Cryopreservation of semen without using egg yolk

Panayiota Siskos ’23

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

Cryopreservation of semen conserves genetic information and allows offspring to inherit the traits of genetically superior organisms via artificial insemination. Egg yolk is an ingredient of bull semen extender, a liquid diluent that acts as a buffer that protects sperm from changes in temperature and environmental conditions. However,  egg yolk is an ingredient that is hard to standardize due to variations between different producers and the possibility of microbial contamination. Cholesterol, a major component of the sperm plasma membrane, strengthens and protects membrane structures by resisting structural changes in phospholipid hydrocarbon chains. Increasing the cholesterol content in sperm plasma membrane increases sperm cryotolerance, or the ability of the sperm to survive freezing temperatures.  The amount of cholesterol present in sperm plasma membranes can be altered by cyclodextrins, oligosaccharides with an hydrophobic interior core and hydrophilic outer surface 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 sperm extender that contains exogenous cholesterol but not egg yolk. Another goal of this study was to compare the post-thaw quality of sperm frozen with the novel extender and varying concentrations of glycerol, a common cryoprotectant that protects sperm from thermal damage at freezing temperatures.A third objective was to assess the in-vitro fertilization ability of sperm frozen using the egg yolk-free extender and sperm frozen using the conventional egg yolk-containing extender.

Semen was first collected from four bulls by an electroejaculation procedure. Semen ejaculates with sperm motility greater than 60% and concentrations greater than 400×106/ml were pooled, and a small amount of the pooled semen was diluted in an extender containing egg yolk and glycerol and subsequently frozen. A large amount of the semen sample was diluted with an extender that did not contain egg yolk, cooled, and treated with a cholesterol-cyclodextrin extender, which served to incorporate exogenous cholesterol into the sperm membranes. =

Sperm total motilities (% of total sperm moving) and progressive motilities (% of sperm moving in a straight line) in sperm frozen with egg yolk-containing extender and sperm frozen with egg yolk-free extender and treated with cholesterol-cyclodextrin were similar.However, the average path velocity, curvilinear velocity, and straight-line velocity of post-thaw sperm were higher in sperm treated with the egg yolk-free extender compared to sperm treated with egg yolk-containing extender. In addition, the sperm protein profiles of the post thaw sperm were relatively similar between the two groups, with 9 common proteins found. The sperm protein profiles of fresh sperm and sperm frozen with egg yolk-free extender were also compared and had 19 common proteins, suggesting sperm were adequately preserved using the egg yolk-free extender. Increasing the glycerol concentration in the egg yolk-free extender resulted in increased post-thaw sperm total motility and progressive motility, with sperm treated with the highest concentration of glycerol, 7% v/v, having the greatest total and progressive motility, respectively.  Finally, sperm frozen with the egg yolk extender and egg yolk-free extender, respectively, had similar cleavage and blastocyst rates, thus 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 caused by 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:


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