Soybean lecithin–based extender preserves spermatozoa
membrane integrity and fertilizing potential during goat
, Valeria Pasciu
, Sara Succu
, Daniela Addis
Giovanni G. Leoni
, Maria E. Manca
, Salvatore Naitana
Department of Veterinary Medicine, University of Sassari, Sassari, Italy
Centro di Competenza Biodiversità Animale, Sassari, Italy
Received 20 June 2014
Received in revised form 3 December 2014
Accepted 6 December 2014
In vitro fertilization
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 ﬁve 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 signiﬁcant 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.
Long-term storage of spermatozoa is one the most
important tool to improve the reproductive technologies
in the ﬁeld of animal and human medicine . 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: email@example.com (F. Berlinguer).
Contents lists available at ScienceDirect
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0093-691X/$ –see front matter Ó2015 Elsevier Inc. All rights reserved.
Theriogenology 83 (2015) 1064–1074
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 ﬁnal mission
of penetrating the oocyte . These membranes contain a
high concentration of polyunsaturated fatty acids, and
therefore are susceptible to oxidative stress, especially
during the freezing and thawing procedures .
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 . Therefore, research efforts are
focusing in ﬁnding a well-deﬁned egg yolk substitute of
nonanimal origin to be used in the constitution of semen
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 ﬁlm around the spermatozoon preventing the
formation of intracellular ice crystals and avoiding the
mechanical damage on the sperm membranes .
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
[14–16], 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
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
facilities meet the requirements of the European Union for
Scientiﬁc 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 artiﬁcial vagina from ﬁve 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 (ﬁve 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
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
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
50 IU/mL) by two consecutive centrifugations (1500gfor
20 minutes ), semen was diluted up to 400 10
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
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
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
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
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) 1064–1074 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. .Brieﬂy, 10 g nigrosin was dissolved in
distilled water by boiling and ﬁltered 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
liter, 25 mM KH
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
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/s, progressive motility greater than
80% straightness. This system has a speciﬁcsetupforgoat
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
L subsample of sperm suspension was
loaded into a prewarmed analysis chamber with a depth
m (Makler Counting chamber; Seﬁ-Medical
Instruments Ltd., Biosigma S.r.l., Italy), and a minimum of
500 spermatozoa per subsample were analyzed in at least
four different microscopic ﬁelds. Sperm motility was
assessed at 37
Cat40 magniﬁcation 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 . Brieﬂy, 50
L of fresh semen and
L of frozen semen (approximately 1.5 10
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
L of 3.5 M K
. After one successive
centrifugation in a Microfuge (3 minutes at 6000g), the
supernatant was analyzed spectrophotometrically with an
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
L from 2 mg/mL)
and glucose-6-phosphate dehydrogenase (2
L from 1 mg/
mL) in the presence of excess glucose (8
L from 18 mg/mL)
and nicotinamide adenine dinucleotide phosphate
L from 20 mg/mL) to perchloric extract (25
and to 400
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-
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 “comet”tail, 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 ﬁeld at
neutral pH , performed according to the method
described by Sakkas et al.  with slight modiﬁcations.
Brieﬂy, sperm suspension (30
L) was diluted in low
melting point agarose at 37
L; 1% wt/vol). A 100-
mixture of sperm-agarose was immediately pipetted onto
1% wt/vol normal melting point agarose–coated 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
g/mL). Incubation was performed for 1 hour at 37
After this, slides were rinsed in PBS and then placed in a
horizontal electrophoresis tank ﬁlled 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
ﬁxed in methanol.
Slides were stained with propidium iodide (PI), moun-
ted with a coverslip, and analyzed under an epiﬂuorescence
microscope. Digital comet images were captured with an
S. Chelucci et al. / Theriogenology 83 (2015) 1064–1074106 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 ﬂuorescein isothiocyanate–conjugated Pisum
sativum agglutinin (FITC-PSA) . The aliquots of sperm
suspension were incubated for 15 minutes at 39
g/mL in PBS, pH 7.4) and PI (14
g/mL in PBS,
pH 7.4). To reduce background ﬂuorescence, unbound PSA
and PI were removed by adding 200
matozoa were washed by centrifugation in a micro-
centrifuge at 800gfor 2 minutes. The supernatant was
aspirated and the pellet resuspended in 100
L of PBS. After
washing, a 10
L sample was put on a slide and coverslipped.
The slide was immediately dried by leaving at 37
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)
epiﬂuorescence 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 ﬁnally 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 : 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-
HCL in dimethylsulfoxide to a ﬁnal 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.
Brieﬂy, 4 10
frozen and thawed spermatozoa were
added to a tube with BAPNA extender containing BAPNA and
L of benzamidine solution; they were incubated at 37
for 90 minutes . Two or three incubations were per-
formed (one for the control and one or two for the sample).
After incubation, 100
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 Scientiﬁc, Rodano, Milano, Italy). Acrosin activity was
deﬁned as the quantity of enzyme that hydrolyzes 1
BAPNA/min at 23
Candspeciﬁc 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 ﬁve 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.
IUÞacrosin106spermatozoa ¼mean Abssample Abscontrol106
½Eincubation time ðminutesÞ=total volumesnumber of sperm ;
S. Chelucci et al. / Theriogenology 83 (2015) 1064–1074 106 7
in which E¼9.9/mM/cm; incubation time ¼180 minutes;
total volumes 1.2 mL. Abs ¼absorbance.
