Cholesterol does not improve cryoprotective efficiency of soybean lecithin-based extenders for bull semen

Design of plant phospholipid based extenders free of animal-derived components is urgent in farm animal semen cryopreservation. However, a complex of plant lipids, unlike the yolk complex, lacks cholesterol which plays an important role in cryoresistance of spermatozoa. Enriching spermatozoa membranes with cholesterol improves their cryotolerance (E. Mocé et al., 2010; E. Mocé et al., 2014; M.H. Fayyaz et al., 2016). We first estimated the impact of different cholesterol concentrations (0, 8, 18, 33 and 50 %) in soybean phospholipid mixtures both on morphology of obtained liposomes or lipid particles, and on their cryoprotective property as judged by the effect on motility when added to the extender at bull sperm cryopreservation. Liposome suspensions were prepared from commercial lecithin LeciPRO 90 («Unite-chem Co., Ltd», China) and purified cholesterol form lanolin («Sigma-Aldrich Co.», USA) by sonication at an ultrasound disintegrator UZDN-2T (NPP «Academpribor», Russia; 22 kHz, 60 W/cm2, 5 min). It was found that cholesterol did not notably influence the obtained liposomes at low concentration (8 %). At 18 and 33 % cholesterol in the lipid mixture the size of the liposomes increased and averaged 66±6 nm and 62±11 nm, respectively. Fifty percent cholesterol changed the lipid suspension completely resulting in complex structures formed by phospholipid-cholesterol lamellae and crystals of cholesterol monohydrate. Eight percent cholesterol did not increase a cryorpotective efficiency of lecithin, while 18-50 % cholesterol decreased its cryoprotective capacity. An increase in cholesterol concentration in the lipid mixture up to 50 % (» 66 mol%) led to somewhat higher spermatozoa motility compared to a minimum score observed at freezing with 33 % cholesterol. The most probable cause of the negative impact of cholesterol on the cryoprotective effectiveness of lecithin suspension is impairment in liposome interaction with a sperm plasma membrane caused by condensation of phospholipid packing in a lipid bilayer.