Cryobiology 51 (2005) 241–249

www.elsevier.com/locate/ycryo

Adding cholesterol to the stallion sperm plasma membrane

improves cryosurvival 夽
Amanda I. Moore, Edward L. Squires, James K. Graham ¤
Department of Biomedical Science, Colorado State University, Fort Collins, CO 80523, USA

Received 1 April 2005; accepted 11 July 2005

Available online 24 August 2005

Abstract

Cryopreservation induces partially irreversible damage to equine sperm membranes. Part of this damage occurs due
to membrane alterations induced by the membrane changing from the Xuid to the gel-state as the temperature is reduced
lower than the membrane transition temperature. One way to prevent this damage is to increase the membrane Xuidity
at low temperatures by adding cholesterol to the membrane. DiVerent concentrations of cholesterol-loaded-cyclodex-
trins (CLC) were added to stallion sperm to determine the CLC concentration that optimizes cryosurvival. Higher per-
centages of motile sperm were maintained after thawing when 1.5 mg CLC was added to sperm from stallions whose
sperm do not survive freezing well, compared to control sperm from those same stallions (67% vs. 50%; P < 0.05). Addi-
tion of CLCs increased the percentages of membrane intact sperm surviving cryopreservation compared to untreated
sperm for all stallions (P < 0.05). The amount of cholesterol that incorporated into the membranes of the sperm cells
increased in a polynomial fashion (R2 D 0.9978) and incorporated into all sperm membranes. In addition, there was a
signiWcant loss of cholesterol from sperm membranes after cryopreservation; however, addition of CLCs to sperm prior
to cryopreservation maintained higher cholesterol levels in the sperm after freezing and thawing than untreated sperm
(P < 0.05). Addition of CLCs also resulted in more sperm binding to the zona pellucida of bovine oocytes after cryopres-
ervation than control sperm (48 vs. 15; P < 0.05). In conclusion, CLCs improved the percentage of post-thaw viability in
equine sperm as well as increased the number of sperm that bind to zona pellucida. Addition of CLCs to stallion sperm
prior to cryopreservation is a simple procedure that increases the cryosurvival of cells.
 2005 Elsevier Inc. All rights reserved.

Keywords: Cholesterol; Cyclodextrin; Equine; Spermatozoa; Cryopreservation; Membrane

The use of cryopreserved equine sperm has

夽
increased in the United States since the large breed
Supported by a grant from the Colorado State Experiment
Station and the Preservation of Equine Genetics Program.
organizations now permit foals produced from fro-
*
Corresponding author. Fax: +1 970 491 7569. zen semen to be registered. However, cryopreserva-
E-mail address: jkgraham@colostate.edu (J.K. Graham). tion induces partially irreversible damage to sperm

