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Theriogenology 67 (2007) 238–248
Cryopreservation of immature and in vitro matured
porcine oocytes by solid surface vitrification
Mukesh Kumar Gupta, Sang Jun Uhm, Hoon Taek Lee *
Department of Animal Biotechnology, Bio-Organ Research Center, Konkuk University,
1 Hwayang-dong, Gwangjin-Gu, Seoul 143 701, South Korea
Received 1 May 2006; accepted 4 July 2006
Abstract
Cryopreservation of normal, lipid-containing porcine oocytes has had limited practical success. This study used solid surface
vitrification (SSV) of immature germinal vesicle (GV) and mature meiosis II (MII) porcine oocytes and evaluated the effects of
pretreatment with cytochalasin B, cryoprotectant type (dimethylsulfoxide (DMSO), ethylene glycol (EG), or both), and warming
method (two-step versus single-step). Oocyte survival (post-thaw) was assessed by morphological appearance, staining (30,60-diacetyl fluorescein), nuclear maturation, and developmental capacity (after in vitro fertilization). Both GV and MII oocytes were
successfully vitrified; following cryopreservation in EG, more than 60% of GV and MII stage porcine oocytes remained intact (no
significant improvement with cytochalasin B pretreatment). Oocytes (GV stage) vitrified in DMSO had lower (P < 0.05) nuclear
maturation rates (31%) than those vitrified in EG (51%) or EG + DMSO (53%). Survival was better with two-step versus single-step
dilution. Despite high survival rates, rates of cleavage (20–26%) and blastocyst formation (3–9%) were significantly lower than for
non-vitrified controls (60 and 20%). In conclusion, SSV was a very simple, rapid, procedure that allowed normal, lipid-containing,
GV or MII porcine oocytes to be fertilized and develop to the blastocyst stage in vitro.
# 2006 Published by Elsevier Inc.
Keywords: Solid surface vitrification; Cryopreservation; Oocyte; Ovary; Porcine
1. Introduction
Cryopreservation of oocytes has important roles in
the preservation and management of genetic resources,
low-cost international movement of selected genetics,
and rapid dissemination of germplasm via in vitro
embryo production, genetic engineering and nuclear
transfer procedures. It has been successfully applied to
livestock including cattle, goats, sheep, and other model
animals, but has met with limited practical success in
pigs, as porcine oocytes are intrinsically very sensitive
* Corresponding author. Tel.: +82 2 4503675; fax: +82 2 4578488.
E-mail address: [email protected] (H.T. Lee).
0093-691X/$ – see front matter # 2006 Published by Elsevier Inc.
doi:10.1016/j.theriogenology.2006.07.015
to cooling [1,2]. The presence of a large number of
intracytoplasmic lipid droplets in the oocytes has been
considered an obstacle to freezing; lipid reduces the
cryotolerance of the oocytes and cause irreversible
damage to membrane structure at temperatures between
+10 and �5 8C [3]. To circumvent this problem,
Nagashima et al. [4] introduced the concept of
delipation before freezing, which could improve the
survival rate of embryos. However, delipation itself
could compromise embryo viability, as intracellular
lipids are a source of oocyte energy and exist as
complexes of ‘‘smooth endoplasmaic reticulum–lipid
globules–mitochondria’’ in cells [5]. In several studies,
in vivo survival rates of dilapidated vitrified embryos
have been fairly low (range, approximately 10–15%),
M.K. Gupta et al. / Theriogenology 67 (2007) 238–248 239
although pregnancy rates of approximately 80% have
been achieved [6,7].
Vitrification is an alternative to traditional freezing
methods to avoid chilling injury and ice crystal
formation [8]. This technique has become the standard
cryopreservation procedure for porcine embryos,
mainly at the blastocyst stage, with several studies
reporting live offspring after the transfer of vitrified
porcine embryos [6–8]. However, it has not given
satisfactory results for porcine oocytes. During the last
several years, important advances in embryo/oocyte
cryobiology optimized vitrification methodology,
including the use of open pulled straws [6], superfine
open pulled straws [10] and microdroplet methods [7].
