Cryopreservation

Cryopreservation

Once genetic material is collected by our herd staff or Tufts veterinarians, it is brought to the laboratory. All processing and freezing takes place in our lab by SVF’s lab director, Dr. Dorothy Roof.

The process begins with the microscopic evaluation of each embryo’s developmental stage and quality. Embryos are washed by transferring them through several different holding mediums. A cryoprotectant solution is added, which allows the embryos to be frozen in liquid nitrogen without the formation of damaging ice crystals. Each embryo is then loaded into a 1/4cc straw, labeled with all pertinent data and placed into a controlled rate freezer. This method of freezing reduces the embryo from room temperature to -35°C at a very precise rate. Once below this temperature, the embryos can be plunged directly into liquid nitrogen at -196°C and placed in our storage tanks. The location, quality, stage, donor and collection method regarding each sample is entered into the FreezerWorks database.

Semen samples are processed and frozen in our lab as well. Immediately after collection, a standard antibiotic solution is added to the raw semen, which protects it from a variety of naturally occurring bacteria in the environment. The sperm concentration is measured by densitometry, and sperm motility (sperm movement) is recorded using our Computer Assisted Semen Analysis (CASA) system.

A commercial soy-based extender is used so that each small semen sample yields a relatively large number of straws to be frozen. The semen is slowly chilled in a refrigerator for a set period, which varies with animal species. Once the semen is chilled, additional extender containing glycerol is added. The glycerol solution, or cryoprotectant, preserves the semen from damaging ice crystals when frozen in liquid nitrogen. The final concentration of fully extended semen is 100 million sperm per 1/2cc straw. These straws are transferred to our controlled-rate freezer, where the temperature is gradually lowered to -140°C. The cryopreservation is completed by plunging the semen straws into liquid nitrogen and transferring them to permanent storage in our cryo tanks.

Research completed by SVF’s Lab team has yielded significant results on the best methods for freezing goat semen. This has led to successful publication of our manuscript,Comparison of two commercial extenders for cryopreservation of goat semen without sperm washing, by the scientific Journal of Theriogenolgy . Please contact us if you are interested in learning more.

The quality of sperm samples are determined using morphology and motility. Morphology determines whether the shape of the sperm cell is normal and functional. The sperm motility reflects how well the sperm moves forward. We record semen motility at three points: fresh, pre-freeze and post-thaw. All of this data is recorded along with the rest of the sample information in our database, FreezerWorks.

Samples which are stored on-site are maintained in Chart MVE 800 series nitrogen freezers. The majority of the tanks are liquid and one is used for vapor storage. In 2008 SVF built a new cryo-facility which houses all the tanks and includes a 1,500 liter bulk liquid nitrogen tank located on-site. Each tank is independently alarmed and monitored for volume, fill rate and frequency as well as temperature. All of this data is logged. The cryoroom is carefully monitored for oxygen content, and tied into a video surveillance system.