0011-2240/$ - see front matter  2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.cryobiol.2005.07.004
242 A.I. Moore et al. / Cryobiology 51 (2005) 241–249
that results in reduced fertility for frozen sperm
from many stallions compared to fresh or cooled
semen. Part of this damage occurs to sperm mem-
branes when the cells are cooled from room tem-
perature (22 °C) to 1 °C. As stallion sperm are
cooled below 18 °C the membrane phospholipids
undergo a phase transition from a liquid to gel-
state [1]. During this phase transition, phospholip-
ids are lost from the plasma membrane leading to
increased membrane permeability, membrane dis-
ruption, and cell death [7,26]. Previous studies indi-
cate that the cholesterol/phospholipid ratio of the
plasma membrane is a major determinant in
plasma membrane Xuidity and stability during
cryopreservation [8,26]. Cholesterol reduces the
transition temperature of membranes, and main-
tains them in a Xuid state at reduced temperatures
thereby reducing the membrane damage that
occurs at low temperatures [1]. Sperm from species
that posses very high cholesterol/phospholipid
ratios, such as human and rabbit sperm, do not
experience this membrane damage when cooled [8].
To prevent phospholipid rearrangement and to
increase membrane Xuidity at low temperatures,
cholesterol can be added to the plasma membrane
of several cell types [12,13,21], including sperm
[5,19]. When cholesterol is added to synthetic liposo-
mal membranes, it reduces the temperature at which
the phase transition occurs and at high concentra-
tions eliminates the phase transition altogether
[13,21]. Cholesterol can easily be incorporated into
or extracted from the plasma membranes of cells
using cyclodextrins. Cyclodextrins, cyclic heptasac-
charides consisting of
(1–4) glucopyranose units,
are water soluble but have a hydrophobic center [19]
and can transport cholesterol into or out of mem-
branes down a concentration gradient [12]. When
cholesterol is loaded into cyclodextrins (CLC) and
the CLCs added to bovine sperm prior to cryopres-
ervation, higher percentages of motile and mem-
brane intact sperm were recovered after thawing
compared to untreated sperm [19]. Similar results
have also been reported for stallion sperm treated
with a single concentration of CLCs [5]. Whereas
this procedure has been optimized for bull sperm
[19] it has not been optimized for stallion sperm.
The objectives of these experiments were to
determine (1) the concentration of cholesterol-
loaded-cyclodextrins that needs to be added to
stallion sperm prior to centrifugation and cryo-
preservation to optimize sperm cryosurvival; (2)
the amount of cholesterol in the sperm plasma
membrane after the addition of CLCs both prior
to and after cryopreservation; and (3) the ability of
equine sperm treated with CLCs to bind to the
zona pellucida.
Materials and methods
Unless otherwise speciWed all chemicals were of
reagent grade (Sigma Chemical, St. Louis, MO).
Preparation of CLCs
Cyclodextrins were prepared as described by
Purdy and Graham [19]. BrieXy, to load cholesterol
into the cyclodextrins, 1 g of methyl-
-cyclodextrin
was dissolved into 2 ml of methanol in a glass test
tube. In a second glass test tube, 200 mg of choles-
terol was dissolved into 1 ml of chloroform and a
450 l aliquot of this solution added to the methyl-