The key to success of these methods was a reduction in
the amount of vitrification medium surrounding an
oocyte/embryo, which allowed them to rapidly pass
through a critical temperature zone in the presence of a
cryoprotective agent [11,12]. Piglets have been success-
fully produced after transferring rapidly vitrified
embryos to recipients [6,7,9]. More recently, we
reported the successful vitrification of embryos using
simple weighing paper, which also resulted in produc-
tion of live calves ([13,14], Lee et al., unpublished data).
However, even these protocols have only limited
success for porcine oocytes, as demonstrated by low
survival after warming, low rates of in vitro maturation
[15,16] and reduced development to blastocysts after
intracytoplasmic sperm injection or parthenogenetic
activation [17,18]. Park et al. [19] reported nuclear
maturation rate of vitrified GV stage oocytes to be 3.7%
without delipation and 15.0% after delipation. How-
ever, no blastocysts were obtained, although a cleavage
rate of 16% was obtained following intracytoplasmic
sperm injection.
Solid surface vitrification (SSV) is a simple method
that involves placing oocytes/embryos in a small
amount of cryoprotectant solution and cooling this as
a small drop directly on a metal surface cooled by liquid
nitrogen or liquid nitrogen vapor [20]. High rates of
survival and development were reported for in vitro-
matured oocytes derived from cattle [21], goats [22],
and monkeys [23] cryopreserved with SSV. Somfai
et al. [17] recently applied SSV to pig oocytes followed
by parthenogenetic activation, but development was
low; therefore, further improvements are required
before the widespread application of this technology
to pigs. Furthermore, since cryopreservation can alter
the meiotic spindle assembly, microtubules, cortical
granule distribution, zona pellucida characteristics, and
cause chromosomal aberrations [24–26], it is important
to determine the development ability of vitrified oocytes
after IVF. However, no data are available regarding
developmental ability after IVF of frozen–thawed
porcine oocytes cryropreserved with SSV. Therefore,
the objectives of this study were to investigate the
effects of SSV method on viability and in vitro
development of porcine oocytes following IVF. The
study evaluated the effect of: (1) nuclear status; (2)
cytochalasin B treatment; (3) warming procedure; (4)
cryoprotectant solutions (ethylene glycol (EG) alone,
DMSO alone or EG + DMSO) on oocyte viability, in
vitro maturation and subsequent developmental capa-
city (after IVF).
2. Materials and methods
2.1. Oocyte retrieval and in vitro maturation
Ovaries from prepubertal gilts were collected from a
local abattoir and transported to the laboratory in saline
maintained at 30–37 8C. Cumulus oocyte complexes
(COCs) were aspirated from follicles (2–6 mm dia-
meter) using a 10 mL syringe fitted with an 18 gauge
needle. The COCs were washed three times in TL-
HEPES media containing 1 mg/mL BSA [27] and
matured in groups of 50 in 500 mL of Tissue Culture
Medium 199 with Earle’s salts (TCM-199; Gibco BRL,
Paisley, Scotland, UK), supplemented with 25 mM
NaHCO3, 10% (v/v) porcine follicular fluid, 0.57 mM
cysteine, 0.22 mg/mL sodium pyruvate, 25 mg/mL
gentamicin sulfate, 0.5 mg/mL p-FSH (Folltropin V;
Bioniche Animal Health, Belleville, ON, Canada),
1 mg/mL estradiol-17b, and 10 ng/mL epidermal
growth factor (EGF; Sigma cat. no. E-4127) under
mineral oil at 39 8C in a humidified atmosphere of 5%
CO2 in air for 42–44 h, as described [28].
2.2. Vitrification of oocytes
Only morphologically high quality oocytes, as
determined by uniform granular, homogeneously
distributed cytoplasm surrounded by compact layers
of cumulus cells (for immature oocytes) or oocytes
having an intact first polar body and a clear cytoplasm
with uniform texture and homogeneous fine granularity
surrounded by >70% homogeneously spread cumulus
cells (for in vitro matured oocytes), were used for
vitrification [29]. Immature (Germinal Vesicle or GV
stage) or in vitro matured (MII stage) were vitrified by
solid surface vitrification, as described by Dinnyes et al.
