Comparison of European eel sperm cryopreservation protocols with standardization as a target

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Herranz-Jusdado, J.G. | Gallego, V. | Morini, M. | Rozenfeld, C. | Pérez, L. | Kása, E. | Kollár, T. | Depince, Alexandra | Labbé, Catherine | Asturiano, J.F.

Edité par CCSD ; Elsevier -

The critical situation of the European eel (Anguilla anguilla) has urged the development of sperm cryopre-servation protocols for reproduction in captivity and cryobanking. In the last years, two research groups havedeveloped their own protocols in Spain and Hungary with positive results, but difficult to compare.Here, a series of experiments were conducted to test the quality of thawed sperm after using both protocols,determining which of them produce the best results and aiming for standardization. The quality of thawed spermwas assessed by studying the motility and kinetic values of thawed sperm from both cryopreservation protocolsusing a computer-assisted sperm analysis (CASA-Mot) system. In addition, a viability analysis was performedusing flow cytometry to test if the cryoprotectants or the freezing-thawing process led to a reduction in sper-matozoa survival. Furthermore, since during cryopreservation the sperm was treated with methylated cryo-protectants (DMSO or methanol) that may induce epigenetic changes in the sperm DNA (cytosine methylation)and could affect the offspring, we conducted a luminometric methylation assay (LUMA) to study the DNAmethylation levels induced by both protocols.In this work, all the above-mentioned parameters were analyzed in fresh and frozen-thawed sperm samples.Our results showed that thawed sperm samples from both protocols presented lower sperm motility and velocity,and lower percentage of live cells than those shown in fresh sperm samples. Furthermore, sperm samples fromthe methanol based protocol showed significantly higher motility, velocity and percentage of live spermatozoathan the same sperm samples treated with the DMSO based protocol. In addition, the DMSO based protocolinduced a hypomethylation of sperm DNA compared to fresh samples whereas the methanol based protocol didnot alter sperm DNA methylation level. Our results indicate that the methanol based protocol is a more suitableprotocol that preserves better the motility and genetic qualities of the European eel sperm.

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