E. Moc and J. K.

Graham
cryosurvival
Cholesterol-loaded cyclodextrins added to fresh bull ejaculates improve sperm
2006, 84:826-833. J ANIM SCI
http://www.journalofanimalscience.org/content/84/4/826
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Cholesterol-loaded cyclodextrins added to fresh bull ejaculates
improve sperm cryosurvival
1
E. Moce and J. K. Graham
2
Department of Biomedical Sciences, Colorado State University, Fort Collins 80523
ABSTRACT: Damage occurring to spermatozoa dur-
ing cryopreservation results in a loss of motile cells and
cells that are functionally normal, compared with fresh
sperm samples. Treating bull sperm with cholesterol-
loaded cyclodextrins (CLC) before cryopreservation re-
sults in increased sperm cryosurvival. However, in pre-
vious studies, CLCwere always addedto spermsamples
that had been highly diluted. The aim of this study was
to develop a procedure for adding CLC to whole bull
ejaculates that would optimize sperm cryosurvival.
Adding 2 or 4 mg of CLC/120 10
6
sperm to sperm
samples ranging in concentration from 120 to 2,000
10
6
sperm/mL resulted in greater (17 to 28 percentage
points; P < 0.05) numbers of live cells compared with
control samples (no CLC treatment), regardless of the
Key words: bull sperm, cholesterol, cryopreservation, cyclodextrin, freezing rate
2006 American Society of Animal Science. All rights reserved. J. Anim. Sci. 2006. 84:826833
INTRODUCTION
Cryopreservation exposes sperm to mechanical and
anisosmotic stresses (Hammerstedt et al., 1990; Med-
eiros et al., 2002) that reduce cell survival and alter
surviving sperm function (Curry, 2000), in turn, reduc-
ing cell longevity and fertility compared with fresh
sperm. Previous research has improved spermsurvival,
but still only about one-half of the sperm survive freez-
ing (Vishwanath and Shannon, 2000). One dairy indus-
try goal is to reduce the number of sperm in each breed-
ing unit so that more breeding units can be made from
each ejaculate (Vishwanath and Shannon, 2000), but
this canonly be achieved by improving cell cryosurvival.
Sperm sensitivity to cold shock damage is deter-
mined by the membrane phospholipid composition and
1
This work was supported by funds from Secretar a de Estado de
Educacion y Universidades y Fondo Social Europeo (Ref. EX 2003-
1028) and the Colorado State University Experiment Station. The
authors thank Leslie Armstrong-Lea for technical help.
2
Corresponding author: jkgraham@colostate.edu
Received August 8, 2005.
Accepted November 9, 2005.
826
sperm concentration, except for samples at 120 10
6
sperm/mL treated with 4 mg of CLC. Incubating sperm
withCLCat 23 or 37Cbefore cryopreservationresulted
in similar sperm cryosurvival. The cooling rate used
to cryopreserve CLC-treated cells did not affect sperm
cryosurvival. Finally, adding CLC to undiluted ejacu-
lates (2 mg of CLC/120 10
6
sperm) resulted in greater
percentages of live sperm compared with the control
samples (62 vs. 45%; P< 0.05), althoughthe percentages
of motile sperm were similar for both CLC-treated and
control samples (58%). In conclusion, bull sperm cryo-
survival can be improved if spermatozoa are treated
with CLC before freezing. In addition, CLC can be
added to fresh ejaculates at either 23 or 37C. This
technique is simple, practical, and can be easily inte-
grated into current cryopreservation protocols.
the membrane cholesterol to phospholipid ratio (Holt,
2000). Sperm possessing high cholesterol:phospholipid
ratios (e.g., rabbit and human) are more resistant to
cold shock damage than sperm having low choles-
terol:phospholipid ratios (e.g., stallion, ram, and bull;
Watson, 1981; Parks and Lynch, 1992; White, 1993).
Cyclodextrins can be used to alter the cholesterol
content of cell membranes (Christian et al., 1997; Vis-
conti et al., 1999), and if cyclodextrins are preloaded
with cholesterol they insert cholesterol into membranes
(Navratil et al., 2003). If stallion (Combes et al., 2000;
Moore et al., 2005a), bull (Purdy and Graham, 2004),
or ram (Morrier et al., 2004) sperm are treated with
cholesterol-loaded cyclodextrins (CLC) before freezing,
they exhibit greater cryosurvival rates than untreated
sperm. However, CLC were added to sperm at sperm
concentrations (120 10
6
sperm/mL) that are imprac-
tical for industry settings. If CLC could be added to
fresh ejaculates and still benet sperm cryosurvival,
this technology could have practical application.
