Cryopreservation of mouse spermatozoa
Division of Reproductive Engineering, Center for Animal Resources & Development, Kumamoto University, 2-2-1 Honjo, Kumamoto 860-0811, Japan
Received: 16 December 1999 / Accepted: 17 December 1999
Abstract. Recently, it has become possible to freeze a large num-
ber of mouse spermatozoa immediately after collection from the
epididymides of a small number of males. The cryopreservation of
spermatozoa is simpler, less time-consuming, and less costly than
that of embryos for maintaining various strains of mice with in-
duced mutations. This chapter attempts to provide a realistic over-
view of the cryopreservation of mouse spermatozoa and to de-
scribe a detailed procedure for mouse sperm freezing.
Recently, a large number of various strains of mice with induced
mutations (i.e., transgenes, targeted mutations, chemically induced
mutations) have been produced in a variety of laboratories world
wide (Jaenisch 1988; Bedell et al. 1997; Simpson et al. 1997;
Hrabe de Angelis and Balling 1998; Brown and Nolan 1998). As
a result, the number of strains of mice with induced mutations is
rapidly expanding, and the maintenance of these strains by stan-
dard breeding colonies is becoming an increasing problem. Em-
bryo freezing is generally used for this purpose (Pomeroy 1991).
However, in the conventional method of embryo freezing, 500
embryos per strain are required from the oviducts of 20–50 mated
females or by in vitro fertilization before the freezing procedure
(Mobraaten 1981). In contrast, 10,000,000–30,000,000 spermato-
zoa can be frozen immediately after collection from the epididy-
mides of each male. If all frozen-thawed spermatozoa from one
male are used for in vitro fertilization, they can fertilize at least 500
oocytes (Nakagata 1996). Therefore, sperm freezing may provide
a much simpler and economical alternative to embryo freezing to
achieve this goal.
The first reports of successful cryopreservation of mouse sper-
matozoa were published in 1990 by three independent groups of
Japanese investigators (Okuyama et al. 1990; Tada et al. 1990;
Yokoyama et al. 1990). We are also subsequently successful in the
cryopreservation of mouse spermatozoa by using an improved
method (Takeshima et al. 1991; Nakagata and Takeshima 1992).
In addition, we demonstrated for the first time that cryopreserved
spermatozoa can fertilize cryopreserved oocytes in vitro and that
two-cell embryos obtained from cryopreserved gametes can de-
velop into normal live offspring after embryo transfer (Nakagata
Since that time, numerous researchers have reported successful
mouse sperm cryopreservation by using various procedures (Pen-
fold and Moore 1993; Tao et al. 1995; Sztein et al. 1997; Song-
sasen and Leibo 1997a, 1997b; Songsasen et al. 1997; Storey et al.
1998). Despite the many different published methods for cryopres-
ervation, the in vitro fertilization rates and rate of development to
fetus and live offspring after transfer of embryos derived from
frozen spermatozoa varies considerably among different research
groups. Moreover, these rates also vary with the strains of frozen
sperm used and the eggs used for in vitro fertilization (Table 1).
However, a comparison of these reports is not possible owing to
the many differences in freezing and thawing and subsequent in
vitro fertilization methods used. These findings clearly indicate
that the underlying critical factors have not yet been elucidated.
Therefore, development of more reliable and effective methods
still appears to be required.
Our group has published numerous papers in which the basic
18% raffinose, 3% skim milk cryopreservation technique has been
refined and applied to variety of different strains and transgenic
stocks of mice (Nakagata and Takeshima 1993; Nakagata et al.
1995, 1997; Nakagata 1996; Okamoto et al. 1998). In addition,
large-scale ENU mutagenesis screening studies have employed our
cryopreservation technique to assess sperm cryopreservation for
mouse mutant archiving and the rapid re-establishment of shelf
stocks (Marschall et al. 1999; Thornton et al. 1999).
In this chapter, I shall restrict myself to a description of the
detailed procedure routinely used for mouse sperm freezing and
the reproductive technologies concerned with the sperm freezing
in my laboratory.
Cryopreservation of spermatozoa
Materials and equipment
1. Male mice (3–6 months old)
2. 50-ml disposable conical tube
3. 20-ml disposable syringe, 18-gauge needle
4. Water bath
5. 2-ml sample tube
6. High-speed microcentrifuge
7. Disposable filter unit (pore size 0.45 m; Millipore Ltd., Cat.
8. 1-ml glass ampules
9. Twin jet ampule sealer
10. Four-well disposable multidish (no. 176740; Nunc, Roskilde,
12. Tip (0.5–10 l, 10–100 l volume)
13. Micro-spring scissors (5-mm blade)
14. Pair of watchmakers #5 forceps
15. 35-mm sterile plastic tissue culture dishes
16. 1-ml disposable syringe
17. Straw connector (2-ml long silicone tube that fits the straw)
18. 0.25-ml insemination straw (no. A-201; IMV, I’Aigle, France)
19. Cellophane tape (12 mm wide)
20. Labels to print mouse number (5 mm × 20 mm)
21. HTF medium (Quinn et al. 1985), sterile, pregassed, plus 4
mg/ml BSA (Takahashi et al. 1995) (Table 2)
22. Impulse sealer
23. Acrylic bar (5 mm × 5 mm × 50 cm)
24. Styrofoam (30 mm thick)
25. 50-ml disposable syringe
26. Cryobiological container
27. Humidified 37°C incubator, 5% CO
, 95% air
Correspondence to: N. Nakagata; E-mail: email@example.com.
Mammalian Genome 11, 572–576 (2000).
© Springer-Verlag New York Inc. 2000