Egg yolk-free cryopreservation of bull semen

Cryopreservation of semen is widely used for conservation of animal genetic resources and exploitation of genetically superior sires through artificial insemination. Egg yolk, an ingredient of bull semen extender, has been extensively used in mammalian semen cryopreservation to protect sperm against initial cold shock. Alternately, milk-based extenders are used for bull semen throughout the world. Unfortunately, egg yolk and milk extenders are difficult to standardize because they vary from producer to producer and introduce microbial contamination. The artificial insemination industry has been facing biosecurity risks through the use of animal products (egg yolk and milk) in semen cryopreservation procedure [10]. The Canadian Food Inspection Agency and World Organization for Animal Health recommended that egg yolk or milk used in semen extender should be free of pathogens or sterilized. Cholesterol, an integral part of the sperm plasma membrane, strengthens and protects membrane structures even below normal temperature. Cholesterol content in sperm plasma membrane determines their cryotolerance. Cyclodextrins bind with exogenous cholesterol, form soluble complexes and deliver cholesterol to cell membranes. Earlier, treatment of sperm with methyl-β-cyclodextrin preloaded with cholesterol before dilution in egg yolk extender improved post-thaw survival in bull, stallion and pig semen. In this study, it was hypothesized that exogenous cholesterol-cyclodextrin (CC) complex can strengthen bull sperm plasma membrane sufficient enough to exclude biosecurity risk egg yolk or milk from a conventional extender. The main goal of this study was to cryopreserve bull semen without adding any exogenous protein in extender. The specific objectives were to compare the post-thaw quality of bull sperm pre-exposed to exogenous cholesterol-cyclodextrin complex and frozen in egg yolk-free tris-glycerol (CC+TG) extender with conventional tris-egg yolk-glycerol (TEYG control) extender, to investigate the role of glycerol in egg yolk-free semen cryopreservation, to determine protein profiles of bull sperm frozen in CC+TG and TEYG control extender and to assess in vitro fertilizing ability of sperm frozen in CC+TG or TEYG control extenders. In first experiment, semen was tris-egg yolk glycerol (TEYG; control extender) extender or first treated with cholesterol-cyclodextrin complex followed by dilution in egg yolk-free tris-glycerol (TG; egg yolk-free) extender (collectively called as “CC+TG”) at 22°C or 4°C, and frozen. After thawing of semen, sperm motion characteristics were similar between CC+TG and TEYG control extenders, and temperature of glycerol addition. In second experiment, semen was frozen in CC+TG extender with different concentration of glycerol (7 to 0%; v/v). Post-thaw sperm quality decreased with the decline in glycerol concentration in TG extender. In third experiment, SDS electrophoresis of proteins from fresh sperm and sperm frozen in CC+TG, and TEYG control extenders was conducted. Protein profiles in fresh sperm and CC+TG frozen sperm were almost similar. Egg yolk proteins bound tightly with sperm plasma membrane. In fourth experiment, in vitro fertilization potentials of sperm frozen in TEYG control and CC+TG extenders were tested. Cleavage and blastocyst rates of semen frozen in CC+TG and TEYG control extenders were similar. In conclusion, cholesterol-cyclodextrin replaced egg yolk from the semen extender.