[20], with some modifications. Briefly, COCs were
partially denuded using 0.1% hyaluronidase and
washed three times in HEPES buffered TCM 199 with
M.K. Gupta et al. / Theriogenology 67 (2007) 238–248240
Earles salt (HTCM; Gibco BRL) supplemented with
20% FBS (FBS; Hyclone, Gibco BRL) and then
suspended in an equilibration medium consisting of 4%
(v/v) ethylene glycol (EG; Sigma cat. no. E-9129) in
TCM 199 + 20% FBS for 10–15 min at 39 8C. Oocytes
were then briefly rinsed three times (<30 s) in
vitrification solution (pre-warmed to 39 8C) consisting
of 35% EG, 5% polyvinyl pyrrolidone (PVP; Sigma cat.
no. P-0930), and 13.7% (w/v) sucrose (Sigma cat. no.
S9378) in HTCM-199 +20% FBS and placed in groups
of �25 oocytes per �1–2 mL droplet on to aluminum
foil kept directly over the liquid nitrogen (LN2). Upon
visual observation of the droplets, those that were
vitrified (completely transparent) were moved with a
liquid-nitrogen-cooled forceps into 1-mL cryovials
(Nunc cat. no. 343958) and plunged in LN2 for storage.
Vitrified oocytes were held in LN2 for at least 1 week
before warming. Warming was performed by dropping
the vitrified droplets from the cryotube directly into a
Petri dish containing 500 mL of 10.3% (w/v) sucrose
solution at 39 8C for 5 min. All solutions in the
microdrops remained transparent during cooling and
warming, which is one indication that the solutions
vitrified. If droplets appeared opaque, they were
discarded. Warmed oocytes were then washed in
HTCM-199 +20% FBS medium three times and kept
in the last HTCM-199 +20% FBS drop for at least 10–
15 min before transferring them into in vitro maturation
or fertilization medium.
2.3. Evaluation of oocyte viability
Oocyte survival was evaluated morphologically
based on the integrity of the oolemma and zona
pellucida; loss of membrane integrity (lysis) was
obvious upon visual inspection as the sharp demarcation
of the membrane disappeared and the appearance of the
cytoplasm changed. Oocytes were also assessed for
viability based on esterase enzyme activity and
oolemma integrity by FDA (30,60-diacetyl fluorescein)
staining, as described by Noto et al. [30]. Briefly,
oocytes were washed in PBS for 1 min, followed by
incubation with 2.5 mg/mL FDA (Sigma cat. no. F-
7378) stain for 1 min. Stained oocytes were washed
again in PBS and observed under ultraviolet illumina-
tion with an epifluorescence microscope with filters at
460–490 for excitation and 515–550 for emission
(Nikon Co., Tokyo, Japan). Live oocytes had green
fluorescence, whereas dead oocytes were non-fluores-
cent. Surviving oocytes were then further incubated for
at least 2 h at 39 8C in 5% CO2 and re-evaluated for
survival.
2.4. In vitro fertilization (IVF)
In vitro fertilization of surviving oocytes was
conducted as previously described [29]. Briefly, oocytes
were washed three times with the fertilization medium
(modified Tris-buffered medium; [31]) containing
1 mM caffeine sodium benzoate and 0.1% BSA and
were placed into groups of 10–15 oocytes per 50 mL
droplets of fertilization medium. Porcine testis were
collected from a local abbatoir and transported to the
laboratory at 30–35 8C in 0.9% (w/v) saline supple-
mented with 75 mg/mL penicillin G and 50 mg/mL
streptomycin sulfate. Sperm were retrieved from cauda
epididymis in TL-HEPES, and pelleted by centrifuga-
tion at 800 rpm for 5 min. The soft pellet was then
subjected to swim-up in Sp-TALP medium [27] for
10 min. The supernatant was collected and washed
twice by centrifugation at 800 rpm for 5 min. At the end
of the washing procedure, the sperm pellet was
resuspended in the fertilization medium and added to
the fertilization droplet to obtain a final sperm
concentration of 5 � 105 cells/mL. Sperm and oocytes
were co-incubated at 39 8C in a humidified atmosphere
of 5% CO2 in air for 6 h.
