When we freeze semen in liquid nitrogen, we are asking the sperm cells to survive a drop in temperature from 37 C to 4 C during the chilling process, followed by a plunge to –196 C during the freezing process. Luckily, canine semen is less sensitive than many other species. We must be meticulous, but we do not require ‘100% clean rooms’ or other high-priced technological aids. Once frozen, semen can be stored indefinitely.
Special extenders are designed to protect the sperm cells during freezing. Glycerol, a cryoprotective agent, is allowed to penetrate the cells after chilling. Buffering compounds maintain an optimum pH of 6.9-7.1 despite hydrogen ion production by sperm metabolic activity (tends to increase the pH). Antibiotics are added to prevent bacterial growth, sugars are included to provide energy, and fresh egg yolk is used to protect cell membranes from cold shock.
The rate of freezing should be fast enough to prevent excessive shrinkage of sperm cells as fluid leaves them (changing osmotic gradient), but slow enough to prevent ice from forming inside the cells. When frozen semen is thawed, the optimal rate is fast enough to prevent ice crystal formation, but slow enough to prevent cells from rupturing as fluid re-enters. We work with Minitube products and technology.
Success with frozen semen depends on consistent attention to detail, good breeding management (ovulation timing), and intrauterine deposition of semen by transcervical insemination or (in rare cases) surgical insemination. Vaginal deposition of frozen semen leads to a success rate of < 25% because the thawed sperm lives for only about 12 hours.