short-time storage of the goat spermatozoa in the between-groups evaluation, and the RJ supplementation did not achieve any success during the liquid storage at 4°C in the TSL extender. RESUMEN El objetivo de este estudio fue evaluar el efecto de la suplementación de diferentes concentraciones de jalea real (RJ) en los diluyentes Tris-yema de huevo (TEY) y Tris-soja (TSL) sobre la calidad del semen refrigerado de machos cabríos Damasco. Los eyaculados se recogieron dos veces por semana durante la temporada reproductiva de cinco machos utilizando un electroeyaculador. Las muestras de semen se agruparon y diluyeron en TEY y TSJ suplementados con RJ (1%, 0,75%, 0,50%, 0,25%), a una concentración final de 150x10 6 espermatozoides/ml y se almacenaron durante 72 horas. Las muestras fueron evaluadas para parámetros de calidad como motilidad, viabilidad, anormalidad, integridad de la membrana, pH y osmolaridad. Cuando las dosis de RJ se compararon entre sí, la concentración de RJ al 1% causó un aumento en el porcentaje de espermatozoides anormales y muertos en el extensor de TSL. En el diluyente TEY, el control y los grupos de concentración de 0,25% RJ tuvieron los valores más bajos de movilidad y viabilidad. En general, se encontró que la adición de dosis medias y altas de RJ en el grupo TEY fue beneficiosa para la motilidad de los espermatozoides y la integridad de la membrana plasmática. Aunque la motilidad se terminó a las 48 horas en el control TEY y en los grupos con 0,25% de RJ, en los otros grupos de prueba la motilidad continuó. En conclusión, se determinó que el diluyente TSL fue superior para el almacenamiento a corto plazo de los espermatozoides de macho cabrío en la evaluación entre grupos, y que la suplementación con RJ no logró ningún éxito durante el almacenamiento de líquido a 4°C en el diluyente TSL. source are credited / Este es un artículo de acceso abierto distribuido bajo los términos de la Licencia Creative Commons Attribution, la cual permite el uso irrestricto, la distribución y reproducción en cualquier medio, dando el crédito correspondiente al autor y la fuente original.
... One of the disadvantages of the previous studies comparing various factors likely to affect cryo-survival, such as egg yolk-and non-egg yolk-containing extenders, or semen collection method, or timing of season collection, is that the fertilizing capacity of the thawed sperm samples was not determined, except in the studies by Dorado et al. [2], Chelucci et al. [6] and Nadri et al. [8]. Interestingly, Dorado et al. [2] found that the effect of these various factors was different for semen samples that achieved a higher pregnancy rate than those that were less successful. ...
... Interestingly, Dorado et al. [2] found that the effect of these various factors was different for semen samples that achieved a higher pregnancy rate than those that were less successful. Chelucci et al. [6] demonstrated that the ability of buck spermatozoa to fertilize sheep oocytes was higher when frozen in 1% lecithin than in egg yolk-containing medium, despite having better motility and membrane integrity in the egg yolk medium. Nadri et al. [8] incorporated an IVF study in their comparison of nano-lecithin, lecithin and egg yolk-based media for buck spermatozoa. ...
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Although several protocols for cryopreserving buck semen are described in the literature, they differ widely in factors such as season and method of semen collection, extender and sperm concentration. Therefore, choosing a protocol that is suitable for a particular on-farm situation can be problematic. In the present study, semen was collected by artificial vagina from seven bucks on a farm located approximately 90 minutes’ drive away from the laboratory, about 6 weeks before the start of the goat breeding season. The semen was immediately extended in warm semen extender containing soy lecithin and was placed in an insulated box with a cold pack for up to 4 h, during semen collection from the remaining bucks and subsequent transport to the laboratory. Following centrifugation at 4 °C and resuspension in the soy lecithin extender to a sperm concentration of 800 × 106 spermatozoa/mL, 0.25 mL plastic straws were filled and frozen in racks 4 cm above the surface of liquid nitrogen. This simple protocol resulted in an acceptable post-thaw quality for all seven bucks, with a mean post-thaw motility of 55 ± 21% and mean fragmented chromatin of 3.27 ± 1.39%. Normal sperm morphology was >90% in all ejaculates. The semen was sent to a gamete bank for long-term storage.
... Spermatozoa have three types of membranes, which are plasma membrane, mitochondrial membrane, and acrosomal membrane. The membranes contain polyunsaturated fatty acids and hence are very susceptible to oxidative stress, especially during freezing procedures (Chelucci et al., 2015). In all mammals, the capacitation and the subsequent acrosome reaction of spermatozoa represent essential steps for successful fertilization and formation of a zygote. ...
