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

J.G Herranz-Jusdado,V Gallego,M Morini,C Rozenfeld,L Pérez,E Kása,T Kollár,A Depincé,C Labbé,Á Horváth,J.F Asturiano

doi:10.1016/j.aquaculture.2018.09.006

Abstract

The critical situation of the European eel (Anguilla anguilla) has urged the development of sperm cryopreservation protocols for reproduction in captivity and cryobanking. In the last years, two research groups have developed 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 sperm was assessed by studying the motility and kinetic values of thawed sperm from both cryopreservation protocols using a computer-assisted sperm analysis (CASA-Mot) system. In addition, a viability analysis was performed using flow cytometry to test if the cryoprotectants or the freezing-thawing process led to a reduction in spermatozoa survival. Furthermore, since during cryopreservation the sperm was treated with methylated cryoprotectants (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 DNA methylation 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 from the methanol based protocol showed significantly higher motility, velocity and percentage of live spermatozoa than the same sperm samples treated with the DMSO based protocol. In addition, the DMSO based protocol induced a hypomethylation of sperm DNA compared to fresh samples whereas the methanol based protocol did not alter sperm DNA methylation level. Our results indicate that the methanol based protocol is a more suitable protocol that preserves better the motility and genetic qualities of the European eel sperm.

Highlights

During the last years, a drastic decrease has been observed in the number of European eels (Anguilla anguilla) returning from Europe and North Africa to the spawning sites in the Atlantic Ocean (Dekker 2000; Jacoby & Gollock, 2014)
The motility results from thawed samples of sperm cryopreserved with the methanol protocol showed higher motility (32.4 ± 1.8%) than those from the dimethyl sulfoxide (DMSO) protocol (10.8 ± 0.9%) (Figure 1)
All the sperm kinetic parameters analyzed showed the same pattern, with higher motility and faster velocities in samples preserved with the methanol protocol than those preserved with the DMSO one (Figure 1)

Summary

Introduction

A drastic decrease has been observed in the number of European eels (Anguilla anguilla) returning from Europe and North Africa to the spawning sites in the Atlantic Ocean (Dekker 2000; Jacoby & Gollock, 2014). The maturation of the European eel in captivity is only achieved by costly and long hormonal treatments (Asturiano et al, 2006; Gallego et al, 2012; Pérez et al, 2000), and still the production of gametes in both sexes can be unsynchronized (Asturiano et al, 2016). The development of cryopreservation protocols for European eel sperm has been considered important for reproduction management, by guaranteeing the availability of both types of gametes when female spawns (Asturiano et al, 2017), besides its application for cryobanking and future broodstock management

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