-cyclodextrin solution. The combined cyclodex-
trin/cholesterol solution was thoroughly mixed,
and the solvents then removed using nitrogen gas.
The resulting crystals were stored in a glass vial at
22 °C until use. To add cholesterol to sperm, a
working solution of CLCs was made by adding
50 mg of CLC to 1 ml of a modiWed Tyrode’s
medium (TALP) [16], mixed vigorously with a vor-
tex, and incubated in a 37 °C water bath until use.
The CLC working solution was mixed again, using
a vortex mixer, prior to any aliquot being removed
for addition to sperm.
Semen collection
Stallions collected for the following experiments
ranged from 5 to 22 years of age; were of Thor-
oughbred, Quarter Horse, Arabian, and grade
breeds; and were on a regular semen collection
schedule (3 times/week). Semen was collected from
each stallion using a Colorado model artiWcial
vagina with an in-line gel Wlter (Animal Reproduc-
tion Systems, Chino, CA) and the semen was used
within 45 min of collection. All stallions were
 A.I. Moore et al. / Cryobiology 51 (2005) 241–249
maintained under the guidelines presented by the
Colorado State University’s Animal Care and Use
Committee.
Experiment 1: EVect of CLCs on frozen-thawed
stallion sperm
Ejaculates from 15 stallions were used for this
experiment. Single ejaculates from stallions with
>40% post-thaw progressive motility (“Good”
freezers, n D 8) and two ejaculates from stallions
with <40% post-thaw progressive motility (“Poor”
freezers, n D 7) were used. Sperm from each ejacu-
late were diluted to a concentration of 120 £ 106
sperm/mL in TALP and divided into seven ali-
quots of 5 ml each (600 £ 106 total sperm). Sperm
were treated with varying levels of CLCs (0, 0.5,
1.5, 3.0, 4.5, 6.0, or 7.5 mg CLC/120 £ 106 sperm)
and the cells incubated at room temperature
(»22 °C) for 15 min. Following incubation the cells
were diluted 1:1 (v:v) with a skim milk, glucose dil-
uent (EZ-Mixin; “BF,” Animal Reproduction Sys-
tems, Chino, CA), and the samples centrifuged at
600g for 10 min. The supernatant was removed,
sperm concentration in the remaining pellet was
determined, and the cells were resuspended to a
Wnal concentration of 200 £ 106 sperm/ml in Lac-
tose–EDTA freezing extender containing 5% glyc-
erol (EZ-Freezin’ “LE,” Animal Reproduction
Systems, Chino, CA). The sperm were then pack-
aged into 0.5 cc straws and frozen in a programma-
ble freezer (Kryo 10 Series III, Planer, Middlesex,
UK) at a rate of ¡10 °C/min from 20 to ¡15 °C
and then ¡15 °C/min from ¡15 to ¡120 °C. At
¡120 °C, straws were plunged into liquid nitrogen
and stored until analyzed for motility and viability.
One straw from each treatment was thawed in a
37 °C water bath for 30 s for assessment of motility
using a computer-assisted sperm analysis system
(CASA) (HTM IVOS; Hamilton-Thorne Biosci-
ences, Beverly, MA). The contents of each straw
was diluted 1:4 (v:v) in EZ-Mixin’ BF to give a
Wnal concentration of 20 £ 106 sperm/ml and each
sample maintained at room temperature for
approximately 10 min before analysis. System
parameters for these analyses were 45 frames
acquired at 60 frames per second; minimum con-
243
trast 70, minimum cell size four pixels; lower aver-
age path velocity (VAP) cut-oV 20 m/s; lower
average straight line velocity (VSL) cut-oV 0 m/s;
VAP cut-oV for progressive cells 50 m/s and
straightness 75%. A 5-l drop of sperm from each
sample was placed on a preheated (37 °C) slide and
a minimum of 200 sperm per sample analyzed.
One straw from each treatment was thawed as
previously described to assess the percentage of
membrane intact sperm, using Xow cytometry. Cells
were diluted to 40 £ 106 sperm/ml in TALP and
100 l (4 £ 106 cells) was placed into 500 l of TALP
containing 10 l of propidium iodine (PI, 1 mg/ml in
H2O) and 20 l of SYBR-14 (Molecular Probes,
Eugene, OR; 10 M in Me2SO). Samples were incu-
bated at room temperature for 10 min prior to anal-
ysis. A minimum of 50,000 sperm per sample were
analyzed using an EPICS V Xow cytometer (Coulter
Electronics, Miami, FL) equipped with Cicero com-
puter software (Cytomation, Fort Collins, CO) as
described by Purdy and Graham [19]. BrieXy, the
Xuorescent probes were excited with a 488 nm argon
laser and the Xuorescence of PI and SYBR-14
detected using a Wlter setup including a 515 nm long-
pass Wlter to block laser light, a 590 nm dicroic beam
splitting Wlter, a 530 nm short-pass Wlter to detect
SYBR-14, and a 630 nm long-pass Wlter to detect PI.
Experiment 2: Amount of cholesterol incorporating
into stallion sperm
Semen from 12 stallions was collected and a
total of 3.5 £ 109 sperm from each ejaculate was