2.5. In vitro culture of presumptive zygotes
At the end of co-incubation period, presumptive
zygotes were cultured in NCSU23 medium supplemen-
ted with 0.4% fatty acid free bovine serum albumin
(BSA; Sigma cat. no. A-6033) for 5 days and in NCSU23
medium supplemented with 10% FBS for the next 2 days,
as described [32]. Cleavage rate was assessed on Day 2
and the blastocyst rate on Day 7 of culture.
2.6. Experimental design
The study comprised five experiments to evaluate the
effect of oocyte nuclear status, cytochalasin B treat-
ment, warming procedure and different cryoprotectant
solutions. In the first four experiments, we evaluated the
effect of above treatments on the viability of oocytes
following vitrification; the best combination was chosen
for the fifth experiment in which developmental ability
of vitrified oocytes was evaluated after IVF. At least
three replicates were performed in each experiment.
2.6.1. Experiment 1: effect of nuclear stage of
oocyte
The first experiment was designed to compare the
viability of immature (GV stage; n = 543) and mature
(MII stage; n = 574) porcine oocytes following vitri-
M.K. Gupta et al. / Theriogenology 67 (2007) 238–248 241
fication. Oocytes from the same lot of abattoir derived
ovaries were partially denuded and vitrified in
vitrification solution (containing EG as cryoprotectant)
before (GV) or after in vitro maturation (MII). After 7
days of storage in liquid nitrogen, vitrified oocytes were
warmed by single-step dilution and evaluated for
viability based on morphology and FDA staining at
two time points: immediately after warming and after
culturing for 2 h in TCM 199 medium supplemented
with 10% FBS.
2.6.2. Experiment 2: effect of cytochalasin B
The second experiment was designed to evaluate
effect of the cytoskeletal stabilizer, cytochalasin B, on
oocyte survival. Partially denuded GV (n = 789) and
MII (n = 963) stage oocytes were pre-treated with or
without 7.5 mg/mL cytochalasin B (Sigma cat. no. C-
6762) in TCM-199 medium supplemented with 20%
FBS at 39 8C for 30 min and vitrified as in Experiment
1, except that equilibration and vitrification media were
also supplemented with cytochalasin B. Oocytes were
assessed for viability as for Experiment 1.
2.6.3. Experiment 3: effect of warming procedure
This series of experiments compared the effect of a
post-warming single-step sucrose based rehydration
method with a two-step method on oocyte survival.
Partially denuded GV (n = 245) or MII (n = 1218) stage
oocytes were vitrified as described in Experiment 1 and
after 7 days of storage in liquid nitrogen, they were
warmed either by dropping each pellet directly into
10.3% sucrose as described above or by dropping each
pellet into sucrose (10.3%; w/v) + 2.5% (v/v) EG for
5 min, followed by 10.3% (w/v) sucrose alone for 5 min
at 39 8C. In both methods, the oocytes were kept in the
Fig. 1. Viability of immature (A and B) and mature (C and D) porcine oo
staining. (A and C) Immediate survival; (B and D) survival after culture fo
HTCM-199 +20% FBS drop for at least 10–15 min
before evaluating the viability. Oocytes were assessed
for viability as for Experiment 1.
2.6.4. Experiment 4: effect of cryoprotectant
Cryoprotectants, such as ethylene glycol, are toxic
and hence, oocytes should not be exposed to these
agents for extended intervals. This series of experiments
therefore, compared the effect of EG with equal
percentage of DMSO or EG:DMSO (1:1) in equilibra-
tion and vitrification solutions on viability and in vitro
maturation rate of GV stage oocytes following
vitrification. Partially denuded GV stage oocytes
(n = 412) were randomly allocated to each of the three
groups and vitrified. After storage for at least 7 days,
they were warmed by the two-step dilution method as
described in Experiment 3. Following warming, oocytes
were evaluated for viability after 2 h of culture in TCM
199 medium supplemented with 10% FBS and viable
GV oocytes were subjected to in vitro maturation.
