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
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.