These studies were conducted to determine if the
spermconcentration alters the benecial effects of CLC
on sperm cryosurvival, and to develop a procedure for
adding CLC to fresh bull ejaculates to improve sperm
cryosurvival.
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Cholesterol addition improves sperm cryosurvival 827
MATERIALS AND METHODS
Four experiments were conducted to develop a proto-
col for adding CLC to fresh bull ejaculates before freez-
ing in an effort to facilitate the use of CLC technology
for improving sperm cryosurvival in a practical setting.
The rst experiment investigated the effects of sperm
concentration on sperm cryosurvival in the presence
of CLC. The second experiment investigated whether
temperature affected the ability of CLC to interact with
the sperm. The third experiment tested whether the
optimal cooling rate for cryopreserving sperm was al-
tered by CLC treatment. Results from these experi-
ments led to the development of a protocol for adding
CLC to undiluted bull ejaculates, and this protocol was
evaluated in the last experiment to determine whether
it was benecial in a practical setting.
Animals
Semen was collected using an articial vagina from
10 mature Holstein bulls housed at the Animal Repro-
duction Laboratory at Colorado State University (Fort
Collins). The bulls were fed a diet that provided 100%
of their nutritional needs (NRC, 2000) and provided
water ad libitum. All animal care and procedures used
to collect semen were approved by the Animal Care and
Use Committee of Colorado State University.
Materials
All chemicals were reagent grade and purchased from
Sigma (St. Louis, MO), except for SYBR-14 and propid-
ium iodide (PI), which were purchased from Molecular
Probes (Eugene, OR).
Cyclodextrin Preparation
Methyl--cyclodextrin was preloaded with choles-
terol as described by Purdy and Graham(2004). Briey,
200 mg of cholesterol was dissolved in 1 mL of chloro-
form. In a separate test tube, 1 mg of cyclodextrin was
dissolved in 2 mL of methanol. A 0.45-mL aliquot of
cholesterol was added to the cyclodextrin, and the mix-
ture was stirred until the combined solution was clear.
The solution was then poured into a glass Petri dish,
and the solvents were removed and allowed to dry for
24 h, after which the resulting crystals were removed
from the dish and stored in a glass container at room
temperature. A working solution of CLC was prepared
by adding 50 mg of CLC to 1 mL of Tris diluent (250
mM tris[hydroxymethyl]aminomethane, 83 mM citric
acid monohydrate, 69 mM D-(+)-glucose, pH = 7.0, 300
mOsm) at 37C and mixing the solution briey using
a vortex mixer (Purdy and Graham, 2004).
Analysis of Spermatozoa
The percentages of motile and progressively motile
spermatozoa in each sample were determined using a
computer-assisted sperm analysis system (IVOS Ver-
sion 10.7s; Hamilton Thorne Research, Bedford, MA)
withsettings of 30 frames acquired to avoid spermtrack
overlapping, minimum contrast = 50, minimum aver-
age pathvelocity =25 m/s, straightness = 80%, nonmo-
tile head size = 5, and nonmotile head intensity = 90.
For eachsample, 6-Lsubsamples were placedonslides
and a minimum of 200 cells per subsample were ana-
lyzed. A sperm was dened as being progressively mo-
tile if the average path velocity was >60 m/s, and
straightness was >80%. Sperm straightness is the ratio
of the straight-line velocity divided by the average path
velocity, as dened by Budworth et al. (1988).
The percentages of viable spermatozoa inthe samples
were determined using ow cytometry, as described by
Purdy and Graham (2004). Briey, spermatozoa were
stained for ow cytometric analysis by transferring a
0.1-mL aliquot fromeach sample into a tube containing
0.45 mLof Tris-BSA(6 mg/mL) diluent, 8.5 Lof SYBR-
14 (10 M solution in dimethyl sulfoxide), and 5 L of
PI (1 mg/mL solution in distilled water). The samples
were incubated for 10 min at 22C and ltered through
a 40-m nylon mesh before being analyzed using an
Epics Vowcytometer (Coulter Electronics, Miami, FL)
equipped with an argon ion laser tuned to 488 nm at
100 mW of power. Fluorescence from 50,000 cells was
measured using a 515-nm long-pass lter and a 525-
nm band-pass lter to detect SYBR-14, and a 590-nm
dichroic mirror and a 630-nm long-pass lter to detect
PI. Using this protocol, all cells stained with SYBR-14,
which permitted the cells to be distinguished from egg
yolk particles, but only nonviable cells stained with PI.
