Birth of mice after in vitro fertilization using C57BL/6 sperm transported within epididymides at refrigerated temperatures

Mochida, Keiichi; Ohkawa, Makio; Inoue, Kimiko; Valdez, Delgado M.; Kasai, Magosaburo; Ogura, Atsuo

doi:10.1016/j.theriogenology.2004.11.013

Cited by 27 publications

(34 citation statements)

References 20 publications

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“…Mochida et al [15] demonstrated that transportation of epididymides preserved in mineral oil at refrigerated temperatures using a commercial courier service was a practical method for exchange of mouse genetic resources. It would be feasible to transport the intact body of a dead mouse under the same conditions for distribution of mouse strains.…”

Section: Discussionmentioning

confidence: 99%

“…An et al [13] stored euthanized male mice at 4-6 C for up to 7 days and demonstrated that the recovered spermatozoa had the ability to fertilize oocytes. Research by others has also shown that high proportions of mouse oocytes can be fertilized in vitro with spermatozoa obtained from the epididymis after storage in mineral oil at low temperature [14,15]. To develop an effective method for short-term, non-frozen storage, Sankai et al [16] compared six simple methods including the above two with respect to the motility of spermatozoa and showed that, either in mineral oil or within the body, a high percentage of progressively motile spermatozoa can be obtained from the epididymis stored for 8 days at 5 C. Thus, it is worthwhile to compare the fertilizing ability of spermatozoa within epididymides stored in mineral oil and in the body of the male.…”

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confidence: 99%

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Piezo-assisted In Vitro Fertilization of Mouse Oocytes with Spermatozoa Retrieved from Epididymides Stored at 4 Degree C

Fan¹,

Li²,

Liu³

et al. 2008

Journal of Reproduction and Development

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Abstract. This study was performed to investigate the effect of partial zona pellucida incision by piezo micromanipulation (ZIP) on the in vitro fertilizing ability of stored mouse spermatozoa. The storage conditions were optimized by storing the mouse epididymides at 4 C in mineral oil or in the mouse body for up to 4 days after death, and the retrieved spermatozoa were used to fertilize fresh oocytes. No significant difference was observed in fertilization rates between the treatments when epididymides were stored for up to 2 days, but the fertilization rates in mineral oil were higher (P<0.05) than those in the mouse body at 3 (41.4 vs. 16.2%) and 4 days (26.0 vs. 15.8%). Spermatozoa retrieved from epididymides stored in mineral oil were then used to fertilize fresh and vitrified oocytes with or without ZIP treatment. The fertilization rates of the ZIP fresh oocytes were higher than those of the zona-intact oocytes at each time point (1 to 4 days). After ZIP, the fertilization rates of spermatozoa stored for 1 and 2 days (91.2 and 86.6%, respectively) were similar (P>0.05) to that of fresh spermatozoa (91.9%). In regard to vitrified oocytes, the fertilization rates of zona-intact and ZIP oocytes using fresh spermatozoa were 46.7 and 84.7%, while the fertilization rates of vitrified ZIP oocytes using spermatozoa stored for 1 to 4 days ranged from 49.3 to 79.6%. When 2-cell embryos derived from ZIP fresh and vitrified oocytes inseminated with 2 day-stored spermatozoa were transferred into recipient females, 47.9 and 15.0% of the embryos developed to term, respectively. These results indicate that storing mouse epididymides at 4 C in mineral oil is more suitable than storage in the mouse body and that the ZIP technique improves the in vitro fertilizing ability of stored mouse spermatozoa in fresh oocytes and significantly increases the fertilization rate of vitrified oocytes with fresh spermatozoa. Key words: Epididymal spermatozoa, In vitro fertilization (IVF), Mouse, Piezo-assisted micromanipulation, Vitrification (J. Reprod. Dev. 54: [107][108][109][110][111][112] 2008) he epididymis is an essential organ for sperm development and storage. The epithelial cells of the epididymis can secrete certain proteins to promote maturation of spermatozoa [1][2][3], and the specific environment within the epididymis, characterized by a low pH and hyperosmotic pressure, allows spermatozoa to survive for several weeks [4,5]. Spermatozoa retrieved from the epididymides of dead animals retain the ability to penetrate and fertilize female gametes and can be used in genetic preservation of endangered male animals. Recently, some studies on preserving the epididymis at refrigerator temperatures in order to retrieve spermatozoa for in vitro fertilization (IVF) or artificial insemination have been carried out in deer [6][7][8] [12]. Research on storage of the mouse epididymis is important since the mouse is most commonly used in biomedical research. Shipping the refrigerated epididymides (spermatozoa) of mouse strains avoids th...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Piezo-assisted In Vitro Fertilization of Mouse Oocytes with Spermatozoa Retrieved from Epididymides Stored at 4 Degree C

Fan¹,

Li²,

Liu³

et al. 2008

Journal of Reproduction and Development

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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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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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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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“…S2) Total RNA was extracted with TRIzol (Invitrogen) from single blastocysts cultured for 96 h. The RNA was then subjected to 2 rounds of linear amplification using TargetAmp Two-Round Aminoallyl-aRNA Amplification Kits (Epicenter) according to the manufacturer's instructions. For control embryos, single blastocysts generated by IVF 37 were subjected to total RNA extraction and 2 rounds of RNA amplification. Amplified RNA was labeled with Cy3 dye (GE Healthcare) and hybridized to a whole mouse genome oligo DNA microarray (4 × 44K; Agilent Technologies) for 16 h at 65 °C according to the manufacturer's instructions.…”

Section: Animalsmentioning

confidence: 99%

Understanding the X chromosome inactivation cycle in mice

et al. 2013

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Birth of mice after in vitro fertilization using C57BL/6 sperm transported within epididymides at refrigerated temperatures

Cited by 27 publications

References 20 publications

Piezo-assisted In Vitro Fertilization of Mouse Oocytes with Spermatozoa Retrieved from Epididymides Stored at 4 Degree C

Piezo-assisted In Vitro Fertilization of Mouse Oocytes with Spermatozoa Retrieved from Epididymides Stored at 4 Degree C

Optimization of a Protocol for Cryopreservation of Mouse Spermatozoa Using Cryotubes

Understanding the X chromosome inactivation cycle in mice

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