Article
Flow cytometric evaluation of acrosomal integrity of buffalo spermatozoa at different stage of cryopreservation for semen diluted with different extenders
... On spermatozoa, the plasma membrane, mitochondrial membrane, and acrosomal membrane contain polyunsaturated fatty acids, and hence, they are very susceptible to OS, especially during freezing procedures [44]. OS is one of the factors that increase cell damage due to ROS. ...
Article
Background and Aim: Lepidium meyenii Walp (Maca) is an herbaceous plant that grows in the Peruvian Andes and it has been widely used as a nutritional supplement and fertility enhancer and has been used in the treatment of a variety of diseases, such as rheumatism, respiratory disorders, and anemia. The most notable feature of Maca is its potent antioxidant capacity, which helps in the scavenging of free radicals and protection of cells from oxidative stress. This study aimed to evaluate the in vitro effect of Maca extract on thawed sperm cells from bulls. Materials and Methods: Three dilutions of 1, 10, and 100 µg/mL of Maca extract were incubated with frozen–thawed bovine semen and analyzed at 1, 3, and 24 h of exposure time, evaluating the activity of the extract on the DNA, motility, morphology, viability, integrity of the membrane and acrosome of spermatozoa. Results: The Maca extract improved the studied sperm parameters of motility, acrosome integrity, vitality, and DNA integrity of sperm cells at a concentration of 10 µg/mL, and at 1 µg/mL, an improvement was observed in the morphology and integrity of the membrane. However, the best activity of the Maca extract was observed on the DNA integrity of the sperm, which was effective at the three concentrations evaluated after 24 h of incubation. Conclusion: The results indicate that L. meyenii can help in maintaining spermatozoa cellular integrity after the frozen–thaw process, especially in the protection against DNA fragmentation. Therefore, Maca would be a feasible supplementation to protect sperm to maintain their fertile ability after thawing.
... Moreover, the need to improve the reproductive efficacy of breeding with cryopreserved semen could involve using better freezing methods to improve post-thaw sperm quality. Sperm cells undergo biochemical and functional changes due to longterm spermatozoa storage, limiting their ability to fertilize [5]. e acrosome, nucleus, mitochondria, axoneme, and plasma membrane are also affected by rapid temperature changes, such as cold shock and the creation and dissolution of ice during the freezing-thawing process [6,7]. ...
Article
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Germplasm storage and transportation in artificial insemination (AI) and other advanced technologies are facilitated by cryopreservation. In reproduction, the cryopreservation of sperm allows it to be transported across vast distances and used even after the sire’s death. However, the technique of cryopreservation might damage sperm and limit their activity. Several cryobiological investigations have reported that the integrity of the sperm membrane is frequently involved in the physical and biological elements that affect sperm survival at low temperatures during the cryopreservation process. However, successful cryopreservation of ram sperm is still a work in progress because a considerable percentage of sperm do not survive the freezing and thawing process. Sperms are destroyed during cryopreservation of semen due to varying concentrations of cryoprotective chemicals and if semen is not cooled at optimal cooling rates. Hence, it is crucial to know the optimum cooling rates with freezing and thawing protocols for maximum recovery of viable and functional sperm cells for a successful cryo-freezing of ram spermatozoa. Therefore, the current study compiled and compared the research on the impact of different cryopreservation procedures, cooling rates, equilibration time, and thawing protocols on post-thaw ram semen quality.
... Sperm has three membrane layers, the plasm, mitochondrial and acrosome. each layer consists of high polyunsaturated fatty acids, thus easily susceptible to oxidative stress in the freezing process (Chelucci et al., 2015). Mitochondrial damage effects are the most detrimental, in which the sperm motility decreased with the slightness of ATP. ...