Experiment 1: Effect of Sperm and CLC
Concentrations on Cell Cryosurvival
Immediately after collection, the concentration of
spermatozoa in each of 10 ejaculates was determined
photometrically, and the spermatozoa were diluted to
5 sperm concentrations (from 120 to 2,000 10
6
cells/
mL) with the Tris diluent. Only 3 ejaculates had a
sperm concentration of at least 2,000 10
6
cells/mL;
therefore, this treatment had only 3 replicates. Each of
the 5 sperm concentrations was then split into 3 CLC-
treatment aliquots (0.5 mL each for the samples at 120
and 360 10
6
cells/mL, 0.4 mL for the samples at 720
10
6
cells/mL, and 0.2 mL for the samples at 1,200 and
2,000 10
6
cells/mL). One of the aliquots was used as
a control (non-CLC treated), 1 was treated with 2 mg
of CLC/120 10
6
sperm, and the other was treated with
4 mg of CLC/120 10
6
sperm. Samples were incubated
for 15 min at 22C, after which each sample was diluted
to 60 10
6
cells/mL with egg yolk-Tris diluent (Tris
diluent with 20% egg yolk).
Spermatozoa were then cooled slowly to 5C over 2 h,
and then diluted 1:1 (vol:vol) with glycerolated (17.5%
glycerol) egg yolk-Tris diluent (resulting in a nal glyc-
erol concentration of 8.75%), and equilibrated for 15
min. Sperm were then packaged into 0.5-mL straws
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Moce and Graham 828
(IMVTechnologies, LAigle, France) and frozeninliquid
nitrogen vapor, with the straws being suspended hori-
zontally at 4.5 cm above the liquid nitrogen for 10 min
before being plunged into the liquid nitrogen for
storage.
Two straws from each treatment were thawed in a
water bathat 37Cfor 30 s before analysis. The contents
of one straw were used to determine the percentage of
motile sperm in each treatment, and that of the second
strawused to determine the percentage of viable sperm
for each treatment.
Analysis of variance (2005 version, SAS Inst. Inc.,
Cary, NC) was used to determine the main effects of
spermconcentration (5 levels), CLC level (3 levels), and
the interaction of these effects. Individual treatment
means were separated using Student-Newman-Keuls
multiple range test (SAS Inst. Inc.). However, we also
were interested in knowing if the sperm concentration
affected cell survival; therefore, ANOVA was used to
determine if different sperm concentrations within a
CLClevel resulted in different cryosurvival rates; these
data are presented in the tables. In this and all experi-
ments, the effects were considered signicant if P <
0.05.
Experiment 2: Effect of Sperm Concentration,
CLC Level, and Temperature of Incubation
on Sperm Cryosurvival
Nine ejaculates were collected as described above,
the concentration of sperm in each ejaculate was deter-
mined photometrically, and each ejaculate was split
into 4 fractions. Three of the fractions were then diluted
to 120, 1,200, or 1,500 10
6
cells/mL (this highest
sperm concentration utilized the fresh ejaculates,
which ranged from 1,500 to 2,000 10
6
cells/mL) with
the Tris diluent. Each of these was then split into 4
treatment aliquots (for 120 10
6
sperm/mL, 0.5 mL
aliquots were used, and for the samples at 1,200 and
1,500 10
6
cells/mL, 0.2 mL aliquots were used). One
set of 2 aliquots was treated with a total of 2 and 4 mg
CLC/120 10
6
cells, and was incubated for 15 min at
22C. The other set of 2 aliquots was treated similarly,
but was incubated at 37C. The fourth fraction was not
diluted, but was maintained as the control ejaculate
(non-CLC treated); it was split and 1 aliquot was main-
tained at 22C, whereas the other was maintained at
37C for 15 min. After incubation, each sample was
diluted to 60 10
6
cells/mL in egg yolk-Tris diluent and
cooled to 5C as described above. Upon reaching 5C,
each aliquot was diluted 1:1 with glycerolated egg yolk-
Tris diluent, frozen, thawed, and evaluated as de-
scribed above.
