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

doi:10.1016/j.aquaculture.2019.03.019

J.G Herranz-Jusdado, V Gallego + Show 4 more

Open Access

https://doi.org/10.1016/j.aquaculture.2019.03.019

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Abstract

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 °C) 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 min), 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 °C) 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.

Highlights

European eel (Anguilla anguilla) is a catadromous fish with a complex life cycle that includes several metamorphoses
The results showed no reduction in motile spermatozoa (MOT) in the first 24 h independently on the dilution ratio, and after 48 h, sperm samples of both dilutions showed a significant decrease in MOT, but the MOT values were significantly higher in samples diluted 1:49 than in sperm samples diluted 1:9
After 48 h, all samples showed a reduction in MOT and progressive motility (pMOT) independently of the storing condition compared to control and only samples stored at 20 oC and still showed lower MOT than samples stored under the other conditions

Summary

Introduction

European eel (Anguilla anguilla) is a catadromous fish with a complex life cycle that includes several metamorphoses. The European eel is very appreciated as food delicatessen with great economical value, reinforcing the need for a program for reproduction in captivity to release the fishing pressure on natural individuals. Maturation in captivity of European eel requires hormonal treatments that last for several weeks in males and even months in females (Butts et al, 2014; Gallego et al., 2012; Mylonas et al, 2017), and frequently there is a maturation asynchrony between genders. Short-term preservation of fresh sperm diluted in extender medium, or cryopreservation in liquid nitrogen is necessary to facilitate artificial fertilization in European eel (Asturiano et al., 2016)

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