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Certain factors may affect sperm motility and integrity in a freezing process. Reactive Oxygen Species (ROS) is essential in damaging the sperm, as the previous study revealed that ROS is a major cause damaging the membrane of spermatozoa. This study aimed to understand ROS production and its interaction within the motility, mitochondrial activity and DNA damage to the cryopreservation process in Indonesian Ongole grade bull. A total of 40 ejaculated semen samples from four different bulls had taken. The ROS intracellularly detected by 2-7-Dichlorodihydrofluorescein Diacetate (DCFH-DA) and propodium iodide (PI). Mito Tracker Red kit was being used to discover the mitochondrial activity, while DNA sperms' integrity was detected by Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL). Examinations were done under a fluorescence microscope. Results of the study demonstrated a significant increase in ROS from fresh semen, diluted or equilibrated before being frozen (at 12°C and 4°C). ROS production showed no differences between frozen-thawed semen and thawed semen before the freezing process. A significant decrease in thawed semen had shown in membrane integrity and mitochondrial activity due to the equilibration process in which the temperature was significantly higher at 12°C and 4°C than the fresh semen temperatures, however, the diluting and freezing process had not given any significant causes to sperm DNA damage. This study concluded that ROS production with its effects might be the main factor that leads to the decreasing of sperm motility and integrity but no effect was discovered on DNA damage as the diluting and freezing process occurred. The results have proven the previous study which stated ROS has different mechanisms in affecting membrane integrity and DNA integrity. It also comes to the new theory that ROS might be affected by the chemical diluter despite the temperature of the freezing and thawing process.
... There is a desire to increase the use and viability of frozen semen doses for small ruminants. However, as in other species, there is currently an association with frozen semen and lower fertility rates in small ruminants even when laparoscopic AI is employed [25]. Sexed semen is beginning to be commercialized for small ruminants for the first time due to a collaborative effort between Animal Breeding Europe and Cogent that began in August of 2020 [26]. ...
Article
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Artificial insemination of livestock has been a staple technology for producers worldwide for over sixty years. This reproductive technology has allowed for the rapid improvement of livestock genetics, most notably in dairy cattle and pigs. This field has experienced continuous improvements over the last six decades. Though much work has been carried out to improve the efficiency of AI, there are still many areas which continue to experience improvement, including semen analysis procedures, sperm selection techniques, sperm sexing technologies, and semen storage methods. Additionally, the use of AI continues to grow in beef cattle, horses, and small ruminants as the technology continues to become more efficient and yield higher pregnancy rates. In this review, AI trends in the various livestock species as well as cutting edge improvements in the aforementioned areas will be discussed at length. Future work will continue to refine the protocols which are used for AI and continue to increase pregnancy rates within all livestock species.
... The sperm comet assay has also been used in pets, namely cats and dogs for the detection of DNA damage in their sperm induced by X-rays as well as for assessing semen quality [149,150]. The sperm comet assay was also conducted on several other domestic animals such as horses [151][152][153][154][155][156][157], donkeys [151,158], bulls [159][160][161][162][163][164], goats [165,166], sheep [167][168][169] and boars [170][171][172] to test semen quality for cryopreservation and artificial insemination. ...
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DNA integrity is considered an important parameter of semen quality and is of significant value as a predictor of male fertility. Currently, there are several methods that can assess sperm DNA integrity. One such assay is the comet assay, or single-cell gel electrophoresis, which is a simple, sensitive, reliable, quick and low-cost technique that is used for measuring DNA strand breaks and repair at the level of individual cells. Although the comet assay is usually performed with somatic cells from different organs, the assay has the ability to detect genotoxicity in germ cells at different stages of spermatogenesis. Since the ability of sperm to remove DNA damage differs between the stages, interpretation of the results is dependent on the cells used. In this paper we give an overview on the use and applications of the comet assay on mature sperm and its ability to detect sperm DNA damage in both animals and humans. Overall, it can be concluded that the presence in sperm of significantly damaged DNA, assessed by the comet assay, is related to male infertility and seems to reduce live births. Although there is some evidence that sperm DNA damage also has a long-term impact on offspring’s health, this aspect of DNA damage in sperm is understudied and deserves further attention. In summary, the comet assay can be applied as a useful tool to study effects of genotoxic exposures on sperm DNA integrity in animals and humans.