2.6.5. Experiment 5: effect of vitrification on
development rate
Based on the results of above experiments, GV and
MII stage oocytes were vitrified in EG + DMSO based
medium without cytochalasin B treatment and after
warming diluted by the two-step protocol. Viable GV
stage oocytes were subjected to in vitro maturation. All
viable oocytes were subjected to IVF and evaluated for
their in vitro developmental ability.
2.7. Statistical analyses
Statistical analyses were carried out using SAS
software (Statistical Analysis System Inc., Cary, NC,
cytes based on morphology (non-fluorescent) and FDA (fluorescent)
r 2 h. Magnification 100�. Arrow indicates dead oocytes.
M.K. Gupta et al. / Theriogenology 67 (2007) 238–248242
Fig. 2. Recovery rate and survival rate (mean � S.E.M.) of porcine
oocytes vitrified at GV (&) or MII (&) stage: (A) morphological and
(B) FDA staining. Three replicates were performed.
USA) for non-paired Student’s t-test or ANOVA, where
appropriate. Data are presented as mean (�S.E.M.).
Differences at P � 0.05 were considered significant.
3. Results
3.1. Experiment 1
The recovery rate of vitrified oocytes ranged from 91
to 94% (Figs. 1 and 2). The viability of vitrified MII
stage oocytes was slightly, but not significantly, higher
than those of GV stage oocytes (14.3%).
Fig. 3. Survival rate (mean � S.E.M.) of porcine oocytes vitrified at GVor M
morphological and (B) FDA staining. Three replicates were performed.
Fig. 4. Survival rate (mean � S.E.M.) of vitrified porcine oocytes at GV o
Viability was assessed based on morphology (&) and FDA staining (&).
3.2. Experiment 2
Treatment of oocytes with cytochalasin B slightly, but
not significantly, improved the survival of GV (13.9%) as
well as MII stage (7.5%) oocytes based on morphology
(Fig. 3). However, upon FDA staining GV stage oocytes
had slightly higher (4.3%) survival, whereas MII stage
had lower (15.9%) survival (not significant).
3.3. Experiment 3
Two-step warming increased the survival of GV
(15.1%) as well as MII stage (5.7%) oocytes (Fig. 4).
Furthermore, when two-step warming was used, GV
stage oocytes had higher (20.0%) viability than MII
stage oocytes (not significant).
3.4. Experiment 4
There was no significant effect of the cryoprotectant
type on the viability of oocytes (Figs. 5 and 6).
However, the nuclear maturation rate of the vitrified GV
oocytes was significantly reduced in DMSO group
(31%) and were similar in EG (51%) and EG + DMSO
(53%) groups (P < 0.05).
II stage after treatment with (&) or without (&) cytochalasin B: (A)
r MII stage after warming by multi- or single-step dilution method.
Three replicates were performed.
M.K. Gupta et al. / Theriogenology 67 (2007) 238–248 243
Fig. 5. In vitro maturation of porcine oocytes vitrified at GV stage: (A) cumulus expansion; (B) PB extrusion; (C) viability based on FDA staining.
Magnification 100�. Open arrow indicate dead oocyte. Closed arrow shows polar body.
Fig. 6. Recovery rate, survival rate and in vitro maturation rate
(mean � S.E.M.) based on cumulus cell expansion and polar body
(PB) extrusion of porcine oocytes vitrified at the GV stage in DMSO
(&) or DMSO + EG (&) or EG ( ). Three replicates were performed.
The asterisk (*) mark indicates a significant difference (P < 0.05).
3.5. Experiment 5
Cleavage rate and blastocyst rates of vitrified GV
stage (20.2 and 9.5%) and MII stage (26.6 and 3.4%)
were significantly lower than those of the control group
(60.5 and 20.1%; Table 1). However, there was a high
degree of variation existed among replicates in vitrified
groups, but not in the non-vitrified control group. The
cell numbers of blastocysts were 10, 22 and 28 for
blastocysts derived from GV stage and 21 and 26 for
blastocysts derived from MII stage porcine oocytes
vitrified by SSV. Control blastocysts had a mean cell
number of 72.1 � 10.8 (Fig. 7).
