Open Journal of Veterinary Medicine, 2012, 2, 98-103 Published Online September 2012 (
Motility and Functional State of the Me mb ra ne of Ca pr ine
Capacitated Spermatozoa under Different Chemical
Alejandra Soberano Martínez1*, José Herrera Camacho2, José Candelario Segura Correa3
1Centro Multidisciplinario de Estudios en Biotecnología, Facultad de Medicina Veterinaria y Zootecnia,
Universidad Michoacana de San Nicolás de Hidalgo, Morelia, Mexico
2Instituto de Investigaciones Agropecuarias y Forestales, Universidad Michoacana de San Nicolás de Hidalgo, Posta Zootecnia,
Tarímbaro, México
3Campus Ciencias Biológicas y Agropecuarias, Universidad Autónoma de Yucatán, Mérida, México
Email: *
Received May 24, 2012; revised June 30, 2012; accepted August 20, 2012
The objective of this study was to test the use of a commercial extender (Triladyl) as a diluent in caprine semen refri-
gerated at 15˚C, using caffeine (CF), heparin (HP), synthetic oviductal fluid (SOF) andtriladyl (TRY) as capacitating
chemical agents at different times. Twenty ejaculates of caprine semen were collected using an artificial vagina. The
ejaculates were diluted and refrigerated by three days. Evaluated the progressive motility (PM) and the functional state
of the sperm plasma membrane trough fluorescent CTC staining, counting 200 spermatic cells (non-capacitated sper-
matozoa NCS, capacitated CS and reacting spermatozoa REA) of caprine in two capacitating agents: CAF and HEP;
one culture medium: SOF and a commercial extender: TRY at 60, 120, 180 and 240 min of incubation, during 24, 48
and 72 hs. PM was high under TRY, and CS was high under the HEP treatment. TRY could be an alternative to capaci-
tate caprine spermatozoa, keeping PM for a longer time than HEP or CAF.
Keywords: Spermatozoa; Capacitation; Caprine; Chemical Agents
1. Introduction
Artificial insemination (AI) is probably the reproductive
technology most widely used, because it is simple and
the highest benefit: Cost ratio when tested bucks are used
for reproduction [1]. Semen from tested bucks could be
used fresh, freeze or frozen. However, fresh semen must
be used inmediately after its collection, because motility
and viability of the spermatozoa is reduced in a short
time. However, frozen semen could be maintained longer
than 48 hs [2], providing a greater flexibility of their use
in AI programs. frozen semen could be carry from the
animal breeding center or a given ranch to the desire one
[3]. Preservation of ovine semen kept at 15˚C [4] capri-
nesemen at 5˚C - 21˚C [5] and canine semen [6] has been
notified. However, it has been found that the production
of reactive oxygen species (ROS) occurs mainly during
the cooling period at 5˚C [7].
In this context, semen has been diluted in Tris base ex-
tenders using as cryoprotector agent non-penetrant lipo-
proteins of yolk egg and milk proteins because both act
against the osmotic effects of the cooling process, pro-
ducing that the hypertonic medium cause the exit of water
of the cells, increasing dehydration and reducing the pos-
sibility of intracellular crystal ice formation. However,
even though this diluent length the lifetime of the sper-
matozoa and allow the sperm capacitation [8], this does
not happen during the cryopreservation process of the ca-
prine spermatozoa, because the interaction of the egg yolk
[9] and descreamed milk [10] with the seminal plasma
produce a set of nocive reactions for the spermatozoa [11],
and irreversible damages in the structure and function of
the membranes during the froze and defroze processes
[12], which limits the use of AI in caprines.