Article
This study investigated the cryoprotectant effects of dimethylformamide (DMF), ethylene glycol (EG), and dimethyl sulfoxide (DMSO) as substitutes for glycerol (GLY) in a soybean lecithin (SL)-based extender in the cryopreservation of buck sperm. In this study, the semen of three Saanen bucks was individually extended in SL supplemented with 5% GLY (control), DMF, EG, or DMSO. After this, the extended semen was cryopreserved and two straws from each group were thawed (37°C for 30 seconds), pooled, and analyzed for sperm motion parameters, plasma membrane integrity (PMI), acrosomal integrity (ACI), and high mitochondrial membrane potential (HMMP). Samples were analyzed after 15 minutes (T0) and after 2 hours of incubation at 37°C (T2). The results revealed higher values of motility (total and progressive) and sperm motion parameters for DMF than the other cryoprotectants (p < 0.0001). PMI and HMMP did not differ (p > 0.05) between GLY and DMF, but ACI was higher (p < 0.01) for DMF compared with GLY. Based on these results, DMF and GLY samples were used in heterologous in vitro fertilization assays by using bovine oocytes (n = 337) obtained from a slaughterhouse. No differences (p > 0.05) were observed between GLY and DMF for unfertilized (GLY: 38.8%; DMF: 25.33%), pronucleus (GLY: 25.68%; DMF: 27.92%), and cleavage rates (GLY: 35.52%; DMF: 46.75%). Based on these results, it is concluded that DMF preserves sperm motion characteristics and ACI better than GLY, EG, and DMSO, and it is the penetrating cryoprotectant of choice for the cryopreservation of buck sperm in SL extender.
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Soybean lecithin is a suitable plant-based cryoprotectant for freezing ruminant sperm. Optimum level of lecithin was not clear for goat semen cryopreservation. The objective of this study was to investigate the effects of different levels of soybean lecithin in semen extender on post-thaw sperm quality including CASA-motion parameters, viability, plasma membrane integrity and lipid peroxidation. Semen samples were collected from 4 Mahabadi bucks using an artificial vagina. Different concentrations of soy lecithin (SL, 0.5, 1, 1.5, 2 and 2.5% w/v) were compared to 15% (v/v) egg yolk-based extender (TR-EY). No significant difference was observed for sperm progressive motility, viability or plasma membrane integrity in 1.5% SL media (33.8%, 66%, and 62.7%, respectively) and TR-EY medium (35.4%, 67.2%, and 64.9%, respectively). Sperm motion characteristics (VAP, VSL, VCL, ALH and LIN) and rapid spermatozoa were improved with extender containing 1 and 1.5% SL, compared to TR-EY extender. Furthermore, egg yolk produced significantly higher malondialdehyde (4.02±0.21) than other groups. Results suggest that the optimal lecithin concentration in the semen extender was 1.5% and also soy lecithin can substitute for egg yolk during cryopreservation for caprine sperm.
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The objective of the present study was to compare the quality of frozen buffalo semen processed in tris-citric egg yolk extender with two different commercially available soybean lecithin-based extenders. Split pooled ejaculates, possessing more than 70 % visual sperm motility were divided in three aliquots and diluted in AndroMed®, Bioxcell®, or tris-citric egg yolk (TCE) extenders. Post-thaw motion characteristics, viability, plasma membrane integrity, acrosome morphology, and DNA integrity of buffalo sperm were studied after thawing and incubation for 4 h. Results demonstrate that total motility in frozen–thawed semen processed in TCE extender were similar when compared with AndroMed® or Bioxcell® extenders (P > 0.05). Kinematic parameters, such as average path velocity and linearity index in the soybean lecithin-based extenders were comparatively superior to TCE extender. After 4 h of incubation, proportions of overall and progressive motility decreased in all extenders but were comparatively superior in soybean lecithin-based extenders than TCE extender (P < 0.05). The proportion of post-thaw sperm viability, plasma membrane integrity and normal apical ridge remained similar in all extenders (P > 0.05). The post-thaw sperm viability was comparatively superior in soybean lecithin-based extenders than TCE after incubation at 37 °C for 4 h (P < 0.05). The type of extender had not been the significant effect on the percentage of spermatozoa with DNA damage after thawing and incubation for 4 h. In addition, our results suggest that soybean lecithin base extenders can be used as a substitute for tris-citric egg yolk extender in cryopreservation of buffalo semen.
