Collection and Cryopreservation of Preimplantation Embryos of Cavia Porcellus
Collection and Cryopreservation of Preimplantation Embryos of Cavia Porcellus
Collection and Cryopreservation of Preimplantation Embryos of Cavia Porcellus
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.
genetically-modified mouse strains necessi- The guineapigs for this study were housed
tates the amplification of maintaining in a controlled environment with 20 + 28C,
capacities for this species. Furthermore, 55 + 5% humidity and a day– night cycle of
because of the decrease in the use of gui- 12 h each (from 18:00 h to 06:00 h light;
neapigs for scientific experiments, solutions 18:00 h to 06:00 h dark). Animals in exper-
for the preservation of the genetic pool of iment were caged in wire-topped Makrolon
these scientifically valuable guineapig type IV cages (Tecniplast, Italy) on auto-
strains must be found. The establishment claved softwood granulate bedding
of a ‘Cryobank’ might solve this problem. (Altromin, Lage, Germany) and autoclaved
However, compared with mice and rats, hay. They received a commercial diet
guineapigs have small- to medium-sized (Ssniff, Soest, Germany) and tap water ad
litters and only few preimplantation embryos libitum. The females used for this study
can be obtained from one female. Induction were 16 + 1 weeks old and had a body
of superovulation by inhibin vaccine as weight of about 550 g. They were all nulli-
described by Shi et al. (2000a,b), or injections parous. For timed mating, females with an
of the pregnant mare’s serum gonadotrophin open vagina were placed into the cage of a
after long-term implantation of progesterone male overnight. Successful mating was
tubing (Kosaka & Takahashi 1989) are not determined by vaginal smears taken the
suitable for routine laboratory use, especially next morning. For this, loose cellular
because these methods are time-consuming material was removed from the vagina by
and quite expensive. For the purpose of gently scraping the dorsal vaginal wall with
establishing a frozen repository, another pro- a sterile loop, dispersed in a drop of saline
tocol using human menopausal gonado- on a slide, air-dried, stained with methylene
trophin (hMG) administered subcutaneously blue solution (Löffleŕŕs methylene blue
on three consecutive days of the oestrus solution, Merck, Darmstadt, Germany)
cycle seems more practicable (Suzuki et al. and examined microscopically. Sperms
2000). However, this method needs to be could be identified as dark blue thread-like
optimized for routine use. Great individual structures. Vaginal plug controls were not
variations in the duration of the oestrus cycle performed. According to our study, the plug
(15 – 19 days) (Terril & Clemons 1998) further fell out of the vagina or was pulled out by
complicate the situation. the female. The next morning the plug
Here, we describe the superovulation of could hardly be found in the bedding.
the strain 2BS using a modified protocol of
Suzuki et al. (2000) and the successful cryo- Superovulation
preservation by a two-step freezing protocol.
The protocol for superovulation was adapted
The suitability of 1,2-propanediol (PROH)
from Suzuki et al. (2000). Females received
and dimethyl sulphoxide (DMSO) as cryo-
an intraperitoneal injection of 5 IU hMG
protectants is compared.
(Sigma-Aldrich; www.sigmaaldrich.com) on
days 14, 15 and 16 after the last vaginal
opening, i.e. oestrus. The females were
Material and methods mated as soon as the vagina opened. Vaginal
Animals and husbandry smears were taken the next morning to
assure successful mating. When sperms were
Guineapigs of inbred strain 2BS were main-
present, the female was considered pregnant.
tained at the Central Animal Facility of the
For comparison, untreated females with open
Hannover Medical School. Maintenance and
vaginas were also mated.
use of the animals were in accord with the
German Animal Welfare Legislation
(Tierschutzgesetz 1998). All experiments were Collecting preimplantation embryos
approved by the local Institutional Animal A total of 53 females with sperm-positive
Care and Research Advisory Committee and vaginal smears were killed on days 2.5, 4.5
permitted by the local government. or 6.5 postcoitum ( p.c.). The oviducts and
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Cryopreservation of guineapig embryos 491
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
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Cryopreservation of guineapig embryos 493
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