Collection and cryopreservation of

preimplantation embryos of Cavia porcellus

M M Dorsch, S Glage and H J Hedrich

Institute for Laboratory Animal Science, Hannover Medical School, Carl-Neuberg-Strasse 1,
30625 Hannover, Germany

Summary
Individual differences and a rather long-lasting reproductive cycle, as well as the relatively
small number of oocytes that mature during one reproductive cycle makes it difficult to
establish a cryopreserved stock of preimplantation embryos of the guineapig (Cavia porcellus)
when compared with other laboratory rodents. Only a few data for superovulation protocols
that can be used for routine laboratory use in guineapigs are available. However, a huge
number of different strains exist for many purposes and the establishment of a frozen
repository makes sense. Here, we describe the successful freezing of preimplatation embryos
of the strain 2BS with a two-step freezing protocol in a freezing medium containing 1,2-
propanediol as cryoprotectant. Human menopausal gonodotrophin induced superovulation
in the embryo donors.

Keywords Guineapig; cryopreservation; preimplantation embryo

The guineapig (Cavia porcellus) is used as a Because of similarities of the immune

laboratory animal since the late 18th century
(Terrill & Clemons 1998). Despite the fact
that this species is still playing an important
role in laboratory animal science, the
number of scientific experiments for which
guineapigs are used is decreasing. In 2004,
guineapigs were used in about 27,000 scien-
tific experiments in the UK, representing
,1% of total animal research. Over half of
these were studies of the respiratory, nervous
and immune systems. The use of guineapigs
has fallen by over three-quarters since 1988,
mostly because of a reduction in their use in
safety testing. A recent contribution to this
reduction has been the introduction of a
milder test for the potential of chemicals to
cause allergic skin reactions (skin sensitiz-
ation), which uses mice instead of guinea-
pigs. However, guineapigs remain essential
in many areas of research.
Correspondence: M M Dorsch.
Email: dorsch.martina@mh-hannover.de
Accepted 5 October 2007
# Laboratory Animals Ltd
Downloaded from lan.sagepub.com
system to that of man, it serves as an excel-
lent model in this field of research (Liang
et al. 2005, Orme 2005, Bowick et al. 2006).
Because of a long gestation period, spon-
taneous ovulation and active corpora lutea,
the guineapig is also an excellent animal
model for the study of reproduction in
humans (Suzuki et al. 2003).
The most common strains in use are
outbred animals of various stocks or strains.
A common outbred stock is the Albino
short-haired Duncan-Hartley English
guineapig. Several inbred strains are also
available. The strain 2, and strain 13
guineapigs are the most widely known and
used strains. New strains for various pur-
poses have been established during the last
decades according to Festing (1993). The
Central Laboratory Animal Facility of the
Hannover Medical School maintains
complement-deficient strains as described
by Bitter-Suermann and Burger (1986, 1990)
as well as the standard inbred strain 2BS,
that was derived from the NIH strain 2
in 1980. The exponential increase of
DOI: 10.1258/la.2007.007011.
Laboratory
at SAGE Publications on June 21, 2016 Animals (2008) 42, 489 –494
490
genetically-modified mouse strains necessi-
tates the amplification of maintaining
capacities for this species. Furthermore,
because of the decrease in the use of gui-
neapigs for scientific experiments, solutions
for the preservation of the genetic pool of
these scientifically valuable guineapig
strains must be found. The establishment
of a ‘Cryobank’ might solve this problem.
However, compared with mice and rats,
guineapigs have small- to medium-sized
litters and only few preimplantation embryos
can be obtained from one female. Induction
of superovulation by inhibin vaccine as
described by Shi et al. (2000a,b), or injections
of the pregnant mare’s serum gonadotrophin
after long-term implantation of progesterone
tubing (Kosaka & Takahashi 1989) are not
suitable for routine laboratory use, especially
because these methods are time-consuming
and quite expensive. For the purpose of
establishing a frozen repository, another pro-
tocol using human menopausal gonado-
trophin (hMG) administered subcutaneously
on three consecutive days of the oestrus
cycle seems more practicable (Suzuki et al.
2000). However, this method needs to be
optimized for routine use. Great individual
variations in the duration of the oestrus cycle
(15 – 19 days) (Terril & Clemons 1998) further
complicate the situation.
Here, we describe the superovulation of
the strain 2BS using a modified protocol of
Suzuki et al. (2000) and the successful cryo-
preservation by a two-step freezing protocol.
The suitability of 1,2-propanediol (PROH)
and dimethyl sulphoxide (DMSO) as cryo-
protectants is compared.
Material and methods
Animals and husbandry
Guineapigs of inbred strain 2BS were main-
tained at the Central Animal Facility of the
Hannover Medical School. Maintenance and
use of the animals were in accord with the
German Animal Welfare Legislation
(Tierschutzgesetz 1998). All experiments were
approved by the local Institutional Animal
Care and Research Advisory Committee and
permitted by the local government.
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M M Dorsch et al.
The guineapigs for this study were housed
in a controlled environment with 20 + 28C,
55 + 5% humidity and a day– night cycle of
12 h each (from 18:00 h to 06:00 h light;
18:00 h to 06:00 h dark). Animals in exper-
iment were caged in wire-topped Makrolon