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The purpose of the present study was to investigate the effects of a chemically defined soybean lecithin-based semen extender as a substitute for egg yolk-based extenders in ram semen cryopreservation. In this study, 28 ejaculates were collected from four Zandi rams in the breeding season and then pooled together. The pooled semen was divided into six equal aliquots and diluted with six different extenders: (i) Tris-based extender (TE) containing 0.5% (w/v) soybean lecithin (SL0.5), (ii) TE containing 1% (w/v) soybean lecithin (SL1), (iii) TE containing 1.5% (w/v) soybean lecithin (SL1.5), (iv) TE containing 2% (w/v) soybean lecithin (SL2), (v) TE containing 2.5% (w/v) soybean lecithin (SL2.5) and (vi) TE containing 20% (v/v) egg yolk (EYT). After thawing, sperm motility and motion parameters, plasma membrane and acrosome integrity, apoptosis status and mitochondrial activity were evaluated. The results shown that total and progressive motility (54.43 ± 1.33% and 25.43 ± 0.96%, respectively) were significantly higher in SL1.5 when compared to other semen extenders. Sperm motion parameters (VAP, VSL, VCL, ALH and STR) were significantly higher in SL1.5 compared to other extender, with the exception of SL1 extender. Plasma membrane integrity (48.86 ± 1.38%) was significantly higher in SL1.5 when compared to other semen extenders. Also, percentage of spermatozoa with intact acrosome in SL1.5 (85.35 ± 2.19%) extender was significantly higher than that in SL0.5, SL2.5 and EYT extenders. The results showed that the proportion of live post-thawed sperm was significantly increased in SL1.5 extender compared to SL0.5, SL2 and EYT extenders. In addition, SL1, SL1.5 and SL2.5 extenders resulted in significantly lower percentage of early-apoptotic sperm than that in EYT extender. There were no significant differences in different semen extenders for percentage of post-thawed necrotic and late-apoptotic spermatozoa. Also, the results indicated that there are slight differences for percentage of live spermatozoa with active mitochondria between extenders. In conclusion, SL1.5 extender was better than other extenders in most in vitro evaluated sperm parameters.
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Two consecutive randomized double-blind field fertility experiments were conducted over a 4-month period and aimed at evaluating the association of two commercial soybean lecithin-based extenders (AndroMed [Minitub, Tiefenbach, Germany] and BioXcell [IMV Technologies, L'Aigle, France]) with pregnancy rates of chilled-stored (CS) and frozen-thawed (FT) ram semen. Semen samples with more than 2 × 10(9) sperm per mL and 70% progressive motile spermatozoa were collected via an artificial vagina from twelve proven fertile Chios rams, split-diluted with the above mentioned extenders, packaged in 0.25 mL straws and either stored at 5 ± 1 °C for 30 to 36 hours or frozen and thawed. Non-lactating multiparous ewes were inseminated in progestagen-synchronized estrus either with CS (AndroMed: N = 212 and BioXcell: N = 206; intracervical AI) or with FT (AndroMed: N = 114 and BioXcell: N = 92; laparoscopic intrauterine AI) semen. Ovulation was confirmed in all ewes based on determination of blood plasma progesterone (>1 ng/mL) 8 days post AI. Ewes were screened for pregnancy diagnosis by transabdominal ultrasonography 65 days post AI. BioXcell was superior to AndroMed in preserving the fertilizing potential of CS (P < 0.05) and FT (P < 0.005) semen. In AndroMed-stored semen, young rams (1.5-2.5 years old, N = 8) had a pregnancy rate (59.1%; 124/210) lower than that (72.4%; 84/116) of mature rams (4.5 to 5.5 years, N = 4; P < 0.025). Compared with AndroMed extender, processing of young ram semen in BioXcell extender improved pregnancy rates of CS (66.7%; 88/132 vs. 83.9%; 94/112; P < 0.005) and FT (46.2%; 36/78 vs. 71.0%; 44/62; P < 0.01) spermatozoa. Both extenders were similarly effective in preserving pregnancy rates of mature ram semen (P > 0.05). Ram-by-extender interactions were significant for pregnancy rates of CS and FT semen. Irrespective of extenders, overall pregnancy rates after intracervical and intrauterine AI were 75.1% and 62.2%, respectively (P < 0.001). In conclusion, BioXcell is a suitable extender for short- and long-term storage of ram semen. Selection of the ewes, farms, and extenders for intracervical AI programs can contribute to satisfactory fertility rates with semen preserved more than 24 hours at 5 °C.
