The influence of Percoll® density gradient centrifugation before cryopreservation on the quality of frozen wisent (Bison bonasus) epididymal spermatozoa

Abstract

Background The wisent (Bison bonasus) is a species that has undergone a population bottleneck. Homozygosity is prevalent within the population and may have a negative impact on semen quality in wisent bulls. Semen samples containing a large amount of functionally and morphologically impaired or dead spermatozoa have lower tolerance for cryopreservation process. Such samples are prone to involve damage acrosomes, to produce and release reactive oxygen which negatively affects proper function of spermatozoas. It is a good practice to select intact and viable gametes before subjecting the sample to cryopreservation to improve the efficiency of this process. The aim of this study was to assess the ability of Percoll® density gradient centrifugation in order to improve the quality of wisent spermatozoa after cryopreservation. Spermatozoa samples were analysed with computer-assisted semen analysis system and flow cytometry. Results Percoll® density gradient centrifugation resulted in increased percentage of motile spermatozoa, higher proportion of spermatozoa with normal morphology and proper functionality but also in a significant reduction of the total number of gametes. Nevertheless, the concentration of frozen spermatoza was still sufficient for obtaining a few complete insemination doses suggested for cattle from each epididymis. Conclusions While creating a high-quality genetic reserve, for in vitro fertilisation purposes, eliminating detritus and improving the overall quality of samples is more important than total number of spermatozoa. For these reasons, the achievement of higher post thaw quality of spermatozoa justifies the purification of samples by centrifugation in a Percoll® density gradient prior to the cryopreservation process.

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

“…Epididymal spermatozoa collected post mortem are considered as a valuable source of gametes for protected animals genome banks [4]. For some species, such as European bison, they are the only source of male gametes that can be cryopreserved and used later in ART [2,8]. The possibility of effective cryopreservation of wisent epididymal spermatozoa has been presented in a few publications [8,9].…”

“…The non-sperm events were gated out based on scatter properties and not analysed. A total of 10,000 events were analysed for each sample [8].…”

“…The staining used were performed according to protocols routinely used in our laboratory, which have been previously described in wisent [8].…”

“…However, despite the fact that the number of European bison individuals is already more than 9,000 [6], their narrow initial genetic pool carries a certain risk for its future. For this reason, currently, wisent conservation programs are enriched by preserving of genetic diversity by gametes banking and implementation of ART [2,5,[7][8]. There are several reports in the literature on the collection and cryopreservation of wisent epididymal sperm [8][9][10].…”

Molecular Markers: A New Paradigm in the Prediction of Sperm Freezability

“…A new insight into the improvement of post-thaw sperm quality brings density gradient centrifugation before cryopreservation. In particular, creating a high-quality genetic reserve for rare species is more valuable than a higher total sperm concentration [ 210 ]. A nanotechnology-based approach may also bring unique accuracy to this field.…”