type IV cages (Tecniplast, Italy) on auto-
claved softwood granulate bedding
(Altromin, Lage, Germany) and autoclaved
hay. They received a commercial diet
(Ssniff, Soest, Germany) and tap water ad
libitum. The females used for this study
were 16 + 1 weeks old and had a body
weight of about 550 g. They were all nulli-
parous. For timed mating, females with an
open vagina were placed into the cage of a
male overnight. Successful mating was
determined by vaginal smears taken the
next morning. For this, loose cellular
material was removed from the vagina by
gently scraping the dorsal vaginal wall with
a sterile loop, dispersed in a drop of saline
on a slide, air-dried, stained with methylene
blue solution (Löffleŕŕs methylene blue
solution, Merck, Darmstadt, Germany)
and examined microscopically. Sperms
could be identified as dark blue thread-like
structures. Vaginal plug controls were not
performed. According to our study, the plug
fell out of the vagina or was pulled out by
the female. The next morning the plug
could hardly be found in the bedding.
Superovulation
The protocol for superovulation was adapted
from Suzuki et al. (2000). Females received
an intraperitoneal injection of 5 IU hMG
(Sigma-Aldrich; www.sigmaaldrich.com) on
days 14, 15 and 16 after the last vaginal
opening, i.e. oestrus. The females were
mated as soon as the vagina opened. Vaginal
smears were taken the next morning to
assure successful mating. When sperms were
present, the female was considered pregnant.
For comparison, untreated females with open
vaginas were also mated.
Collecting preimplantation embryos
A total of 53 females with sperm-positive
vaginal smears were killed on days 2.5, 4.5
or 6.5 postcoitum ( p.c.). The oviducts and
Cryopreservation of guineapig embryos
part of the consecutive uterus horns were
excised. Preimplantation embryos were
flushed from the oviduct with phosphate-
buffered saline (PB1, Whittingham 1971)
through the infundibular opening using a
blunt end 33 gauge needle, attached to a
syringe filled with PB1 medium. The
embryos were washed three times in fresh
PB1. Only morphological intact embryos
were kept for further use.
Cryopreservation and thawing
Preimplantation embryos were frozen by
a modified two-step method using either
1.5 mmol/L PROH or 1.5 mmol/L DMSO
as cryoprotectant in PB1 as described by
Hedrich and Reetz (1990) for the rat. The
freezing medium was precooled to 48C.
Plastic straws (Minitübw; www.minitube.de),
with one end closed using a metal bulb, were
loaded with 250 mL freezing medium. The
embryos were deposited in the centre of the
medium and the other end of the straw was
sealed with a glass bulb. The straws were
then transferred to a programmable auto-
matic ethanol cooling bath (Thermo-Haake
P2-C75P; Thermo, Karlsruhe, Germany)
equilibrated at 08C. After 5 min, cooling was
started with a velocity of 18C/min to 268C.
Seeding was induced manually and cooling
was continued at 0.48C/min to 2328C. The
same temperature was held for 5 min and
then the straws were directly transferred to
liquid nitrogen for storage.
Thawing at a rate of about 3008C/min was
achieved by warming the straws at room
temperature for about 40 s. After melting,
the freezing medium with the embryos was
flushed into a 30 mm Petri dish and an equal
volume of fresh PB1 supplemented with
0.1 mmol/L sucrose (Merck) was added
immediately to dilute the cryoprotectant.
After 10 min, another volume of PB1
(without sucrose) was added. This step was
repeated twice. Embryo viability was verified
by staining with fluorescein diacetate (FDA,
Sigma) (Mohr & Trounson 1980, Niemann
et al. 1981). Viable blastomeres showed green
fluorescence (Zeiss-Axiovert 135, UV-filter
for green fluorescence at 546 nm; Carl Zeiss
AG, Germany).
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491
Statistical analysis
For descriptive statistics, we used the Stat
View 5.0 program (2002).
Results
Effect of superovulation
The epithelial structure sealing the vagina
ruptured the day after the last hormone
injection, indicating that all treated females
were in rut (oestrus). On day 2.5 p.c. more
embryos were collected than on day 4.5 p.c.
from untreated as well as from superovulated
females. The total number of embryos on
both days of collection was higher in the
superovulated females. On day 6.5 p.c.
morulae and blastocysts were found in
untreated females, no embryos could be
flushed from the oviducts of superovulated
females.
Different developmental stages were found
on the same day of pregnancy. On day 2.5 of
pregnancy, we found one- to eight-cell stages
in both groups. On day 4.5 of pregnancy
four-cell to morula stages were found in
untreated females and eight-cell to
blastula-stages in superovulated females.
On day 6.5 p.c. mainly early blastocyst
stages were collected from untreated females,
preimplantation stages were never found in
the oviducts of superovulated animals. Upon
microscopic examination all embryos
seemed to be morphologically intact. Table 1
summarizes the results.
The strain 2BS used in this study, normally
gives birth to two pups, occasionally to three.
This is in contrast to the number of embryos
that can be collected from untreated
females.
Cryopreservation
We cryopreserved four- to eight-cell embryos
collected on day 2.5 of pregnancy of untreated
and superovulated females. Most of the
embryos appeared to be morphologically intact
upon thawing when normal light was used for
microscopy. FDA staining however, showed
that most of the blastomeres were not viable
when 1.5 mmol/L DMSO was used (Figure 1).
Nearly all blastomeres fluoresced after FDA
Laboratory Animals (2008) 42
492 M M Dorsch et al.