As in Exp. 1, ANOVA (SAS Inst. Inc.) was used to
determine the main effects of sperm concentration (3
levels), CLC level (3 levels), and temperature of incuba-
tion (2 levels), and all of their interactions. Individual
treatments within each main effect were separated us-
ing Student-Newman-Keuls multiple range test (SAS
Inst. Inc.). In this study, we were interested in the
sperm concentration CLC level treatment differences
within each incubation temperature. Therefore, the
data are presented as such in the tables.
Experiment 3: Effect of Freezing Rate
Immediately after collection, the concentration of
spermatozoa in each of 9 ejaculates was determined
and each ejaculate was split into 2 aliquots. One aliquot
was used as a control (no CLC treatment) and the other
was treated with 2 mg of CLC/120 10
6
sperm, and
incubated for 15 min at 22C. After incubation, 60
10
6
cells were added to 1 mL of the egg yolk-Tris diluent
and then cooled to 5C, diluted 1:1 with glycerolated
egg yolk-Tris diluent, and packaged into 0.5-mL plas-
tic straws.
Spermwere frozeneither inthe liquidnitrogenvapor,
or in a programmable freezer (Planer Kryo 10 Series
III, TS Scientic, Perkasie, PA). Straws were frozen in
the programmable freezer at 8 freezing rates (5, 8,
10, 12, 15, 25, 35, or 50C/min) from 5C until
they reached 80C, and then were stored in liquid
nitrogen. For the samples frozen in liquid nitrogen va-
por, straws were suspended horizontally for 10 min at
8 heights (1.5, 2.5, 3.5, 4.5, 5.5, 6.5, 7.5, or 8.5 cm) above
the liquid nitrogen before being plunged into the liquid
nitrogen for storage. Straws were thawed and evalu-
ated as described above.
The cooling rates used for the straws frozen in liquid
nitrogen vapor were determined by measuring the tem-
perature inside a strawat each level using a thermocou-
ple, and calculating the initial cooling rate.
For this experiment, ANOVA (SAS Inst. Inc.) was
used to evaluate the main effects of CLC treatment and
cooling rates, and the interaction between them. The
Student-Newman-Keuls multiple range test (SAS Inst.
Inc.) was used to separate the cooling rate means.
Experiment 4: Addition of CLC
to Undiluted Ejaculates
Immediately after collection, the concentration of
spermatozoa in each of 61 ejaculates was determined,
and each ejaculate was split into 2 aliquots. One aliquot
was used as a control (no CLC treatment) and the other
was treated with 2 mg of CLC/120 10
6
sperm, and
incubated for 15 min at 22C. After incubation, 60
10
6
cells were added to 1 mLof the egg yolk-Tris diluent,
cooled to 5C, diluted 1:1 with glycerolated egg yolk-
Tris diluent, frozen, thawed, and evaluated as de-
scribed above.
In this experiment, ANOVA(SAS Inst. Inc.) was used
to determine if the cryosurvival rate of CLC-treated
sperm was different from control (nontreated) sperm.
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Cholesterol addition improves sperm cryosurvival 829
RESULTS
Experiment 1: Effect of Sperm and CLC
Concentrations on Cell Cryosurvival
The initial concentrationto whichspermwere diluted
before CLC treatment and subsequent cryopreserva-
tion did not affect the percentage of motile sperm after
thawing. However, sperm concentration at the time of
CLCadditiondid affect the percentages of progressively
motile spermand live spermafter thawing. Progressive
motility was less for CLC-treated sperm diluted to 120
10
6
sperm/mL (14%) than for spermtreated with CLC
at 2,000 10
6
sperm/mL (28%; P < 0.05; data not
shown). Similarly, the percentage of live spermwas less
for samples diluted to 120 10
6
sperm/mL before CLC
treatment (37%) than for samples treated with CLC at
greater sperm concentrations (49 to 56%; P < 0.05; data
not shown). Analysis for the maineffect of CLCrevealed
that samples treated with 2 or 4 mg of CLC/120 10
6
sperm exhibited greater percentages of live cells (61
and 48%, respectively) than control (38%) sperm (P <
0.05; data not shown). In addition, a signicant interac-
tion between sperm concentration and CLC level was
observed in the percentages of live cells. This interac-
tion was specically due to a detrimental effect of CLC
treatment to samples diluted to 120 10
6
sperm/mL
before CLCaddition, whereas this same CLCtreatment
beneted sperm at all other sperm concentration
treatments.