Table 1
Developmental ability (mean � S.E.M.) of in vitro fertilized porcine oocyte
Group No. of oocytes
Control 443
GV vitrified 153
MII vitrified 231
Values in parentheses indicate number of embryos. Three replicates were p
number of cleaved embryos. Values with different letters differ (P < 0.05)
4. Discussion
Our objective was to develop a simple and effective
method to cryopreserve porcine oocytes. Vitrification
was carried out by SSV, as described by Dinnyes et al.
[20], and modifications that aimed to circumvent the
cellular disruptions during vitrification and might lead
to the improved survival and embryonic development
were tested. Up to 80% of the oocytes had normal
morphology following SSV and up to 70% had normal
esterase enzyme activity and oolemma integrity, as
determined by FDA staining (Fig. 1). Therefore, SSV
was effective for the cryopreservation of porcine
oocytes [17]. The viability rate following SSV in the
present study seemed higher than those reported by
Somfai et al. [17], which might be ascribed to
differences in protocols or to technical differences
[22,33] and quality of oocytes [34]. In our study, to
simplify the vitrification procedure, we utilized floating
aluminum foil, similar to Sagirkaya et al. [35] who
reported morphological viability of 98.5% for SSV of
pronuclear stage mouse embryos, compare to a survival
rate of �90% utilizing a partially immersed metal cube
[36]. It appears that the floating aluminum foil yields a
better survival rate than a partially immerged metal
cube utilized by Somfai et al. [17]. Nevertheless, it must
also be emphasized that use of aluminum foil might add
a technical variable and would require technical skill in
s vitrified by solid surface vitrification (SSV) at the GV or MII stage
Cleavage rate (%) Blastocyst rate (%)
60.5 � 15.2 a (328) 20.1 � 12.7 a (40)
20.21 � 1.09 b (31) 9.5 � 3.3 b (3)
26.6 � 1.6 b (62) 3.4 � 1.1 b (2)
erformed. The blastocyst rate was calculated as the percentage of the
.
M.K. Gupta et al. / Theriogenology 67 (2007) 238–248244
Fig. 7. Hoechst 33342 stained porcine blastocysts derived from in vitro fertilization of porcine oocytes vitrified at GV stage (A) or MII stage (B). (C)
Expanded blastocyst from control oocytes; (D) hatched blastocysts from control oocytes. Magnification 200�.
controlling the size of the droplets formed on the cold
surface, to keep the metal surface dry and to avoid
settling of liquid nitrogen vapor on the surface of
droplet while being placed on the metal surface.
Moreover, we have used sucrose instead of trehalose in
the vitrification solution, as sucrose has been reported to
be equally effective as trehalose for SSV without any
significant effect on the outcome [37]. Other modifica-
tions such as the use of a different base medium may
also account for differences in viability; in that regard,
we used HTCM-199 [22], whereas others have used
diverse media [17,36]. Furthermore, in our study, we
used only morphologically high quality oocytes, i.e.
uniform granular, homogeneously distributed cyto-
plasm surrounded by compact layers of cumulus cells
(for GV oocytes) or oocytes having intact first polar
body and showing clear cytoplasm with uniform texture
and homogeneous fine granularity, surrounded by
>70% homogeneously spread cumulus cells (for MII
oocytes). Given that ability of an embryo to withstand
freezing and thawing has been used in the past as a
useful indicator of quality [37–40], it is plausible that
use of best quality oocytes in our study might have
influenced cryotolerance and hence the viability of the
oocytes following warming.
Several authors had reported that viability of vitrified
oocytes/embryos decreases after culture [17,22]; there-
fore, we re-evaluated the vitrified-warmed oocytes after
2 h of in vitro culture. Many vitrified oocytes lysed after
culturing for 2 h (Fig. 1), suggesting an increased
sensitivity of vitrified oocytes to the culture conditions
[17].
