Cryopreservation of Mouse Spermatozoa from Inbred and F&lt;SUB&gt;1&lt;/SUB&gt; Hybrid Strains

Nakagata, Naomi; Takeshima, Tsutomu

doi:10.1538/expanim1978.42.3_317

Cited by 87 publications

(60 citation statements)

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“…Epididymal spermatozoa were collected from mature ICR (Jcl:ICR, CLEA Japan, Tokyo, Japan) and B6 (C57BL/6JJcl, CLEA Japan) males aged 70-128 days, and sperm suspensions were prepared as described previously [25,26]. In brief, the epididymides were incised with fine scissors, and the spermatozoa were allowed to disperse in 100 µl sperm freezing medium comprising 18% raffinose (cat.…”

Section: Methodsmentioning

confidence: 99%

“…We first examined the effect of the position of the cryotube during freezing on the survival of spermatozoa. In this experiment, the volume of sperm suspension in the cryotube was set at 10 µl, and the frozen cryotubes were thawed at 37 C following our conventional sperm cryopreservation protocol using plastic straws [25,26]. The position of the cryotubes, but not the mouse strain, had a significant effect on sperm survival, and there was no interaction between the factors (position of cryotubes and mouse strain) ( Table 1).…”

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Optimization of a Protocol for Cryopreservation of Mouse Spermatozoa Using Cryotubes

Hasegawa

Yonezawa

Ohta

et al. 2012

Journal of Reproduction and Development

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Abstract. The rapid increase in the number of genetically modified mouse strains has produced a high demand for their frozen spermatozoa from laboratories and mouse banking facilities. Historically, plastic straws have been used preferentially as containers for frozen mammalian spermatozoa because spermatozoa frozen in plastic straws have a high survival rate after thawing. However, plastic straws are more fragile and are used less often than the cryotubes used for conventional cell freezing. In this study, we sought to develop a new protocol for sperm freezing using cryotubes as the container to increase the accessibility of mouse sperm cryopreservation. Epididymal spermatozoa were collected from mature ICR or C57BL/6J (B6) males and were suspended in 18% raffinose and 3% skim milk solution. We then optimized the following conditions using the sperm survival rate as an index: 1) distance of cryotubes from the surface of the liquid nitrogen at freezing, 2) volume of the sperm suspension in the cryotube and 3) temperature of warming sperm during thawing. The best result was obtained when cryotubes containing 10 µl of sperm suspension were immersed 1 cm below the surface of the liquid nitrogen and then thawed at 50 C. The fertilization rates using spermatozoa frozen and thawed using this method were 63.1% in ICR mice and 28.2% in B6 mice. The latter rate was increased to 62.3% by adding reduced glutathione to the fertilization medium. After embryo transfer, 68% and 62% of the fertilized oocytes developed into normal offspring in the ICR and B6 strains, respectively. These results show that cryotubes can be used for cryopreservation of mouse spermatozoa under optimized conditions. This protocol is easy and reproducible, and it may be used in laboratories that do not specialize in sperm cryopreservation. Key words: C57BL/6, Glutathione, Mouse, Spermatozoa (J. Reprod. Dev. 58: [156][157][158][159][160][161] 2012) L arge-scale mutagenesis/knockout and phenotyping programs using mouse strains have been launched to provide the research community with a long-lasting resource for the study of mammalian gene function [1,2]. In addition to these large organized programs, individual laboratories generate mice with gene modifications of specific interest using conventional transgenic and knockout methods [3,4]. These uses have increased the demand for the efficient and safe preservation of invaluable mouse strains. Since the first success by Whittingham in 1972 [5], embryo cryopreservation has been the main strategy for preserving mouse genetic resources because of its high reproducibility and technical ease.From the practical viewpoint, however, preservation of mouse strains as embryo stocks has several technical disadvantages because of the steps required before the production of embryos, including superovulation of females, in vitro fertilization (IVF), and in vitro embryo culture. All of these steps should be well controlled for the best results. In addition, the number of oocytes collected from a single female is limi...

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Optimization of a Protocol for Cryopreservation of Mouse Spermatozoa Using Cryotubes

Hasegawa

Yonezawa

Ohta

et al. 2012

Journal of Reproduction and Development

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“…[10] 35-36 -** -** Okuyama et al [9] 42 -** -** Takeshima et al [12] 34-36 -45 Nakagata & Takeshima [15] 71-95 -51 Tada et al [16] 85 57 -ICR (CD-1) Storey et al [25] 33-58 -** -** <hybrid> B6CF1 (C57BL/6 X BALB/c) Yokoyama et al [11] 19-37 -17-75 B6C3F1 (C57BL/6N X C3H/He) Nakagata & Takeshima [15] 59 -56 B6D2F1 (C57BL/6J X DBA/2J) Sztein et al [20] 89-93 37 38 Songsasen and Leibo [21] 64-73 -** -** Songsasen and Leibo [22] 61 -42 Songsasen et al [23] 26-39 -24-47 M.m.musculus Nakagata et al [18] 48-63 -24-51 *PZD oocytes were inseminated with frozen-thawed spermatozoa with low motility. **Fertilized oocytes were not transferred.…”

Section: -Review-mentioning

confidence: 99%

“…Since that time, numerous researchers have reported successful mouse sperm cryopreservation by means of various procedures [15][16][17][18][19][20][21][22][23][24][25][26]. Nevertheless, 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 the different research groups (Table 1).…”

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Mouse Spermatozoa Ctyopreservation.