The concentration of sperm to which cells were ini-
tially diluted before adding CLCdid not affect postthaw
percentages of total motile and live cells, except for
sperm treated with 4 mg of CLC/120 10
6
sperm. At
this CLC concentration, sperm diluted to 120 10
6
/mL
before CLC addition exhibited lower percentages of live
cells, total motile cells, and progressively motile cells
than samples at greater initial sperm concentrations
(P < 0.05; Table 1).
Experiment 2: Effect of Sperm Concentration,
CLC Level, and Temperature of Incubation
on Sperm Cryosurvival
Similar to Exp. 1, sperm concentration affected the
percentages of total motile and live sperm, after cryo-
preservation(P< 0.05). Again, the percentages of motile
sperm and live sperm were less for samples diluted to
120 10
6
sperm/mL (47 and 40%, respectively; data
not shown) than for samples at greater sperm concen-
trations (56 to 63% and 51 to 52%, respectively; P <
0.05). In addition, adding CLC to the samples improved
the percentages of total motile (52 to 58%), and live
cells (44 to 51%) compared with control samples (44
and 21%, respectively; P < 0.05; data not shown). No
interactions between the main effects of sperm concen-
tration, CLC concentration, and temperature were
found for any sperm variable.
Temperature of incubation did not affect the percent-
ages of total motile or live sperm except for sperm di- T
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Moce and Graham 830
Table 2. Percentages of motile and live sperm after cryopreservation when bull sperm diluted to different cell
concentrations were treated with 0, 2, or 4 mg of cholesterol-loaded cyclodextrins (CLC; per 120 10
6
cells) and
incubated at 22 or 37C before cryopreservation (n = 9)
0 mg of
CLC (control)
2 mg of CLC 4 mg of CLC
Sperm concentration Fresh Pooled
(millions/mL): ejaculate 120 1200 1500-2000 120 1200 1500-2000 SEM
Sperm count
Total motile sperm, %
22C 47
b
63
a
* 55
ab
62
a
46
b
46
b
65
a
6
37C 42
ab
45
ab
58
ab
65
b
34
b
59
ab
61
ab
6
Progressively motile sperm,
1
%
22C 4
b
11
a
* 7
ab
8
ab
7
ab
5
ab
10
ab
2
37C 18 6 8 7 5 7 7 5
Live sperm, %
22C 23
c
49
ab
56
a
55
a
37
b
47
ab
52
a
4
37C 18
b
44
a
52
a
49
a
30
b
48
a
51
a
4
ac
Means with different superscripts within a row differ, P < 0.05.
1
Progressively motile sperm had an average path velocity >60 m/s and straightness (straight line velocity/average path velocity) >80%.
*Indicates a difference between temperatures within a column, P < 0.05.
luted to 120 10
6
/mL and treated with 2 mg of CLC
(Table 2). Cells diluted to 2,000 10
6
sperm/mL and
treated with CLC exhibited greater percentages of both
motile and live spermafter thawing than control sperm
(P < 0.05; Table 2). However, sperm at lower concentra-
tions generally showed greater percentages of live cells
when treated with CLC, although percentages of motile
cells were often not different from controls (Table 2).
Experiment 3: Effect of Freezing Rate
Similar to previous experiments, samples treated
with CLCbefore freezing exhibited greater percentages
of total motile, progressively motile, and live sperm(72,
14, and 33%, respectively) than control sperm (55, 10,
and 21%, respectively; P < 0.05; data not shown).
The cooling rates for sperm frozen at different dis-
tances above the liquid nitrogen level ranged from 51
to 114C/min (Table 3). However, samples frozen at
Table 3. Percentages of motile and live sperm after cryopreservation of bull sperm frozen at different levels above
the liquid nitrogen and treated with 0 or 2 mg of cholesterol-loaded cyclodextrins (CLC; per 120 10
6
cells) before
cryopreservation (n = 9)
Freezing rate (height and cooling rate)
1.5 cm, 2.5 cm, 3.5 cm, 4.5 cm, 5.5 cm, 6.5 cm, 7.5 cm, 8.5 cm, Pooled
Sperm count 114C/min 109C/min 97C/min 91C/min 86C/min 65C/min 56C/min 51C/min SEM
Total motile sperm, %
No CLC 45 62 53* 57 59 52 59 56 8
2 mg of CLC 61 72 77 75 79 71 72 73 6
Progressively motile sperm,
1
%
No CLC 8 14 10 11 10 10 9 9 2
2 mg of CLC 14* 17 15 16* 14 14 11 13 2
Live sperm, %
No CLC 16 20 21 21 22 24 22 21 3
2 mg of CLC 21
b
26
ab
30
ab
32
ab
36
ab
* 38
a
* 39
a
* 41
a
* 4
a,b
Means with different superscripts within a row differ, P < 0.05.