Until recently, attention has focused on the
cryopreservation of mature oocytes, but this can induce
disruption of the spindle, chromosomes, microfilaments
and cortical granule distribution [24,25]. Such problems
are not associated with immature oocytes cryopreserva-
tion. Therefore, in the present study, experiments were
conducted to compare the viability of immature and
mature porcine oocytes vitrified by SSV method.
Immature pig oocytes were more sensitive to SSV
vitrification than in vitro matured oocytes. However,
when oocytes were treated with cytochalasin B or a
multi-step dilution was used, the GV-stage oocytes had
slightly higher viability than MII stage (Fig. 2), but this
increase was statistically non-significant. It can be
M.K. Gupta et al. / Theriogenology 67 (2007) 238–248 245
speculated that the cumulus cells enclosing the oocytes
could have influenced the cryoprotectant or water
permeability and hence affected the survival. There are
however, conflicting reports regarding the effects of
cumulus cells surrounding the oocyte for subsequent
development of matured oocytes after vitrification and
warming [41,42]. Fujihira et al. [16] reported a
significant relationship between the presence of
cumulus cells surrounding the oocytes and the
cryoprotectant permeability. Several earlier studies
have documented the GV stage to be more sensitive
than any other nuclear stages [43,44] for probable
reasons such as impaired intercellular communication
between the oocyte and the cumulus cells and
substantial disruption of microfilaments [43].
Osmotic stress during cryopreservation may alter the
physicochemical properties and integrity of cytoskeletal
elements, leading to abnormal progression of meiotic
division and the retardation of embryo development
[9,43,44]. Stabilizing the cytoskeleton system during
vitrification could be beneficial for improving post-
thawed survival and subsequent development of
vitrified oocytes [15,45]. Cytochalasin B has a specific,
reversible effect on depolymerization of cytoskeletal
elements, making them more flexible and hence, less
susceptible to cryo-damage [46]. However, controver-
sial results have been reported with the effect of
cytochalasin B on oocyte vitrification [15,16,47]. In our
study, pre-treatment of oocytes with cytochalasin B did
not significantly improve the proportion of surviving
oocytes (Fig. 3), consistent with Somfai et al. [17]
during SSV of porcine oocytes and Silvestre et al. [47]
for open pulled straw vitrification of sheep oocytes.
To avoid formation of ice crystals, a high concentra-
tion of cryoprotectants are included in the vitrification
solution, which must be removed after warming. Multi-
step dilution may be beneficial by avoiding sudden
osmolarity changes that may damage the oocyte,
whereas a single-step dilution minimizes the steps
required for oocyte washing, thereby substantially
reducing the time needed for warming and special
manipulations [48]. In the present study, the multi-step
dilution was found to be more effective (Fig. 4),
consistent with a previous report [15].
Different cryoprotectants can have different effects
on the viability and developmental potential of vitrified
oocytes and embryos. Recently, EG has gained
importance as an effective cryoprotectant for vitrifica-
tion of oocytes [17,49] as it is less toxic and is rapidly
permeable (into and out of oocytes) during cryopre-
servation, due to its low molecular weight. A balance
between the toxicity and cryoprotective action of the
cryoprotectant is critical for developing an efficient
cryopreservation method. Inclusion of a second
cryoprotectant can be used to reduce the concentration,
and hence the toxicity, of each cryoprotectant. In that
regard, a combination of EG with DMSO was reported
to give better survivability than EG alone [50], whereas
others have reported no added benefit of the combina-
tion [16]. In the present study, there was no beneficial
effect of the combination of DMSO and EG versus EG
per se on oocyte viability, as well as nuclear maturation
after vitrification of immature porcine oocytes. The EG-
based cryoprotectant medium was superior to DMSO
alone (Fig. 6). Although DMSO is a common
cryoprotectant used for freezing of cells and has
yielded good success in vitrification of human oocytes
by the open pulled straw method [51], we obtained the
worst results when DMSO alone was used. This
outcome was similar to those of Bagis et al. [36]
who compared three different cryoprotectants for SSV
of pronuclear stage mouse embryos; in that study, EG
was better than DMSO or propylene glycol for
embryonic development after vitrification. In a com-
parative study with bovine oocytes, Cestin and Bastan
[52] reported that EG or EG + DMSO was superior to
DMSO alone for vitrification. The exact cause for these
variable results is not clear. Trounson and Kirby [53]
documented that mouse oocytes exposed to DMSO at
temperatures from 4 to 37 8C incurred serious
irreversible changes in microtubules, pericentriolar
material and chromosomes of unfertilized oocytes;
they suggested a relationship between the toxicity of
DMSO with temperature and duration of exposure to
oocytes. Better results were obtained when oocytes
were exposed to DMSO at lower temperatures [53,54].