diluted to 200 £ 106 sperm/ml with TALP. The
sperm sample was then divided into seven aliquots
and each aliquot treated with 0, 0.5, 1.5, 3.0, 4.5, 6.0,
or 7.5 mg CLC/120 £ 106 sperm. The samples were
incubated as described previously prior to being
centrifuged through a 45% Percoll gradient at
600 g for 25 min and the resulting sperm pellet sus-
pended in 3 ml of phosphate-buVered saline (PBS;
171 mM NaCl, 3 mM KCl, 10 mM Na2HPO4, and
2 mM KH2PO4, pH 7.4). The sperm were washed a
second time (600g for 10 min), resuspended in
0.5 ml of PBS and the sperm concentration deter-
mined. The samples were stored at ¡20 °C until
cholesterol analysis was performed.
244 A.I. Moore et al. / Cryobiology 51 (2005) 241–249
The amount of cholesterol in each sample was
determined using the Cholesterol Liquicolor enzy-
matic assay (Stanbio, Boerne, TX) as described by
Navratil et al. [17]. BrieXy, cells were diluted 1:1
(v:v) with lysate buVer (0.4% Triton X-100 in PBS)
for 1 h to solubilize the plasma membranes. Sam-
ples were diluted 1:5 (v:v) with reagent and allowed
to incubate for 25 min at 37 °C. Samples were then
centrifuged to remove the cells and the supernatant
was analyzed for cholesterol content via spectro-
photomer (Spectronic Genesys 5, Phoenix, AZ)
with wavelength set to 500 nm. Cholesterol concen-
trations of the unknown samples were compared
to a standard curve from known amounts of cho-
lesterol.
Experiment 3: Cholesterol concentration in the
sperm plasma membrane before and after
cryopreservation
Semen from 14 stallions was extended to
120 £ 106 in TALP and each ejaculate divided into
two sub-groups (fresh and frozen). Fresh samples
were again divided into two aliquots and treated
with 0 or 1.5 mg CLC/120 £ 106 sperm, as described
previously. After incubation, the cells were centri-
fuged through a 45% Percoll gradient at 600 g for
25 min and then washed free of Percoll, as described
above and the total number of sperm in each sample
determined. Samples to be frozen were similarly
divided into two aliquots and treated with 0 or
1.5 mg CLC/120 £ 106 sperm, as described. The sam-
ples were then processed for cryopreservation as
described in experiment 1, with the exception that
the sperm were frozen at a Wnal concentration of
400 £ 106 sperm/ml. Five straws (1 £ 109 sperm)
were thawed, the contents pooled and then centri-
fuged through 45% Percoll and prepared as the
fresh samples. The cholesterol content of the sam-
ples was determined as described above.
Experiment 4: Fluorescent imaging of cholesterol
incorporation into stallion sperm
Semen from three stallions was extended to
60 £ 106 sperm/ml in TALP and incubated with
3.0 mg CLC/120 £ 106 sperm in which the cyclo-
dextrin had been pre-loaded with cholesterol in
which 20% of the cholesterol was labeled with 22-
(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino)-23,
24-bisnor-5-cholen-3
-ol (NBD; Molecular Probes,
Eugene, OR). After incubation, 5-l drops of sam-
ple were placed onto pre-heated glass slides (37 °C)
and examined using a Nikon Optishot-2 Xuores-
cent microscope equipped with a 450–490 nm exci-
tation Wlter, a 505 nm dichroic mirror and a 520 nm
long-pass Wlter, to determine which sperm mem-
branes contained Xuorescence.
Experiment 5: Zona binding assay of CLC-treated
stallion sperm
Sperm preparation
Straws of cryopreserved sperm for six of the
stallions in experiment 1 were used to determine
the ability of cryopreserved stallion sperm treated
with CLCs to bind to the zona pellucida of bovine
oocytes.
For each replicate, a single straw from each of
two diVerent stallions was thawed as described
above and the contents of each straw diluted 1:3
(v:v) with TALP. The sperm were then washed by
centrifugation at 400g for 5 min, suspended in 1 ml
of TALP containing 35 g/ml Hoechst 33342 (ICN
Biomedical, Aurora, OH) and incubated for 15 min
at 37 °C. The sperm were then washed again (400g
for 5 min) and suspended to a Wnal concentration
of 2 £ 106 sperm/ml in TALP. Five microliters ali-
quots (10,000 sperm) from each sperm suspension
were added to the droplets containing 10 oocytes.
Oocyte preparation
Immature bovine oocytes were recovered from
ovaries obtained from a local abattoir and trans-
ported to the laboratory in saline at 37 °C within
4–5 h of collection. Follicles ranging from 2 to
6 mm were aspirated with a 20 g needle and the fol-
licular Xuid searched for oocytes under a stereomi-
croscope and oocytes placed into TALP after
which the cumulus cells were removed using a
vortex mixer at maximum speed for 2 min. The
 A.I. Moore et al. / Cryobiology 51 (2005) 241–249 245