1
Progressively motile sperm had an average path velocity >60 m/s and straightness (straight line velocity/average path velocity) >80%.
*Indicates a difference between CLC levels within a column, P < 0.05.
different levels above the liquid nitrogen level exhibited
similar percentages of total and progressively motile
sperm at all cooling rates and CLC levels (0 or 2 mg/
120 10
6
). Differences in the percentages of live sperm
were observed among cooling rates when CLC-treated
sperm were frozen 1.5 cm above the liquid nitrogen
level. This rapid cooling rate resulted in lower percent-
ages of live cells (21%) than samples frozen at 6.5 cm
(38 to 41%; P < 0.05; Table 3). Control and CLC-treated
sperm exhibited a difference in percentage of total mo-
tile sperm only when cells were frozen 3.5 cm above
the liquid nitrogen (53 vs. 77%, respectively; P < 0.05;
Table 3). For the other cooling rates, similar percent-
ages of motile control and CLC-treated sperm were ob-
served, although the percentages of total motile sperm
were between 10 and 24% greater for the CLC-treated
samples than for the control samples (Table 3). Sperm
treated with CLC exhibited greater percentages of pro-
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Cholesterol addition improves sperm cryosurvival 831
Table 4. Percentages of motile and live sperm after cryopreservation of bull sperm frozen
in a programmable freezer at different freezing rates and treated with 0 or 2 mg of
cholesterol-loaded cyclodextrins (CLC; per 120 10
6
cells) before cryopreservation (n = 9)
Freezing rate, C/min
Pooled
Sperm count 5 8 10 12 15 25 35 50 SEM
Total motile sperm, %
No CLC 62 70 70 68 66 69 70 57 6
2 mg of CLC 76 71 73 77 76 76 80 68 5
Progressively motile sperm,
1
%
No CLC 11 14 13 13 14 16 13 10 2
2 mg of CLC 13 13 13 14 16 16 16 13 2
Live sperm, %
No CLC 31 31 31 29 32 31 26 20 4
2 mg of CLC 44 45* 41 44* 43 40 34 34* 4
1
Progressively motile spermhad an average path velocity >60 m/s and straightness (straight line velocity/
average path velocity) >80%.
*Indicates a difference between CLC levels within a column, P < 0.05.
gressively motile sperm than control samples when
spermwere frozen 1.5 and 4.5 cmabove the liquid nitro-
gen level (P < 0.05; Table 3). Percentages of live cells
were signicantly greater (P < 0.05) for CLC-treated
sperm than for control samples (between 14 and 20%;
Table 3) when sperm were frozen 5.5, 6.5, 7.5, and 8.5
cm above the liquid nitrogen level.
When sperm were frozen in the programmable
freezer, the percentages of total motile, progressively
motile, or live sperm were similar for control and CLC-
treated samples across all cooling rates. However, the
percentages of live cells were greater for CLC-treated
sperm samples cooled at 8, 12, and 50C/min than
for control samples cooled at the same rates (P < 0.05;
Table 4).
Experiment 4: Addition of CLC
to Undiluted Ejaculates
When undiluted ejaculates were treated with 2 mg of
CLC/120 10
6
sperm before cryopreservation, greater
percentages of live cells were observed (62%) than for
control sperm (45%; P < 0.05; Table 5), although the
percentages of total motile spermand progressively mo-
Table 5. Percentages of motile and live sperm after cryo-
preservation of undiluted bull ejaculates with 0 or 2 mg
of cholesterol-loaded cyclodextrins (CLC; per 120 10
6
cells), before cryopreservation (n = 61)
Treatment
Control 2 mg Pooled
Sperm count (no CLC) of CLC SEM
Total motile sperm, % 58 58 3
Progressively motile sperm,
1
% 22 19 2
Live sperm, % 45
b
62
a
2
a,b
Means with different superscripts within a row differ, P < 0.05.
