Optimization of a Protocol for Cryopreservation of Mouse Spermatozoa Using Cryotubes

Abstract

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...

High Osmolality Vitrification: A New Method for the Simple and Temperature-Permissive Cryopreservation of Mouse Embryos

“…Here, we have optimized the vitrification method developed by Jin et al [3] for efficient embryo cryopreservation using cryotubes so that the embryos could be cryopreserved more conveniently and transported for long distances more safely. Similarly, we have developed a freezing method for mouse spermatozoa optimized for cryotubes instead of plastic straws [13]. Nakao et al [14] previously developed an embryo vitrification method using cryotubes, but it needed a specific combination of a cooling device with a cryotube to keep the temperature inside the tubes at 0°C during storage.…”

Microdroplet <i>In Vitro</i> Fertilization Can Reduce the Number of Spermatozoa Necessary for Fertilizing Oocytes

“…Recently, we used cryotubes as containers and found that the volume of the sperm suspension may critically affect the survivability of spermatozoa [11]. In this experiment, we used spermatozoa that had been frozen and stored in a 50 or 100 µl suspension, a volume larger than the optimal volume (10 µl).…”

“…Indeed, we were able to increase the fertilization rates from 7% to 25% by using this method when spermatozoa had very poor motility (< 10% motility rate) because of inadequate freezing in cryotubes. We have previously reported that sperm cryopreservation using cryotubes was critically affected by the volume of the sperm suspension in a cryotube [11]. …”

“…The addition of methyl-β-cyclodextrin (MBCD) to the sperm preincubation medium greatly increased the fertilizing ability of frozen-thawed spermatozoa [8], and the addition of reduced glutathione (GSH) [9] to the IVF medium significantly promoted penetration of fertilizing spermatozoa through the zona pellucida of oocytes. Later, it was found that these two techniques synergistically improved the fertilization rates of IVF using frozen-thawed spermatozoa, and therefore, this combination is now routinely used in many laboratories and mouse archive centers [10,11,12]. …”

Devising Assisted Reproductive Technologies for Wild-Derived Strains of Mice: 37 Strains from Five Subspecies of Mus musculus

“…Spermatozoa from the epididymal caudae of male mice (3–15 months old) from each strain were suspended in 200 µl of sperm preincubation medium (HTF containing 0.4 mM methyl-β-cyclodextrin [17] , [18] and 0.1 mg/ml polyvinyl alcohol instead of BSA) and incubated at 37°C under 5% CO 2 in air for 45–60 min. Spermatozoa that had been frozen in plastic straws were thawed and preincubated as described previously until used for IVF [19] . At the time of insemination, the preincubated spermatozoa were transferred into 80 µl drops of fertilization medium containing cumulus-enclosed oocytes at a final concentration of 200–500 spermatozoa/µl.…”