Freezing sperm in short straws reduces storage space and allows transport in dry ice

Abstract

Efficient cryopreservation and transportation of mouse sperm are among the most desirable strategies for current and future research on mouse genetics. However, the current method for sperm cryopreservation uses an 11-cm plastic straw, which is a bulky and fragile container. Developing an alternative to overcome the limitations associated with this method would accelerate biomedical research. Here, we developed the ST (sperm-freezing in ShorT STraw to reduce STorage space) method for cryopreserving mouse sperm in short 3.8-cm plastic straws. Up to nine short straws can be stored in a cryotube, reducing storage space. We further show that sperm frozen by the ST method can be transported in liquid nitrogen or dry ice without any detrimental effects on subsequent fertilization and the birth rate. Our findings suggest that this sperm-freezing method is beneficial not only for individual laboratories but also for large-scale mutagenesis/knockout and phenotyping programs.

Improvement of short straws for sperm cryopreservation: installing an air-permeable filter facilitates handling

“…3E ), comparable to the values for the CARD method and our previous ST method. Furthermore, the embryos from the iST method gave birth to live born pups at practical efficiencies (27.2 ± 20.3%), which is comparable with the CARD method, 31.5 ± 12.1% [ 10 ] and 27.9 ± 18.7% (this study). The efficiencies are also comparable to the efficiency obtained by our previous ST method (37.5 ± 6.9%) [ 10 ].…”

“…We previously developed the ST (sperm freezing in ShorT STraw to reduce STorage space) method for cryopreserving mouse sperm in short straws (ST straws hereafter), which dramatically reduced the storage space needed for frozen sperm (less than 16% of the conventional method) [ 10 ]. Although the ST straw and method have the benefit of efficiently using storage space, the ST method has some drawbacks, including the need for careful operation during freezing and thawing and the potential loss of sperm-containing straws during storage in a liquid N 2 tank.…”

“…First, we refined the container to store straw. In our previous ST methods, sperm-containing ST straws were stored in a 2-ml cryotube (outer cap type, MS-4603G, Sumitomo Bakelite Co., Ltd., Tokyo, Japan), and the cap of the 2-ml cryotube was loosened and stored in a liquid N 2 tank to lower the risk of cryotube breakage during thawing [ 10 ]. However, loosening the cap may increase the risk of the cap being removed, followed by the loss of sperm-containing straws.…”

“…Our concept was to broaden the working space to lower the potential risk of loss of sperm, which could reduce the care needed during freeze and thaw procedures. Importantly, cryopreserved sperm frozen using the new method should demonstrate comparable performance in in vitro fertilization (IVF) to sperm frozen by the conventional CARD method [ 11 ] and our previous ST method [ 10 ]. In our previous report, sperm frozen using the CARD method and the ST method showed 63.7 ± 12.8% and 68.5 ± 15.6% fertilization rates after IVF with C57BL/6J oocytes, respectively [ 10 ].…”

Is sperm cryopreservation in absence of permeable cryoprotectants suitable for subfertile donkeys?

“…Cryopreservation of semen is an important tool to preserve genetic material and avoid the undesirable effects of inbreeding (Rota, Panzani, Sabatini, & Camillo, ), thanks to the possibility of national and international exchange of genetic resources through shipping of frozen semen among others (Kaneko, Kakinuma, Sato, & Jinno‐Oue, ). However, permeable cryoprotectants used are known to exert a toxic effect in the sperm cell (Fahy, ), mostly due to the intrinsic toxicity and osmotic stress (Macías García et al., ).…”