1
Progressively motile sperm had an average path velocity >60 m/
s and straightness (straight line velocity/average path velocity) >80%.
tile sperm were similar for the control and the CLC-
treated sperm (Table 5).
DISCUSSION
Treating sperm with CLC resulted in increased per-
centages of live cells (from 17 to 26 percentage points
greater than the control samples) regardless of sperm
concentration, when sperm were treated with 2 mg of
CLC/120 10
6
sperm. Previous studies using diluted
bull or stallion sperm reported similar increases in the
percentage of live sperm (between 9 and 20 percentage
points greater compared with the control samples)
when similar concentrations of CLC were added to
sperm before cryopreservation (Combes et al., 2000;
Purdy and Graham, 2004; Moore et al., 2005a). Regard-
less of sperm concentration, 2 mg of CLC/120 10
6
sperm increased both the percentages of motile sperm
and live sperm at all sperm concentrations, including
when CLC were added to fresh ejaculates. However,
when the sperm concentration was adjusted to 120
10
6
sperm/mL before CLC treatment, treatment with
4 mg of CLC/120 10
6
cells reduced spermcryosurvival.
This could be due to a reduced egg yolk concentration
in the nal sample when the sperm concentration was
adjusted to 120 10
6
cells/mL (15% egg yolk) with re-
spect to the other treatments (between 18.3 and 19.75%
egg yolk), or because this CLC concentration is detri-
mental for sperm survival when added to low sperm
concentrations. However, neither possibility explains
this result completely, because Graham and Ham-
merstedt (1992) did not observe differences in bull
spermcryosurvival when spermwere frozen in diluents
containing either 1% or 20% egg yolk, and Purdy and
Graham (2004) did not observe a decrease in sperm
cryosurvival when sperm were treated with CLC until
CLC concentration reached >6 mg/120 10
6
cells. Even
then, cryosurvival of CLC-treated sperm remained
greater than that of control samples. Therefore, the
reason for decreased sperm cryosurvival for samples
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Moce and Graham 832
containing this low sperm concentration when treated
with 4 mg CLC in this particular experiment is not
known.
The temperature at which sperm were incubated
with CLC had no effect on subsequent spermcryosurvi-
val. Only when samples were adjusted to 120 10
6
sperm/mL before treatment with 2 mg of CLC, did incu-
bation temperature affect cryosurvival. In this situa-
tion, sperm incubated at room temperature exhibited
greater percentages of total and progressively motile
sperm than did the sperm incubated at 37C. These
results are similar to those reported previously by
Purdy and Graham (2004) for bull sperm. Although we
did not observe differences between incubation temper-
atures for the control samples, the trend was the same
(spermincubated at 22C exhibited 5 percentage points
greater motile and live spermthan the spermincubated
at 37C). Because sperm can be incubated with CLC
at either of the temperatures tested, this protocol is
sufciently exible with respect to the incubation tem-
perature to make its use feasible for industry appli-
cation.
When sperm are frozen at suboptimal freezing rates,
sperm cryosurvival decreases. A cooling rate that is too
slow exposes spermatozoa to the solution effects of
increasing salt concentration(osmolality increases) and
changing pH. Moreover, a cooling rate that is too fast
does not allow intracellular water sufcient time to
leave the cell, whichleads to intracellular ice nucleation
(Vishwanath and Shannon, 2000). This experiment was
conducted to determine if CLC-treated sperm require
a different cooling rate than that for control sperm.
However, sperm cryosurvival was similar for all freez-
ing rates tested for sperm frozen in a programmable
freezer. For sperm frozen at different heights above the
liquid nitrogen level, differences were only observed in
the percentages of live cells for CLC-treated sperm,
which exhibited greater percentages of live cells when
samples were frozen at 6.5, 7.5, and 8.5 cm above the
liquid nitrogen level (estimated cooling rates between
51 and 65C/min). Previous studies report contradic-
tory results with respect to the optimal rate for freezing
bull sperm. Thus, optimal freezing rates for bull sperm
have been reported between 15 to 52C/min (Robbins
et al., 1976; Chen et al., 1993; Kumar et al., 2003) and
40 to 140C/min (Woelders et al., 1997). Differences
in the percentages of live cells between CLC-treated
and control spermwere only observed when spermwere
frozen at a rate of 8, 12, or 50C/min, for sperm
frozen in a programmable freezer, whereas differences
in the percentages of live cells between CLC-treated
and control spermwere only observed when spermwere
frozen at 5.5, 6.5, 7.5, and 8.5 cm above the liquid nitro-
gen level. However, the percentages of motile and live
cells were greater at all the freezing rates for CLC-
treated sperm, compared with control sperm.
