Nuestro último artículo, aceptado en Aquaculture

European eel sperm storage: optimization of short-term protocols and cryopreservation of large volumes

J.G. Herranz-Jusdado, V. Gallego, C. Rozenfeld, M. Morini, L. Pérez, J.F. Asturiano

Maturation in captivity of European eel (Anguilla anguilla) requires long and costly hormonal treatments that often lead to asynchronic maturation between sexes. Therefore, optimization of sperm short-term storage methods and cryopreservation protocols can be a key factor for successful artificial fertilization. Two experiments were carried out to optimize the existing protocols.

For the short-term storage experiment, sperm was diluted in P1 extender and then stored at different dilution ratios (1:9 and 1:49). The best outcome was then tested at different temperatures (4 and 20 ºC) and in constant agitation or still. In the cryopreservation experiments, large sperm volumes (cryotubes of 2 and 5 ml), different cooling rates (freezing tubes 1 or 3 cm above liquid nitrogen during 15 and 20 minutes), and different extender compositions (methanol 10% was used as cryoprotectant, and complemented with FBS 20%, BSA 5% or egg yolk 5%) were tested. Sperm kinetic parameters were analyzed with a CASA-Mot system both in fresh and short- or long-term stored samples.

In the short-term storage trial, sperm quality did not show significant differences in the first 24 h after sperm collection between the different storage conditions tested. For longer time, 1:49 dilution ratio showed significantly better results than 1:9, and low temperature (4 ºC) was better for sperm preservation after 3 days.

Cryopreserved sperm samples showed good motility results when they were frozen in cryotubes of 2 and 5 ml, with no significant differences compared to samples cryopreserved in lower volumes (straws of 0.5 ml). Furthermore, the combination of methanol (10%) and egg yolk (5%) as freezing medium, induced significant higher post-thawing motility values (over 50%) than the control (methanol 10%), whereas the addition of FBS (20%) and BSA (5%) led to a significant reduction of the sperm motility. The establishment of these storage and cryopreservation protocols will be important for the improvement of European eel artificial reproduction programs.