Table 1 Mean number + standard error of preimplantation embryos collected 2.5, 4.5 and 6.5 days post-
coitum ( p.c.) from untreated and superovulated females
Untreated females (44) Superovulated females (9)
Developmental stage 2.5 p.c. 4.5 p.c. 6.5 p.c. 2.5 p.c. 4.5 p.c. 6.5 p.c.
One-cell embryo 0.40 + 0.18 0 0 0.33 + 0.33 0 0
Two-cell embryo 0.57 + 0.19 0 0 0.33 + 0.33 0 0
Four-cell embryo 0.67 + 0.22 0.22 + 0.22 0 5.00 + 3.22 0 0
Eight-cell embryo 0.33 + 0.33 0.44 + 0.29 0 1.33 + 1.33 0.17 + 0.17 0
Morula 0 0.56 + 0.38 0.20 + 0.20 0 2.67 + 1.26 0
Blastocyst 0 0 1.60 + 0.75 0 2.83 + 0.95 0
Deg. embryos 0.07 + 0.07 0 0 0 0 0
Total number 1.73 + 0.24 1.00 + 0.41 1.80 + 0.80 7.00 + 2.65 5.66 + 1.99 0

staining when 1.5 mmol/L PROH served as Discussion

the cryoprotectant (Figure 2). There was
Effect of superovulation
no difference whether embryo donors had
been superovulated or not. Table 2 The hormone treatment considerably
compares the results of the freezing methods increased the number of oocytes that
used. matured during one ovulatory cycle.

