Preservation of mouse sperm by convective drying and storing in 3-O-methyl-D-glucose

Abstract

With the fast advancement in the genetics and bio-medical fields, the vast number of valuable transgenic and rare genetic mouse models need to be preserved. Preservation of mouse sperm by convective drying and subsequent storing at above freezing temperatures could dramatically reduce the cost and facilitate shipping. Mouse sperm were convectively dried under nitrogen gas in the Na-EGTA solution containing 100 mmol/L 3-O-methyl-D-glucose and stored in LiCl sorption jars (Relative Humidity, RH, 12%) at 4°C and 22°C for up to one year. The functionality of these sperm samples after storage was tested by intracytoplasmic injection into mouse oocytes. The percentages of blastocysts produced from sperm stored at 4°C for 1, 2, 3, 6, and 12 months were 62.6%, 53.4%, 39.6%, 33.3%, and 30.4%, respectively, while those stored at 22°C for 1, 2, and 3 months were 28.8%, 26.6%, and 12.2%, respectively. Transfer of 38 two- to four-cell embryos from sperm stored at 4°C for 1 year produced two live pups while 59 two- to four-cell embryos from sperm stored at 22°C for 3 months also produced two live pups. Although all the pups looked healthy at 3 weeks of age, normality of offspring produced using convectively dried sperm needs further investigation. The percentages of blastocyst from sperm stored in the higher relative humidity conditions of NaBr and MgCl(2) jars and driest condition of P(2)O(5) jars at 4°C and 22°C were all lower. A simple method of mouse sperm preservation is demonstrated. Three-O-methyl-D-glucose, a metabolically inactive derivative of glucose, offers significant protection for dried mouse sperm at above freezing temperatures without the need for poration of cell membrane.

Figure 1. Drying chamber and storage jar.

A) Drying chamber (Patent pending). It is made of aluminum and held together by two sets of press-to-seal clamps instead of 10 screws to provide increased ease of operation and a reliable system for long term reproducible drying process. The chamber is also designed to hold a 25 mm×25 mm square glass slide with a 10-mm diameter etched ring in the center of the slide. B) LiCl storage sorption jar. Each sorption jar contains a plastic system consisting of a lower tri-pot and a removable upper rack to accommodate eight 25 mm×25 mm glass slides.

Figure 2. Regressions of the percent blastocyst on time of storage.

Computed by transformation of the following logit responses on storage time to percentages, where p is the proportion of blastocysts: 4°C: logit(p) = −0.108 t+0.3338 22°C: logit(p) = −0.514 t−0.247.

Figure 3. Mice produced by ICSI.

Mice produced from sperm dried with 100 mmol/L 3-OMG for 6 minutes and stored in LiCl jars at 4°C for one year.