Improvement of canine semen vitrification using sucrose and soy lecithin extender


   

Improvement of canine semen vitrification using sucrose and soy lecithin extender 
Maja Pipan,a Margret Casal,b Janko Mrkun,a Irma Klunc 

aClinic for Reproduction and Large Animals, Veterinary Faculty, University of Ljubljana, Slovenia 
 bClinical Sciences and Advanced Medicine, University of Pennsylvania School of Veterinary Medicine, PA 

cUniversity Medical Centre Ljubljana, Ljubljana, Slovenia 
 
 Major challenge in freezing semen for short or long term availability is avoiding damage to intact 
spermatozoa caused by freezing. Freezing-induced impairment becomes particularly problematic for low-
quality semen. Most freezing methods involve tedious slow cooling, which requires increased processing 
time and specialized media. Although vitrification protocols are faster (no slow cooling) and provide 
improved results, they are not commonly used for canine semen. Our objective was to improve vitrification 
methods for canine semen by adding sucrose to a basic canine semen extender not containing animal 
proteins. Twenty ejaculates were obtained from 19 dogs. Semen volume, concentration, sperm motility, 
progressive motility, morphology, tail membrane integrity and acrosomal defects were determined on the day 
of collection. Each ejaculate was divided into 9 aliquots and diluated in semen extenders labeled A - G. 
Control extender consisted of TRIS, citric acid, fructose and antibiotics, with soy bean lecithin and sucrose 
added to create test extenders. For Extender A, only soy lecithin (1%) was added. Extenders B, C, and D 
contained 0.2, 0.25, and 0.3 M of sucrose, respectively, but no soy lecithin, whereas Extenders E, F, and G 
contained corresponding concentrations of sucrose, plus 1% soy lecithin. From each group, a 33 µl 
suspension of cells was dropped directly into liquid nitrogen. After at least 48 hours, frozen drops were 
placed in 5 ml of devitrification medium (control medium containing 1% bovine serum albumin) that has 
been prewarmed to 37ºC. The after thaw suspension was maintained at 37ºC for 10 minutes and was then 
centrifuged at 300 g for 5 minutes. The cell pellet was finally resuspended in 50 µl of devetrification medium 
only for sperm evaluation. After thaw evaluation included  motility, progressive motility, morphology, 
viability, plasma membrane modifications, acrosomal defects, and DNA fragmentation. Neither soy bean 
lecithin (A) or sucrose alone (B, C, D) were sufficient for preserving thawed sperm quality. However, 
extenders containing soy lecithin and various concentrations of sucrose (E, F, G) resulted in significantly 
better semen quality than all other extenders. In particular, Extender F (2.5 M sucrose) had a higher 
proportion of motile (p < 0.001), progressively motile (p < 0.001), morphologically normal (p < 0.05) and 
viable (p < 0.01) spermatozoa compared to Extenders E and G. There was also a higher proportion of 
spermatozoa with intact DNA (p < 0.01) and normal plasma membrane integrity (p < 0.05) in group F. 
Vitrification of canine spermatozoa in extenders containing soy bean lecithin and sucrose preserved 
important physiological attributes, including viability, motility, progressive motility, sperm tail membrane 
integrity and DNA integrity during. We concluded that this method was simple, fast and effective for 
vitrification of canine semen. Use of soy lecithin in lieu of animal proteins (e.g. egg yolk) facilitates ease of 
shipping to countries with strict import requirements.  

Keywords: Vitricification, semen preservation, canine 
 

 

515 Clinical Theriogenology • Volume 11,  Number 3 • September 2019



516Clinical Theriogenology • Volume 11,  Number 3 • September 2019



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