Addition of CLC to fresh ejaculates resulted in
greater percentages of live cells after thawing, com-
pared with the non-CLC treated samples, although the
percentages of motile spermwere similar betweenCLC-
treated and control sperm. As discussed above, the in-
crease in the percentages of live cells is similar to that
reported for spermfrombulls and stallions treated with
CLC (Combes et al., 2000; Purdy and Graham, 2004;
Moore et al., 2005a). However, previous studies gener-
ally report an increase in the percentages of motile
sperm after cryopreservation (Combes et al., 2000;
Purdy and Graham, 2004; Moore et al., 2005a) as well.
The reason we did not observe a similar increase in
motile sperm is not known. However, the motility anal-
yses for stallion sperm were performed after cryopre-
served sperm were further diluted 2- to 4-fold after
thawing (Combes et al., 2000; Moore et al., 2005a), simi-
lar to the dilution performed for the viability analysis
in this study (dilution 1:4.5). Perhaps differences in
motility between CLC-treated and nontreated sperm
are more easily separated for sperm subjected to os-
motic stress. This might be expected because CLCtreat-
ment increases the osmotic tolerance limits of stallion
spermcompared with the control samples (Moore et al.,
2005b). However, Purdy and Graham (2004) reported
an increase in the percentage of motile sperm after
thawing for bull sperm that had been treated with CLC
before cryopreservation, when motility analyses were
performed on samples that were not diluted further
after thawing. The fact that treating sperm with CLC
before cryopreservation resulted in increased percent-
ages of live cells is very important, because these cells
have experienced all the processes that sperm have to
undergo osmotically when inseminated into a female.
The exact mechanismby whichadded cholesterol pro-
tects spermmembranes is not known. As stated earlier,
species with high cholesterol to phospholipid ratios
(rabbit and human) are resistant to cold shock. At least
part of the sperm damage induced from cold shock is
due to the lipid phase transition that the membrane
experiences during the cooling process. Human sperm
are resistant to cold shock, and an abrupt lipid phase
transition has not been detected when they are cooled
(Drobnis et al., 1993). High cholesterol levels stabilize
membranes during cooling. The cholesterol content in
the membranes of bull and stallion sperm increased 2-
to 3-fold compared with control sperm after treatment
with CLC (Purdy and Graham, 2004; Moore et al.,
2005a), and it remains greater than in control sperm
after cryopreservation (Moore et al., 2005a). This in-
creased cholesterol content in bull and stallion sperm
raises the cholesterol to phospholipid ratio in these
spermto values similar (0.82 to 0.9; Purdy and Graham,
2004; Moore et al., 2005a) to those of sperm that are
cold-shock resistant (0.88 and 0.99 for rabbit and hu-
man sperm, respectively; Watson, 1981). It is likely that
the lipid phase transition is eliminated or the tempera-
ture at which it occurs is lower for CLC-treated bull
and stallion sperm than for control sperm. Supporting
this idea, Purdy et al. (2005) demonstrated increased
membrane uidity at lower temperatures for bull sperm
treated with CLC than for untreated sperm. On the
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Cholesterol addition improves sperm cryosurvival 833
other hand, cholesterol could be increasing spermmem-
brane permeability to cryoprotectants and lessening os-
motic cell damage, because CLC treatment increases
the osmotic tolerance of stallion sperm (Moore et al.,
2005b). This is very important because sperm experi-
ence large volume changes when cryoprotectants are
added or removed, and their membranes can suffer
damage during these processes (Graham, 1996). Fur-
ther studies need to be conducted to determine the exact
mechanism by which cholesterol affects sperm mem-
branes during cooling.
IMPLICATIONS
Treating fresh bull ejaculates with 2 mg of choles-
terol-loaded cyclodextrins/120 10
6
sperm increased
the cryosurvival rate of the cells. The improved cryosur-
vival was similar regardless of whether sperm were
incubated with cholesterol-loaded cyclodextrins at 22
or 37C. These sperm can be frozen using a wide range
of cooling rates without reducing sperm cryosurvival.
Therefore, treating fresh ejaculates with cholesterol-
loaded cyclodextrins caneasily be incorporated into cur-
rent cryopreservation techniques for practical appli-
cation.
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