Nakagata

2000

J.Mamm.Ova.Res.

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“…At present, the 18% raffinose plus 3% skim milk method [12] is relied upon in most laboratories. However, with this technique, the fertility of cryopreserved sperm is far less than that of fresh sperm, especially with inbred mice [13][14][15][16]. In particular, fertility decreases markedly in C57BL/6J, a principal inbred strain used for transgenesis studies.…”

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confidence: 99%

Marked Improvement of Fertility of Cryopreserved C57BL/6J Mouse Sperm by Depletion of Ca2+ in Medium

Suzuki-Migishima

Hino

Takabe

et al. 2009

Journal of Reproduction and Development

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Abstract. Cryopreservation of mouse sperm is useful for maintaining various strains. However, fertility generally decreases after freezing. In particular, the fertility of cryopreserved C57BL/6J sperm is very low. To improve the fertility of frozen sperm, we examined the efficiencies of various media used for sperm preincubation (SP) and in vitro fertilization (IVF) in frozen C57BL/6J sperm. In this study, SP medium was examined for efficiency of fertility with respect to content, especially calcium (Ca 2+ ), phosphate (PO4 3-) and lactate. In all media containing no Ca 2+ , including medium lacking Ca 2+ , lacking Ca 2+ and PO4 3-, lacking Ca 2+ and lactate and lacking Ca 2+ , PO4 3-and lactate, high IVF rates were obtained (79, 69, 76 and 71%, respectively). On the other hand, the rates for media containing Ca 2+ were significantly lower (30-38%, P<0.05). After transfer, 41-50% of newborns were obtained in all media containing no Ca 2+ .In conclusion, preincubation of thawed sperm in medium containing no Ca 2+ markedly improved the fertility of cryopreserved C57BL/6J sperm. These results indicate that the present method of IVF using medium with no Ca 2+ is practical for use in cryopreserved C57BL/6J sperm. Key words: C57BL/6, Calcium, Cryopreservation, In vitro fertilization, Sperm (J. Reprod. Dev. 55: [386][387][388][389][390][391][392] 2009) ryopreservation of mouse sperm is useful because it is simple, rapid and inexpensive. A large number of sperm can be frozen immediately after collection from a male. Females are required only when the frozen sperm are thawed for fertilization. Successful cryopreservation of mouse sperm was reported in 1990 using raffinose with glycerol [1], ME2SO [2] or skim milk [3]. Since then, cryoprotectant solutions and freezing methods for mouse sperm have been investigated [4][5][6][7][8][9][10][11]. At present, the 18% raffinose plus 3% skim milk method [12] is relied upon in most laboratories. However, with this technique, the fertility of cryopreserved sperm is far less than that of fresh sperm, especially with inbred mice [13][14][15][16]. In particular, fertility decreases markedly in C57BL/6J, a principal inbred strain used for transgenesis studies. Frozen C57BL/6J sperm exhibit acrosome damage with loss of contents caused by the freeze-thaw procedure [17], and almost no sperm can penetrate the zona pellucida. When partial zona pellucida nicking [18,19] or intracytoplasmic sperm injection (ICSI) [20,21] is performed to increase fertility, post-thawed C57BL/6J sperm can fertilize a high percentage of eggs. Improved IVF rates with intact oocytes have rarely been reported using motile sperm separated from thawed suspension [16,22] or methyl-beta-cyclodextrin (MBCD) stimulating cholesterol efflux from thawed sperm [23]. Successful IVF with fresh sperm depends on the media used. Various IVF media have been developed, and the effects of media on capacitation and IVF have been investigated. However, the effects of media on frozen mouse sperm have not been investigated.In this s...

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Cryopreservation of Mouse Spermatozoa from Inbred and F<SUB>1</SUB> Hybrid Strains

Cited by 87 publications

References 9 publications

Optimization of a Protocol for Cryopreservation of Mouse Spermatozoa Using Cryotubes

Optimization of a Protocol for Cryopreservation of Mouse Spermatozoa Using Cryotubes

Mouse Spermatozoa Ctyopreservation.

Marked Improvement of Fertility of Cryopreserved C57BL/6J Mouse Sperm by Depletion of Ca2+ in Medium

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