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

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 computerassisted 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.

Improvement of techniques for sperm evaluation and cryobanking in European eel.

“…Furthermore, the use of methylated cryoprotectants is known to induce the production of reactive oxygen species (ROS) that can cause several damages, such as cytosine methylation in fish spermatozoa DNA (Kawai et al, 2010), which is one of the principal epigenetic mechanisms (Bird, 2002), and have been suggested to be a good indicator for sperm quality (Herráez et al, 2017), affecting consequently the success of a cryopreservation protocol. In this comparative study, Herranz-Jusdado et al (2019a) showed that the protocol using methanol, initially developed by the Hungarian group (Müller et al, 2012;Szabó et al, 2005), was better in terms of higher spermatozoa viability and motility than the protocol with DMSO developed by the Spanish group (Asturiano et al, 2016;Peñaranda et al, 2009). Furthermore, the protocol with DMSO induced a hypo-methylation of them spermatozoa DNA, whereas no changes in DNA methylation were observed when sperm was cryopreserved with the protocol with methanol.…”

“…The traditional hormonal treatment with gonadotropins to induce maturation in Japanese eel and European eel males typically consists of weekly injections of human chorionic gonadotropins (hCG) (Ohta et al, 1997;Pérez et al, 2000) and has been used as the preferred method to obtain high quality sperm for cryopreservation trials in the eels (Asturiano et al, 2004;Garzón et al, 2008;Herranz-Jusdado et al, 2019a;Müller et al, 2004;2012;Peñaranda et al, 2009;Szabó et al, 2005;Tanaka et al, 2002a). However, application of heterologous hormonal treatments with hCG have been observed to produce low rates of fertilization and hatching due to low gamete quality (Tanaka et al, 2002a), and a new line of studies focuses on the development of homologous gonadotropic hormones to induce eel maturation.…”

“…Despite the fact that cryopreservation protocols developed by the groups from Spain and Hungary were proved to succeed in fertilization trials, they differed in many aspects (Table 2) and a need of standardization of the protocol was evident. With this aim, both groups conducted together a joined study (Herranz-Jusdado et al, 2019a), where both protocols were tested using the same sperm samples. In this study, in addition to analysis of viability and motility in frozen-thawed samples, epigenetic effects of cryopreservation on spermatozoa DNA was also tested.…”

“…The most recent work on the European eel sperm cryopreservation aimed at the improvement of the protocol by using sperm membrane protection additives and to adapt the protocol to larger volumes (Herranz-Jusdado et al, 2019b) as done with Japanese eels (Nomura et al, 2018). In this latest work, using the protocol described by Herranz-Jusdado et al (2019a), the researchers successfully scaled up the volume of sperm cryopreserved using 2 and 5 mL cryotubes, by adapting the cooling rate. Furthermore, adding egg yolk to the extender solution, they improved the frozen-thawed sperm quality, reaching motility values over 50%, which are the highest motility ever reported in cryopreserved European eel sperm.…”

“…In European eel, several researchers have developed different sperm cryopreservation protocols (Asturiano et al, 2003;Herranz-Jusdado et al, 2019a;Müller et al, 2004;Peñaranda et al, 2009;Szabó et al, 2005). Moreover, cryopreserved European eel sperm have been successfully used in fertilization trials (Asturiano et al, 2016) and in hybridization trials with Japanese eel (Anguilla japonica) eggs (Müller et al, 2012;, although an increased rate of larval deformities were observed when fertilizing with cryopreserved sperm (Müller et al, 2018), evidencing the need of refinement of the protocols.…”