The in vivo sperm capacitation occurs during migra-
tion in the reproductive tract of the female, whereas in
vitro capacitation requires the exposition of fresh or
freeze semen to specific capacitating agents [13]. The
beginning of this process implies the remotion of choles-
terol and probably other sterols from the plasmatic mem-
brane of the spermatozoa, changes in the membrane, flux
and redistribution of the proteins modulacion in the in-
tracellular ion concentration (Ca2+, HCO3
, K+ and Na+),
hyperpolarization of the plasmatic membrane associated
with K+ flux, pH increase and increase of phosphoryla-
*Corresponding author.
opyright © 2012 SciRes. OJVM
tion of the protein tyrosine [14].
Many methods have been applied to measure the
changes that occur in the membrane, during the in vitro
capacitation process. The most common method used is
chlortetracycline staining (CTC) [15]. This fluorescent
antibiotic binds to the membrane linking cations, spe-
cially Ca+2 and then shows an increase of fluorescence on
the segments of themembrane where these cations acu-
mulate. CTC staining has demonstrated to interact with
spermatozoa of different species of mammals, that pre-
sent different fluorescent patterns on the spermatozoa
head, which are though to express different stages of the
capacitation process [16].
In vivo the oviductal fluid of the reproductive tract of
the female is used as cholesterol-acceptor because is rich
in albumins and high density lipoproteins (HDL), capa-
ble of withdrawing cholesterol from the membrane of the
spermatozoa [17]. Similarly, the pass through the repro-
ductive tract cause that the spermatozoa get ride of the
seminal plasma keeping only those proteins or desca-
pacitating factors (muco-polysaccharides and proteins)
that cover the spermatic surface [18], which are removed
in the oviductal crypts [19] and by progesterone recep-
tors ones initiated the capacitation process [20]. In vitro
studies, glycosaminoglycans (GAGs) have been identi-
fied as efficient inductors of the spermatozoa capacita-
tion. This group of carbohydrates (polysaccharides)
formed by repetitive units of disaccharides include the
heparin, heparin sulphate, chondroitin sulphate, keratan
sulphate and hyaluronic acid [21]. GAGs promote the
capacitation binding and removing the proteins of the
seminal plasma fixed by adsortion to the plasma mem-
brane of the spermatozoa and inhibin the capacitation
[22]. Heparin has been the most potent inductor of ca-
pacitation, because it attaches to the spermatozoa through
union proteins situated in the cell membrane, driving to
the affluence of Ca+2, which increases the synthesis of
AMPc and consequentely produce the protein phos-
phorylation [23]. Caffeine is an alkaloid compose of the
metilxantin group used in vitro as capacitating agent be-
cause it inhibit the phosphodisterasa nucleotide which is
responsible of the degradation of AMPc; inducing an
increase in its concentration [24]. This has conducted to
find new ways to improve the spermatozoa capacitation
results. Bergqvist et al., [16] have pointed out the use of
oviductal fluid collected in vivo, as spermatozoa capaci-
tation medium for bovines. The use of this medium based
on synthetic oviductal fluid (SOF) of ovine has not been
used in caprines. The use of only the extender triladyl on
caprine frozen semen and spermatozoa capacitation with
caffeine, heparin, SOF and extender triladyl have also
not been evaluated.
The objective of this study was to test the use of a
commercial extender (Triladyl) as a diluent in caprine
semen refrigerated at 15˚C, using caffeine (CF), heparin
(HP), synthetic oviductal fluid (SOF) andtriladyl (TRY)
as capacitating chemical agents at different times.
2. Materials and Methods
Twenty ejaculates from a buck (Saanen) were collected
using an artificial vagina. The semen was evaluated in
terms of its micro and macroscopic characteristics and
thereafter diluted 9:1 in a commercial base TRIS (Tri-
ladyl, Minitüb, Tiefenbach, Germany) extender and re-
frigerated at 15˚C. In order to evaluate the spermatozoa
capacitation of the cool semen under different conditions,
the experimental protocol was carried out during three
consecutive days.
Semen samples (3 ml) diluted in TRIS were divided in
four aliquots, which were incubated at 37˚C by 15 min.