2.11. Quantiﬁcation 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 , with some modiﬁcations. After thawing,
semen was centrifuged for 7 minutes at 7000gand 100
of supernatant was added to 100
L glacial acetic acid 33%,
L SDS 10%, 100
L Tris-HCl 50 mM, pH 7.4, and 250
thiobarbituric acid 0.75%. The mixture was then incubated
for 1 hour at 100
C and immediately cooled on ice. After
10 minutes, 200
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 Scientiﬁc), 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 Dulbecco’s PBS at a temperature between
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 Earle’s 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
(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 ﬁve 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) 1064–1074106 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 of cysteamine. Cumulus-
oocyte complexes were put in groups of 30 to 35 in
L of the maturation medium in a four-well Petri dish
(Nunclon; Nalgene Nunc International, Roskilde, Denmark)
layered with 300
L mineral oil and cultured for 24 hours in
in air at 39
After maturation, the COCs were partially stripped of the
cumulus cells and fertilized in vitro at 39
C and 5% CO
, and 90% N
atmosphere in four-well Petri dishes (Nun-
clon; Nalgene Nunc International). Synthetic oviduct ﬂuid
(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
g/mL heparin, and 1
(IVF-SOF). Percoll gradients were prepared as described by
Rosenkrans et al. . In brief, 100% Percoll solution was
mixed with a 10salt solution (NaCl 2.889 g; KCl 0.238 g;
0.116 g; CaCl
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
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
a humidiﬁed atmosphere at 5% CO
in air for 15 minutes.
Swim-up derived motile spermatozoa were diluted in IVF-
SOF at a 1 10
spermatozoa/mL ﬁnal concentration and
incubated for 45 minutes at 39
. 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 ﬁxing solution to evaluate chromatin conﬁguration
. Brieﬂy, oocytes were denuded and ﬁxed 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 classiﬁed 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 signiﬁcance. Dif-
ferences in sperm parameters were studied by performing
an ANOVA analysis. Differences in fertilization rates per
extender were evaluated by performing a
test. All the
data are expressed as the mean SEM.
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 signiﬁcantly 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 ﬁve 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) 1064–1074 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 signiﬁ-
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 signiﬁcant 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 quantiﬁcation 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 signiﬁcantly
affected when buck spermatozoa were cryopreserved in the
Tris-based extender supplemented with egg yolk (P <0.05;
Fig. 4,panelsCandD).Nosigniﬁcant 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 ﬂuorescent 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 signiﬁcantly higher fertilization rates
compared with the other media (P <0.01; Fig. 4, panel G).
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
Finally, the signiﬁcantly higher fertilization rates ob-
tained with spermatozoa cryopreserved in a soybean leci-
thin extender compared with the other media conﬁrmed
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
conﬁrms similar ﬁndings in goat  and other species,
such as ram , human , and cat . 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 . Therefore,
possibly the optimal amount of lecithin needed to protect
sperm membranes and increase tolerance to the freezing
process, without signiﬁcantly increasing extender viscosity,
can be obtained from 1% soybean lecithin.
The results of this study reveal that viability rates and
motility parameters decreased signiﬁcantly 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
lecithin–based extenders [21,34], whereas other authors
found that lecithin-based extenders let to higher viability
rates compared with egg yolk–based extenders .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 .
In this study no signiﬁcant 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 lecithin–based extenders compared with the
EXT CM and the extender alone, but differences did not
reach statistical signiﬁcance. Energy metabolism is a key
factor supporting sperm function. However, in a previous
study in goat we did not ﬁnd any predictive value of
spermatozoa energetic status, as evaluated by ATP intra-
cellular concentration before and after thawing, on IVF
outcome . 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. 
reported no signiﬁcant difference in the percentage of
live spermatozoa with active mitochondria, as evaluated
by ﬂow cytometry analysis, between lecithin and egg
yolk–based extenders. Another study found that lecithin
induced serious mitochondrial damage that clearly
affected the inner mitochondrial membrane, and conse-
quently, sperm motility , but it used a higher lecithin
concentration (3.5%) and did not investigate sperm
S. Chelucci et al. / Theriogenology 83 (2015) 1064–10741070
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
ﬂuidity and ﬂexibility needed to engage in membrane
fusion events associated with the fertilization.
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 .
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
ﬂuorescent 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 ﬁve 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 (C–F): P <0.05. sptz, spermatozoa.
S. Chelucci et al. / Theriogenology 83 (2015) 1064–1074 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
yolk–based extender. These results are in agreement with
recent studies in goat  and bull , which reported
that in egg yolk–based extenders the production of MDA
was signiﬁcantly 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 .
Lipid peroxides are also capable of inducing DNA damage
and decrease in fertility during storage of semen .
In the present study, the high levels of LPO end products
(MDA) found in the egg yolk–based 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 .
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 . In the present study, the better preservation of
DNA integrity in spermatozoa frozen in the soybean leci-
thin–based 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 . 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 . 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 yolk–based
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 .
Proacrosin activation strongly relies on intra-acrosomal pH
. 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 . Thus, the higher acrosin activity in the egg
yolk–based extender and extender alone would suggest
membrane damage, as conﬁrmed 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 efﬂux of cholesterol from
the sperm plasma membrane and resulted in the change in
ﬂuidity that increases the sensitivity to cold shock .
Results of the heterologous IVF test support this hypothesis.
Spermatozoa cryopreserved in the lecithin-based extender
lead to signiﬁcantly 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 ﬂuidity of the membrane,
antigens to relocate over the plasma membrane .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
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 yolk–based extender may
be explained by the presence of high-density lipoproteins,
which are regarded as one factor that increases the
sensitivity to cold shock.
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).
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