denuded oocytes were washed in TALP and stored then analyzed by paired t test for the pair-wise
in a hyperosmotic salt solution until use [1]. comparison between treatments [22]. For all exper-
iments, untransformed means are reported.
Zona binding assay

Prior to adding sperm, the oocytes were washed Results

several times in TALP and then incubated in
TALP for approximately 1 h at 38.5 °C in an atmo- Experiment 1
sphere of 5% CO2 in air. After incubation, 10
oocytes were randomly placed into 45 l droplets The percentages of total and progressively
of TALP (1 droplet/treatment) to which sperm motile sperm were similar for control samples and
were added. Oocytes and sperm were incubated samples containing 0.5, 1.5, 3.0, or 4.5 mg CLCs
together at 38.5 °C in an atmosphere of 5% CO2 in (P > 0.05; Table 1). When data for stallions were
air for 2 h after which the oocytes were washed grouped into “poor” and “good” categories, the
four times in TALP, using a small-bore Wre pol- percentages of motile sperm from “poor freezer”
ished glass pipette to remove loosely bound sperm. stallions after cryopreservation, was signiWcantly
Groups of Wve oocytes were placed onto glass higher when the sperm were treated with 1.5 mg
slides and covered with a cover slip supported by a CLC (67%) compared to control samples (50%;
mix of paraYn wax and petroleum jelly. Oocytes P < 0.05; Table 1).
were viewed using an epiXuorescence microscope Sperm frozen at each concentration of CLCs
(Eclipse E800, Nikon Instruments, Melville, NY) exhibited signiWcantly higher percentages of mem-
equipped with a 360/40 nm band pass excitation brane intact sperm compared to sperm frozen in
Wlter and a 460/50 nm band pass emission Wlter. the absence of CLCs for all stallions regardless of
The total number of sperm bound to each zona freezing category (P < 0.05; Table 1).
pellucida was determined at 400£ magniWcations.
Table 1
Statistical analysis The percentage of total, progressively motile, and membrane
intact stallion sperm treated with various levels of cholesterol-
Data for experiments 1–4 were analyzed by loaded cyclodextrins (CLC) prior to cryopreservation
analysis of variance (ANOVA) and treatment CLC dose (mg/120 £ 106 cells)
diVerences separated by Student–Newman–Keuls 0 0.5 1.5 3.0 4.5 6.0 7.5 SEM
(SNK) mean separation technique [22]. Treatments
All stallions (n D 15)
were considered diVerent if P < 0.05. Data from Total 62ab 70a 72a 64ab 55b 43c 38c 4
experiment 1 were transformed to minimize varia- Progressive 45ab 49a 50a 39b 29c 20d 16d 3
tion due to stallions. Data were transformed within Membrane intact 47a 55b 56b 55b 56b 55b 52b 2
each ejaculate by subtracting each CLC treatment “Good” freezers
(n D 8)
from its own control value. A regression analysis
Total 73a 76a 76a 63ab 53b 37c 29c 4
was preformed on data from experiment 3 to deter- Progressive 56a 56a 55a 40b 29c 19d 12d 3
mine the Wt of the regression line. In experiment 5, Membrane intact 50a 57a 58a 57a 58a 56a 52a 4
data were transformed by square root transforma- “Poor” freezers
tion to adjust for unequal variances. To Wnd the (n D 7)
Total 50b 64ab 67a 65ab 58ab 51ab 48b 4
binding potential of an individual sperm cell data
Progressive 32abc 42ab 45a 37ab 29bc 22c 19c 4
were also normalized to take into account the per- Membrane intact 43a 52b 53b 52b 55b 54b 52b 2
centage of motile sperm added to each droplet of a,b,c,d
DiVerent superscripts within rows indicate treatment
oocytes. To Wnd the number of motile sperm diVerences (P < 0.05). Stallions were divided into “Good” freez-
bound to the zona pellucida the average of the ers (n D 8) and “Poor” freezers (n D 7) based on post-thaw pro-
total number of sperm bound for each stallion was gressive motility, with two ejaculates being collected from
divided by the percent motile sperm. Data were “Poor” freezers and the average mean reported.
246 A.I. Moore et al. / Cryobiology 51 (2005) 241–249

1.4
*
1.2 *
*

Cholesterol (µg)
1
*
0.8
0.6 *

0.4
0.2 2
R = 0.99
0
0 2 4 6 8
CLC (mg)
Fig. 1. The concentration of cholesterol (g/106) within the sperm plasma membrane when increasing levels of CLCs (mg/120 £ 106
sperm) were added to fresh cells (n D 12). * diVerence from control value (P < 0.05).

0.8
0.7
Cholesterol (µg)

0.6
0.5
0.4
0.3 c d
0.2
0.1 a b
0
Control CLC

Fig. 2. The concentration of cholesterol (g/10 sperm) in the sperm plasma membrane of cells treated with 0 or 1.5 mg CLC/120 £ 106
6

sperm. The concentration of cholesterol was determined both before (䊐) and after (䊏) cryopreservation (n D 14). a,b,c,dDiVerent labels
denote treatment diVerences (P < 0.05).

Experiment 2 (0.28 vs. 0.20 g cholesterol/106; Fig. 2; P < 0.05).