Samples were swim-up three times consecutively. Semen
was centrifuged at 750 rpm by 5 min at room tempera-
ture. The semen fluid was decanted and reposed twice
with equal volume of mDM medium and homogenerized
gently. In the third wash the pill was incubated with
mDM for 45 min to allow to up and select the spermato-
zoa with greater movility [25].
The supernatant was separated and placed in inde-
pendent vials to get the different treatments of the study:
Treat 1) upper fraction + mDM (Vol/vol) + Caffeine
(concentration 5 mM; CA); Treat 2) upper fraction +
mDM (Vol/vol) + Heparin (50 µg/ml; HE); Treat 3) up-
per fraction + synthetic oviductal fluid (SOF; Vol/vol;
and Treat 4) upper fraction + Triladyl (TR; Vol/vol).
Evaluation of the functional state of the spermatic
membrane: It was done trough fluorescent CTC staining,
counting 200 spermatic cells. The evaluation of the func-
tional state of the membrane and PM of the spermatozoa
was carried out in 150 µl of each treatment sample at 60,
120, 180 and 240 min. At all times the treatments stayed
in incubation at 37˚C. The protocol was repeated 24 hs
(day 2) and 48 hs (day 3) with diluted semen and refri-
gerated in Triladyl.
The functional state of the membrane was classified
according to staining portion in the following categories:
a) Non-capacited spermatozoa, with uniform fluores-
cencein the head and intact acrosome; b) Capacitated
spermatozoa, with fluorescence concentrated in the acro-
somal region, a band without fluorescence in the post-
acrosomal region and the presence of intact acrosomes; c)
Spermatozoa with acrosomal reaction, without fluores-
cence in the head, except by a thin band in the equatorial
region [26].
Statistical analysis: The effect of chemical agent caf-
feine (CF), heparin (HP), synthetic oviductal fluid (SOF)
and triladyl (TRY) extender, culture time (60, 120, 180
and 240 min), day of evaluation (1, 2 and 3) and simple
Copyright © 2012 SciRes. OJVM
interactions among them on progressive motility (PM),
non-capacitated spermatozoa (NCS), capacitated sper-
matozoa capacitated (CS) and reacting spermatozoa
(REA) was tested. Data were analyzed using the general
linear model procedure of SAS [27].
3. Results
The statistical analysis showed effects of treatment (P >
0.0001), day (P > 0.0001) and culture time (P > 0.0001).
However, there was no effect of the treatment* day ( P>
0.476) and treatment* time interaction (P > 0.995).
Treatment effect: PM, CS, NCS and REA were af-
fected by treatment. A greater PM and NCS spermatozoa
was observed for TRY and SOF treatments (P < 0.01),
respectively (Tabl e 1). CS was better for HEP and CAF;
whereas better REA was found for CAF.
Time effect: PM was greatest at 120 min and CS at
240 min as compared with other culture times (Table 2 ).
No significant difference was found with respect to REA.
Effect of day (Storage time): Evaluation day had a
significant effect on PM and CS; as day of evaluation
increased PM decreased but CS increased (Table 3).
Table 1. Effect of treatment on progressive motility and
functional state of the plasmatic membrane of caprine
Variable (%) Caffeine Heparin SOF Triladyl SEP-value
PM 29.2ª 34.8ª 59.9b 65.9b 1.670.0001
CS 59.5ab 63.2ª 55.7bc 54.2c 0.950.0011
NCS 31.2ª 29.2ª 37.1b 37.8b 1.090.0002
REA 9.3ª 7.7ab 7.2ab 7.8b 0.320.04
abcDifferent literals in the same row mean statistical significant effect. PM =
progressive motility; CS = capacitated spermatozoa; NCS = non-capacitated
spermatozoa; REA = reacting spermatozoa.
Table 2. Effect of incubation time with different chemical
agents on progressive motility and functional state of the
caprine spermatozoa membrane.