Figure 1 Frozen-thawed guineapig embryos at Figure 2 Frozen-thawed guineapig embryos at

magnification x80. Cryoprotectant used was 1.5 mmol/
L dimethyl sulphoxide. Embryos have been collected on
day 2.5 of pregnancy after superovulation. (a) Normal
light illumination, (b) Same embryos as in Figure 1a
after fluorescien diacetate A staining. An ultraviolet
filter for green fluorescence at 546 nm was used. Viable
blastomers show green fluorescence
magnification 380. Cryoprotectant used was
1.5 mmol/L propanediol. Embryos have been collected
on day 2.5 of pregnancy after superovulation. (a)
Normal light illumination, (b) Same embryos as in
Figure 2a after fluorescein diacetate staining. An
ultraviolet filter for green fluorescence at 546 nm was
used. Viable blastomers show green fluorescence

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Cryopreservation of guineapig embryos 493

Table 2 Comparison of dimethyl sulphoxide (DMSO) seemed to be necessary to evaluate the

and propanediol (PROH) as cryoprotectant in a viability by FDA staining, as it was difficult
1.5 mmol/L concentration
to judge the morphological integrity by
1.5 mmol/L 1.5 mmol/L normal light microscopy. The cytoplasm
Cryoprotectant PROH DMSO
of the blastomeres in guineapig embryos
Intact embryos (%) 64.81 0 has a granulated appearance, because it con-
Number of frozen 54 36
embryos tains many lipid droplets. Therefore, preim-
Viability was confirmed by fluorescein diacetate staining after
plantation embryos of the guineapig are more
thawing like swine embryos than mouse or rat
embryos, despite the evolutionary
relatedness.
Moreover, it seemed to variably accelerate
For swine, it was shown that the high lipid
the embryo development. Although morulae
content was responsible for hypothermic
and blastocysts were still in the oviducts on
sensitivity (Dobrinsky 1997, 2000), and
day 6.5 in untreated females, these develop-
freezing the embryos of this species is
mental stages were already found on day 4.5
still difficult. Surprisingly, viability was
in the treated group. We never found preim-
maintained in guineapig embryos although a
plantation embryos on day 6.5 in treated
method was used that was originally adapted
females, suggesting that implantation has
for mouse embryos which contain nearly no
occurred before. These findings are in
lipid.
accordance with the findings of Ueda et al.
For successful cryopreservation, it is
(1994) who found preimplantation embryos
important that most of the intracellular
from day 1 to day 5 of pregnancy, but not on
water is removed while at the same time an
day 6 after treatment with gonadotrophins.
osmotic shock is avoided. We conclude that
The best day for collecting preimplantation
the guineapig embryos dehydrate nearly
embryos for cryopreservation from super-
completely during the first phase of our
ovulated guineapigs was day 2.5 of pregnancy
freezing protocol. Thus, there is no
according to our study.
intracellular water that can form
Different developmental stages were found
deleterious ice crystals during the fast
in the same individuals after superovulation.
thawing process (3008C/min) and cause
There are still too few results for statistical
freezing injury.
analysis.
Till now, there is no report of a successful
The modified protocol of Suzuki et al. (2000)
embryo transfer of frozen-thawed embryos
for superovulation was successful in inducing
and only one of the transfer of freshly col-
oestrus and vaginal opening. Superovulated
lected embryos in guineapigs (Suzuki et al.
females also produced considerably more
2000). To demonstrate that viability of
embryos than untreated animals. This
guineapig embryos can be preserved during
protocol, therefore, is a reliable, fast and
a whole cycle of freezing and thawing,
inexpensive method suitable for routine
frozen-thawed embryos have developed to
laboratory use.
term when transferred to surrogate dams.
This will be our next challenge.
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