The amount of cholesterol that became incor-
porated into the stallion sperm increased in a
polynomial fashion (Fig. 1; g Cholesterol D
¡0.0149 (mg CLC)2 + 0.246 (mg CLC) + 0.2291;
R2 D 0.9978). The amount of cholesterol within
the sperm when 0, 0.5, 1.5, 3.0, 4.5, 6.0, or 7.5 mg
CLC are added was 0.23, 0.34, 0.56, 0.86, 1.03,
1.14, and 1.26 g cholesterol/106 sperm. When
71.5 mg CLC was added, the amount of choles-
terol in the sperm was higher than control sperm
(Fig. 1; P < 0.05).
Experiment 3
The concentration of cholesterol in control and
CLC-treated cells decreased after cryopreservation
However, the addition of CLCs to the sperm
increased the cholesterol in sperm both before and
after cryopreservation (0.63 vs. 0.52 g cholesterol/
106; Fig. 2; P < 0.05).
Experiment 4
The cholesterol became incorporated into all
sperm membranes. However, the mitochondria
and acrosomal compartments appeared to be more
heavily labeled (Fig. 3).
Experiment 5
Treatment with CLCs prior to cryopreservation
resulted in more sperm binding to the zona pellu-
cida of bovine oocytes compared to control cells
 A.I. Moore et al. / Cryobiology 51 (2005) 241–249 247

crystals and plasma membrane damage [14,24].

