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Sperm cryopreservation

Vivarium and Transgenics Core

Overview

Investigators interested in the mouse sperm cryopreservation service should have two fertile males, two to six months of age. The service begins once VARI receives a completed request form via iLab and the male mice. The males are housed individually for a week before the service begins.

Cryopreservation method

Sperm is isolated from the cauda epididymis and vas deferens of the male mice and placed into warmed cryoprotective media supplemented with an antioxidant. Sperm is allowed to “swim-out” for 10 minutes, and then four, 1 mL aliquots are aspirated into each straw. Approximately 20 straws are used. Each straw is heat sealed and placed into a cassette (five straws/cassette). Loaded cassettes are statically frozen in liquid nitrogen vapor for 10 minutes, then plunged into liquid nitrogen and moved to two different liquid nitrogen tanks for long-term storage. Offsite triple banking is available for an additional cost.

Quality control

Core personnel perform two levels of quality control. The first level is a fertilization capability check (in vitro). The second level is a developmental capability check of live-born mice (in vivo).

To assess the viability of the frozen sperm, one straw is thawed and the sperm assessed according to the quality control level chosen by the investigator. Personnel routinely thaw one straw of sperm and perform in vitro fertilization to the 2-cell stage as a quality control protocol for no additional cost.

If requested and for additional cost, Core personnel will perform in vivo quality control by implanting fertilized, 2-cell stage embryos into pseudopregnant females to produce live pups. The offspring will be tail clipped to obtain DNA for genotyping, which will confirm the gene of interest has been successfully cryopreserved.