Variable (%) Incubation time (min) SEP-value
60 120 180 240
PM 57.4ª 49.6b 42.7c 40.1c 1.730.0001
CS 52.9a 56.3ab 60.1bc 63.3c 1.710.002
NCS 40.1a 35.9ab 31.4bc 27.9c 1.650.0001
REA 6.9a 7.8a 8.4a 8.7ª 0.530.08
abcDifferent literals in the same row mean statistical significant effect. PM =
progressive motility; CS = capacitated spermatozoa; NCS = non-capacitated
spermatozoa; REA = reacting spermatozoa.
Table 3. Effect of incubation day with different chemical
agents on progressive motility and functional state of the
caprine spermatozoa membrane.
Evaluation day
Variable (%)1 (0 h) 2 (24 h) 3 (48 h) SE P-value
PM 55.6a 49.2b 37.7c 1.45 0.0001
CS 56.1a 55.3a 62.3b 0.82 0.002
NCS 37.2a 35.8a 28.4b 0.95 0.0001
REA 5.8a 8.1b 9.3b 0.28 0.0001
abcDifferent letters in the same row mean statistical significant effect. PM =
progressive motility; CS = capacitated spermatozoa; NCS = non-capacitated
spermatozoa; REA = reacting spermatozoa.
4. Discusion
Capacitation process is a prerequisite step for sperm to
bind to the zona pellucida, it experiment acrosomic reac-
tion in response to natural agonistics and express hyper-
motility, a special movement that allow spermatozoa to
move in the viscose fluid of the oviduct and get into the
zona pellucida [28].
The results of this study indicate that caprine semen
diluted in Triladyl could be preserve at 15˚C until 48 hs
maintaining an acceptable motility (P > 0.0001). Probably
the presence of lipoproteins of egg yolk and milk may not
be required because the extender per se, compose of TRIS,
sugar, glycerol and citric acid, is capable of providing the
nutrients that the spermatozoa need for its metabolism. It
also gives protection against pH variation [26], an ade-
quate osmotic pressure for the spermatozoa, and avoids-
dehydration damage through the stabilization of the lipid
bilayer. In vivo the pH of luminal fluid in the reproductive
tract has effects on the whole process of reproduction,
including spermatogenesis, sperm capacitation, fertiliza-
tion, and early stage embryo development. The epithelia
lining along the reproductive tract wall of both males and
females actively perform considerable transepithelial
transport of acid-base equivalents to tightly control the pH
of luminal fluid milieu along the entire reproductive tract
[29]. Because sugars (sucrose, raffinose, trehalose and
lactose) are non-penetrating cryoprotectors of high mo-
lecular weight they are useful when applied at high cool-
ing speeds, because the cryoprotector action is associated
with its dehydrate activity and its specific interaction with
the phopholipid membrane [30]. In this context, the type
and sugar concentration used can modify not only the
sperm motility but also the viability and acrosomal integ-
rity [31]. In general the monosaccharides in addition to
act as cryoprotector play other functions. They provide a
source of energy for the spermatic cell and maintain the
osmotic pressure [32].
The glycerol has been used as a penetrating cryopro-
tector agent since its discovery by Polge et al. [33]. It has
Copyright © 2012 SciRes. OJVM
been show that their presences in the extenders reduces
damage in the spermatozoa, because it avoids the build
of intracellular crystal ice, as well as the excessive dehy-
dration caused by the slow cooling [34], and improve fer-
tility [8]. The differences between studies respect to the
use of glycerol, are due to alterations in the organization
and viscosity of the cytoplasm of the spermatozoa, to the
permeability and stability of the membrane, and altera-
tion of the protein and phospholipids organization [35].
However, the previous could be due to many factors such
as glycerol concentration, the use of different diluents,
different cryopreservation protocols, and the use of other
cryoprotectors; as well as the different criteria and
methods used to evaluate sperm quality [36].