That the temperature of equilibration and vitrification
solution was 39 8C in the present study might have
contributed to the lower success obtained with DMSO.
Alternatively, DMSO might have caused spindle
polymerization with increased potential for polyploidy
[55] and therefore, had higher adverse effect on spindle
configuration than the combination of EG and DMSO
[56].
The presence of a large number of intracytoplasmic
lipid droplets in the oocytes has been considered an
obstacle to cryopreservation of porcine oocytes as lipid
reduces the cryotolerance of the oocytes and causes
irreversible damage to membrane structure at low
temperatures [3]. To circumvent this problem, Naga-
shima et al. [4] had introduced the concept of delipation
before cryopreservation. However, in the present
experiments using the SSV method to preserve porcine
oocytes that had not undergone delipation, these
M.K. Gupta et al. / Theriogenology 67 (2007) 238–248246
oocytes were not only capable of resuming meiosis, but
also developed in vitro after vitrification (Fig. 5),
consistent with previous reports [17,19].
The EG + DMSO combination allowed both GV and
MII vitrified oocytes to develop to blastocysts following
IVF. However, the development rate and cell number of
blastocysts was much lower than that obtained with fresh
oocytes (Table 1; Fig. 7). Furthermore, there was
individual variation among replicates for formation of
blastocysts, indicating that further improvements in the
technical aspects of the methodology are needed. The
low blastocyst rate and cell number can be ascribed to the
damages caused by cryopreservation, e.g. damage to
microtubules, pericentriolar materials and chromosomes
[53], aberrant spindle polymerization and polyploidy
[54], meiotic spindle disassembly, abnormal cortical
granule distribution and chromosomal aberrations [24–
26] and alterations in zona pellucida characteristics
[55,56]. Larman et al. [26] reported that DMSO as well as
EG used in vitrification solutions caused large transient
increases in intracellular calcium concentration in mouse
metaphase II oocytes, comparable to the initial increases
triggered at fertilization. This rise in calcium level can
negatively affect several physiological processes within
oocytes during oocyte vitrification and thus, might
explain the current poor efficiency of vitrification.
Successful production of blastocysts from vitrified
porcine oocytes has been reported by intracytoplasmic
sperm injection [16], parthenogenetic activation [17] and
following delipation [6]. However, to our knowledge, the
present study is the first to report the IVF blastocyst
production from porcine GV- or MII-stage porcine
oocytes, preserved with SSV, without any micromani-
pulation or delipation procedure.
In conclusion, SSV, a simple and rapid procedure,
allowed normal, lipid-containing, porcine oocytes
vitrified at either the GV or MII stage to be fertilized
and develop into blastocysts in vitro. The study also
demonstrated that: (1) GV and MII stage were equally
tolerant to the vitrification protocol; (2) pre-treatment
with cytochalasin B had no significant effect on the
vitrification of porcine oocytes; (3) two-step warming
was superior to single-step warming; (4) the use of EG or
EG and DMSO as a cryoprotectant were advantageous
for in vitro maturation of vitrified immature porcine
oocytes; (5) vitrified–warmed porcine oocytes matured
after IVM, could develop to the blastocyst stage, albeit at
a low efficiency, following IVF. We do not regard this
vitrification protocol as a definitive protocol; rather, we
regard it as a direction for further investigations into the
cryopreservation of porcine oocytes by SSV, which may
lead to more effective protocols.
Acknowledgment
This work was supported by the Research Project on
the Production of Bio-Organs (no. 200503030201),
Ministry of Agriculture and Forestry, and Biogreen 21,
RDA, Republic of Korea.
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