The damage to the sperm membranes occurs as
these membranes undergo a phase transition from
a liquid to crystalline state resulting in the mem-
brane becoming rigid and porous to extracellular
molecules [1]. It has been shown that increasing the
cholesterol/phospholipid ratio within the plasma
membrane of many cell types can reduce the tem-
perature at which the phase transition occurs as
well as maintain a more Xuid membrane at lower
temperatures [12,13,21].
This study reports similar Wndings to Combes
et al. [5] in that cholesterol-loaded into methyl-
-
cyclodextrin improves both the percentages of
motile and membrane intact sperm after cryopres-
ervation. When individual stallions were analyzed,
stallions considered to be “poor” freezers and
treated with CLCs had an increase in the percent-
age of motile cells after cryopreservation, while all
stallions exhibited an increase in the percentage of
Fig. 3. Stallion sperm incubated with 3.0 mg CLC/120 £ 106
membrane intact cells after freezing indicating a
sperm with 20% of the cholesterol-loaded into the cyclodextrin
labeled with NBD. protective eVect of cholesterol on the plasma mem-
brane during cryopreservation. The optimal level
Table 2
of CLC for cryopreservation of equine sperm was
The total number of stallion sperm bound (#SB) and total 1.5 mg CLC/120 £ 106 sperm, which is similar to
number of motile stallion sperm bound (#MSB) to bovine zona that reported for bull sperm [19].
pellucida The concentration of cholesterol that was incor-
Sperm treatment porated into stallion sperm increased in a polyno-
Control CLC mial fashion with CLC level (R2 D 0.9978). It is
a likely that as CLC concentration increased beyond
#SB 15 § 3 48 § 18b
#MSB 0.23 § 0.02a 0.64 § 0.20b 3.0 mg, the sperm became saturated with choles-
terol. A similar phenomenon is seen for bull sperm
Sperm were cryopreserved with 0 or 1.5 mg CLC/120 £ 106
sperm for analysis (n D 6). Data presented as means § SEM. a,b treated with CLCs [19]. This indicates that there is
DiVerent superscripts within rows indicate treatment diVer- a Wnite amount of cholesterol that can incorporate
ences (P < 0.05). into the sperm. The addition of 1.5 mg CLC
increases the cholesterol concentration within the
stallion sperm membranes approximately 2.5
(48 vs. 15; P < 0.05; Table 2). When data were nor- times. This increase in cholesterol, therefore,
malized to determine the binding potential of an increases the cholesterol/phospholipid ratio from
individual sperm, CLC-treated sperm were 2.76 0.36 [18] to approximately 0.82–0.89. This increase
times more likely to bind to the zona pellucida in cholesterol increases the cholesterol/phospho-
than control sperm (Table 2). lipid ratio of stallion sperm to values reported for
human and rabbit sperm, both of which are resis-
tant to cold-shock damage [8]. The actual concen-
Discussion tration of cholesterol within the membranes of
stallion sperm has not been reported. However, the
Equine sperm are damaged during the cryopres- values of cholesterol reported in this study lie
ervation process by formation of intracellular ice between reported values of boar and ram sperm
248 A.I. Moore et al. / Cryobiology 51 (2005) 241–249
(0.16 and 0.28 g/106 sperm, respectively) [8,4]. This
also coincides with the cholesterol/phospholipid
ratios of 0.26, 0.36, and 0.38 for the boar, stallion,
and ram [18]. When Xuorescent microscopy was
used to visual the incorporation of cholesterol into
the stallion sperm, cholesterol could be visualized
throughout the entire sperm. Areas containing
more membranous structures (acrosomal cap and
mitochondria) appeared to Xuoresce brighter.
Cerolini et al. [4] reported that boar sperm lose
approximately 50% of the cholesterol from the
plasma membranes after the cells have been cryo-
preserved. This may be one of the primary causes
of a “pre-mature” capacitation phenotype seen in
cryopreserved sperm cells. It is known that a loss
of cholesterol from the plasma membrane is one of
the Wrst stages of capacitation which decreases the
stability of the membrane [25]. This “pre-mature”
capacitation state of cryopreserved sperm is
thought to be one major reason why cryopreserved
cells do not remain viable in the female reproduc-
tive tract as long as fresh sperm, and therefore, why
cryopreserved sperm must be inseminated closer to
the time of ovulation [2,27]. Data from experiment
3 show that stallion sperm also lose a signiWcant
amount (28%) of cholesterol from the plasma
membranes during the cryopreservation process.
When 1.5 mg CLC was added to the sperm prior to
freezing, the cholesterol concentration was main-
tained at a higher level after cryopreservation. This
higher cholesterol concentration during cryopres-
ervation may inhibit these cells from undergoing
“pre-mature” capacitation and allow the sperm to
remain viable for a longer period of time. This may
reduce the need for intense mare management
when frozen semen is being used.
The zona binding assay determined if sperm
treated with CLCs prior to cryopreservation could
undergo capacitation and bind to the zona pellu-
cida (ZP). Previous studies have shown that equine
sperm bind equally well to equine as bovine ZP
[6,23]. Sinowatz et. al. [23] reported that equine
sperm bound tightly to bovine ZP, underwent the
acrosome reaction and penetrated the ZP after 4 h
of incubation. This suggests that there are similar
ZP protein ligands and sperm receptors between
bovine and equine species. A sperm’s ability to
bind to the ZP has been correlated with fertility of
several species, including stallions [3,9–11,15]. In
this study, there was a signiWcant increase in the
total number of sperm bound to the ZP when
sperm were treated with CLCs. This increase was
also observed when the data were normalized to
account for the number of motile sperm co-incu-
bated with the oocytes. It is thought that sperm
that have been treated with CLCs are protected
from the acute membrane damage and/or loss of
sperm/oocyte receptors that occurs during cryo-
preservation. This may explain why more CLC-
treated sperm were able to bind to the ZP than
control sperm. Although it was not speciWcally
observed in this experiment, previous reports sug-
gest that the sperm that have bound to the ZP will
undergo an acrosome reaction and are capable of
penetration [23]. When CLC-treated bovine sperm
were used for in vitro fertilization, no diVerences
were observed between the ability of CLC-treated
or control sperm to fertilize oocytes [20]. However,
there was a slight increase in pregnancy rates of
CLC-treated sperm (59% vs. 50%) [20]. In vitro fer-
tilization assays are not practical using equine
gametes and further experiments including a fertil-
ity trial need to be preformed to determine the
actual fertilizing potential of CLC-treated equine
sperm.
Conclusions
Adding cholesterol to stallion sperm prior to
cryopreservation increases the cryosurvival rates
of stallion sperm. This is a simple procedure that
alters the cholesterol/phospholipid ratio of sperm
that are aVected by cold shock and cryopreserva-
tion damage. The data reported in this study show
the positive results of CLC addition to stallion
sperm by using several in vitro laboratory assays
including increasing the percentages of motile and
membrane intact sperm, to maintaining higher
cholesterol levels within the sperm after cryopres-
ervation, as well as increasing the ability of the
sperm to bind to the zona pellucida. This technique
may prove beneWcial to cryopreserving sperm from
all stallions in general, but more importantly from
stallions whose sperm routinely do not freeze in
acceptable condition.
 A.I. Moore et al. / Cryobiology 51 (2005) 241–249
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