Sugar and glycerol act in a way that their hydrogen
bind to the polar group of the head of the membrane li-
pids, which have the ability to replace the water mole-
cules normally found in the polar groups, which help to
stabilize the membrane during the temperature transition
On the other hand, the results of CS (56.1%) and PM
(55.6%) here obtained indicated that the spermatzoa can
be conserved during one day only, this is due to that cit-
ric acid and sugar compounds provide a source of energy
enough for the sperm metabolism and to increase moti-
lity [37] but can decrease viability. Previous studies indi-
cate that the use of Triladyl can provide substrates such
as citric acid, which through a series of chemical reac-
tions favor sperm motility due to an increase of ATP
production [38]. Spermatozoa require a permanent pro-
duction of ATPs in order to maintain the cell structure,
the composition of intracellular ions and motility. It has
been hypothezised that composition of ions participate in
the capacitation process [39].
The decreasing effect of HEP on PM and the increas
ing effect of CAF on CS agree with the results of Zhou et
al. [40], who evaluating the effect of heparin in fresh
semen during caprine spermatic capacitation by 120 min
observed a significant reduction of motility and mem-
brane integrity. El Gaafary et al. [41] using CAF ob-
tained acceptable results of sperm motility of fresh bo-
vine semen during 1 h; however, motility decreased as
incubation time increased (up to 6 hs).
Previous research suggested that the reduction of the
motility of in vitro capacitated spermatozoa treated with
heparin or caffeine may be due to reduction of the gly-
cosylable substrates like glucose and/or fructose, and
also to a reduction in the availability of pyruvate and lac-
tate, substrates that during the cell metabolism are pri-
mordial to produce ATPs [42]; which make difficult its
conservation and use for longer times.
Respect to the medium known as synthetic oviduct
fluid (SOF) which composition is based in the bio-
chemical analysis of ovine oviduct fluid plus HEPES and
polyvinyl alcohol (PVA). The incubation results with the
SOF medium showed a greater PM than for CAF and
HEP but the capacitation percentages was similar. The
mechanism by which the motility vary has not been es-
tablished. However, it is known that motility is particu-
larly dependent of the mitochondrial function [43]. Mito-
chondrias are strategically distributed around the mid-
piece of the spermatozoa to provide energy to dynein that
propel the microtubules. The mitochrondia provides the
main source of oxidative energy throughout the produc-
tion of ATPs via the electrons transport chain. In the
other hand, the response to the capacitation could be due
to the fact that the medium is a source of intracellular
ions (NaCl, KCl, KH2PO4, MgCl2, NaHCO3, CaCl2) py-
ruvate and lactate, that cause changes in the properties of
the plasmatic membrane, particularly of the ionic con-
ductance which has been shown to be powerful regulator
of the metabolism and consequently of capacitation and
sperm motility [44]. Also, it is known that bicarbonate
plays an important role in the capacitation and fertiliza-
tion processes both in vivo and in vitro, because it in-
creases the distabilization of the lipid membrane [45],
and regulates and stimulates the adenylcyclase activity
that increases the concentration of AMPc and speed up
the A1 cyanase protein (PKA1), which phosphorylate
some proteins [46]. In the other hand, Huo et al. [47]
made reference to the fact that the elimination of the
seminal plasma by centrifugation and sperm dilution
could cause destabilization of the spermatic membrane.
Therefore the hydrophobic an adjustment among mem-
branes of the proteins and the “lipidic rafs” cause small
changes in the membrane thickness [48] and protein in-
teractions with lipidic rafs. This may allow the exterior-
ization of receptors of the ionic channels that participate
in the activation of the mechanism of calcium transduc-
tion flux, AMPc synthesis, and protein phosphorylation
diphosphorylation [49].
5. Conclusion
In summary, TRY without egg yolk and milk could be an
option for refrigerating caprine sperm and flexible option
to use in caprine AI programs. The use of SOF medium
and TRY extender as chemical agents maintained an ac-
ceptable motility and sperm capacitation for longer time
compared with HEP or CAF. However, further FIV test
are required to determine the fecundate capacity of the
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