The first report on the use of a zona pellucida binding assay to compare the effects of European bison (Bison bonasus) epididymal spermatozoa cryopreservation in two different extenders

Abstract

The wisent (or European bison) Bison bonasus is a species that has gone through a bottleneck, resulting in a narrow gene pool of the current population. Presently, the protection program for this species, in addition to protecting the habitat, is aimed at preserving gene pool. One of its elements is creating a bank of gametes with the intention of using them in constantly developed Assisted Reproductive Techniques in this species. In order to use the potential of the gametes stored in the bank as effectively as possible, it is extremely important to assess their post-freezing quality. Wisent epididymal spermatozoa stored in the bank are evaluated using basic semen analysis methods, the CASA system and flow cytometry. So far, functional tests of spermatozoa have not been introduced to the evaluation protocol of spermatozoa obtained post mortem from European bison. This article, for the first time describes the use of the zona pellucida binding test to assess the quality of cryopreserved wisent epididymal spermatozoa. Due to the close relationship between the species, the Zona binding Assay protocol developed for cattle was used. To exclude the influence of the composition of the extender used for cryopreservation, the spermatozoa were cryopreserved in the Extender based on Tris buffer, egg yolk and glycerol and in the commercial extender Andromed(Minitube GMBH). Regardless of the diluent used, 100% of the oocytes had sperm attached. The average number of spermatozoa attached to the oocyte was 66.26 for the Tris-based diluent group and 27.37 for the Andromed (Minitube GMBH) - frozen group. The advantage of the Tris based extender resulted from ZBA seems to coincide with the results of the basic and advanced semen evaluation. The conducted experiment showed that the ZBA protocol developed for cattle is suitable for the evaluation of wisent epididymal spermatozoa.