Article
Two experiments were conducted to compare the effectiveness of different extenders conventionally used for semen cryopreservation to maintain the viability and fertility of cooled bull semen. In Experiment 1, sperm samples obtained from 20 Nellore bulls were preserved at 5 °C for 48 hours using two extenders containing 20% of egg yolk [Tris (TRIS-R) and Botu-Bov® (BB)] and another composed of 1% soy lecithin [Botu-Bov®-Lecithin (BB-L)] as substitutes for animal origin products. The samples were evaluated at 6, 24 and 48 hours for plasma and acrosomal membrane integrity, quantification of thiobarbituric acid reactive substances (ng of TBARS/108 cells) and sperm motility parameters by computer-assisted semen analysis (CASA). In Experiment 2, pregnancy rate (P/AI) of 973 fixed-time artificially inseminated Nellore cows were compared when cows were inseminated with conventionally cryopreserved semen in TRIS-egg yolk glycerol (TRIS-C Control, n = 253) or semen cooled for 48 hours in TRIS-R (n = 233), BB (n = 247) or BB-L (n = 240). Although none of the extenders used was effective on maintaining total progressive motility and cellular integrity throughout the 48-hour of the refrigeration period (P < 0.01), BB-L conferred greater protection against oxidative stress (P < 0.05) than egg yolk-based medias. The P/AI for semen samples preserved in TRIS-C, TRIS-R, BB and BB-L were 39.92a, 25.32b, 26.32b and 33.33ab, respectively. These results demonstrate that the three conventional extenders used for semen cryopreservation do not provide the protection required to maintain bull semen fertility under refrigeration for a 48-hour period, resulting in reduced pregnancy rates. However, the use of lecithin-based medium instead of egg yolk results in greater protection against lipid peroxidation, producing P/AI results comparable to those obtained using frozen semen.
Article
the extender supplemented with 6% soybean lecithin, with values of 59.7% for the percentage of total motile sperm (TM%), 44.3% for motility, 45.3% for plasma membrane integrity and 61.9% for acrosome integrity. TM%, motility, acrosome integrity and plasma membrane integrity in the extender containing 6% soybean lecithin were significantly higher than that of other concentrations of soybean lecithin and 20% egg yolk (P < 0.05). However, the percentages of TM, acrosome integrity and plasma membrane integrity decreased with the increasing concentration of soybean lecithin in extender. In summary, the effect of soybean lecithin on spermatozoa quality was superior and the effective concentration of soybean lecithin in extender was 6% (w/v). Soybean lecithin might replace egg yolk in extender in the cryopreservation of boar semen.
Article
The aim of this study was to evaluate the effect of different concentrations of soybean lecithin (SL) in extenders for sperm goat cryopreservation. Sexually mature male Saanen goats (n = 4) were used, and the ejaculates were obtained using an artificial vagina method. The semen samples were pooled and diluted in a skim milk-based extender (control group; CG) or Tris extender supplemented with SL at different concentrations (G1 = 0.04%, SL G2 = 0.08% SL and G3 = 0.16%) for a final concentration of 240 × 106 spermatozoa/mL. The semen samples were packed in straws (0.25 mL), frozen using an automated system and stored in liquid nitrogen (−196 °C). After thawing (37 °C/30 s), the samples were evaluated for sperm quality parameters, including sperm motility, membrane integrity, acrosome integrity and mitochondrial activity. No significant difference was observed among the experimental and control groups for all of the parameters (P > 0.05). However, even though the control group presented a significantly lower mitochondrial membrane potential compared to fresh semen (P < 0.05), the same did not occur for the extender supplemented with soybean lecithin, that is, it did not differ from fresh sperm (P > 0.05). The extender containing soybean lecithin at different concentrations preserved the sperm quality parameters in a manner similar to the conventional skim milk-based extender. Thus, it is concluded that an extender containing soybean lecithin as the lipoprotein source can be used for freezing goat semen.
Article
The objective of this study was to evaluate the effect of addition of egg yolk (EY) or soy lecithin (SL) based extenders on dog sperm parameters during 10days storage at 5°C. Four ejaculates of pooled semen from three Mongrel dogs were divided into three aliquots and extended to a final concentration of 200 million sperm/mL using following non-commercial extenders: egg yolk extender (EYE) group - semen+20% Tris-EYE; soy lecithin (SL) group 1 - semen+SL extender with 0.04% SL; SL group 2 - semen+SL extender 0.4% SL. The extended semen samples were stored at 5°C and were evaluated for sperm mitochondrial membrane potential (MMP), plasma membrane integrity (%PMI) and computer assisted sperm analyzer (CASA) sperm motility parameters on days 0, 1, 2, 3, 4, 7 and 10. The extender, days of storage and extender×days of storage affected the MMP, %PMI and CASA sperm motility parameters (P<0.05). The addition of 0.4% SL extender reduced the speed of deterioration of sperm parameters evaluated in this study compared to EY and 0.04% SL extenders (P<0.05). In conclusion, the addition of 0.4% SL extender enhanced sperm preservation based on the variables evaluated in the present study compared to EYE and 0.04% SL extenders and plausibly preserves sperm quality longer.