Cryopreservation of semen without using egg yolk

Panayiota Siskos ’23

Figure 1: Egg yolk is used to protect sperm from cold shock in semen cryopreservation.

Cryopreservation of semen conserves genetic information and allows fertilization via artificial insemination. Egg yolk is an ingredient of bull semen extender, which buffers sperm from temperature and environmental stressors. However, egg yolk composition is very variable between different producers. Cholesterol is a molecule that strengthens membrane structures. Increasing cholesterol content in sperm plasma membrane increases cryotolerance, allowing sperm to survive freezing temperatures. The amount of cholesterol present in sperm plasma membranes can be altered by cyclodextrins, oligosaccharides that bind to cholesterol. The main purpose of this study, led by scientists from the University of Saskatchewan, was to cryopreserve bull semen with a novel egg yolk-free extender containing exogenous cholesterol. Another goal was to compare the qualities of sperm frozen with the novel extender and varying concentrations of glycerol, a cryoprotectant that protects sperm from freezing temperatures. A third objective was to assess the in-vitro fertilization ability of sperm frozen using the egg yolk-free extender.

Semen was first collected from bulls by an electroejaculation procedure. Some sperm was diluted in an extender containing egg yolk and glycerol, and subsequently frozen. The rest of the sperm was diluted in an egg yolk-free extender, cooled, and treated with a cholesterol-cyclodextrin extender to incorporate exogenous cholesterol into the sperm membranes. 

The percentage of total sperm moving in sperm frozen with egg yolk-containing extender and egg yolk-free extender/cholesterol-cyclodextrin, respectively, were similar. However, sperm velocity was higher in the egg yolk-free group compared to the egg yolk-containing group. In addition, 9 common proteins were found in the post-thaw sperm between the two groups. Fresh sperm and sperm frozen with egg yolk-free extender had 19 common proteins, suggesting adequate preservation using the egg yolk-free extender. Additionally, increasing the glycerol concentration in the egg yolk-free extender resulted in increased post-thaw sperm motility. Finally, sperm frozen with the egg yolk extender and egg yolk-free extender, respectively, had similar cleavage and blastocyst rates, suggesting similar in-vitro fertilizing ability. 

This study is important because it showed the success of a novel cryopreservation technique using egg yolk-free extender and exogenous cholesterol, which avoids the biosecurity risks associated with using egg yolk. Future studies could use cholesterol-cyclodextrin complex diluted in glycerol as an extender to study changes in proteins after cryopreservation, which may allow researchers to isolate membrane proteins and improve freezing abilities. In addition, in-vivo fertility trials of semen frozen without egg yolk are currently in progress and will be reported in the future. 

Works Cited:

[1] A. Muhammad, et al., Egg yolk-free cryopreservation of bull semen. Plos One 14, (2019). doi:

[2] Image retrieved from:


Leave a Reply

Fill in your details below or click an icon to log in: Logo

You are commenting using your account. Log Out /  Change )

Facebook photo

You are commenting using your Facebook account. Log Out /  Change )

Connecting to %s