Abstract:
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[EN] Contents European eel, Anguilla anguilla, is a target species for future captive breeding, yet best methodology to estimate sperm density for application in in vitro fertilization is not established. Thus, our objectives ...[+]
[EN] Contents European eel, Anguilla anguilla, is a target species for future captive breeding, yet best methodology to estimate sperm density for application in in vitro fertilization is not established. Thus, our objectives were to evaluate methods to estimate European eel sperm density including spermatocrit, computer-assisted sperm analysis (CASA) and flow cytometry (FCM), using Neubauer Improved haemocytometer as benchmark. Initially, relationships between spermatocrit, haemocytometer counts and sperm motility were analysed, as well as the effect of sperm dilution on haemocytometer counts. Furthermore, accuracy and precision of spermatocrit, applying a range of G-forces, were tested and the best G-force used in method comparisons. We found no effect of dilution on haemocytometer sperm density estimates, whereas motility associated positively with haemocytometer counts, but not with spermatocrit. Results from all techniques, spermatocrit, CASA and FCM, showed significant positive correlations with haemocytometer counts. The best correlation between spermatocrit and haemocytometer counts was obtained at 6000xg (r=0.68). Of two CASA variants, one or three photographic fields (CASA-1 and CASA-2), CASA-2 showed a very high accuracy to haemocytometer counts (r=0.93), but low precision (CV: CASA-2=28.4%). FCM was tested with and without microfluorospheres (FCM-1 and FCM-2), and relationships to haemocytometer counts were highly accurate (FCM-1: r=0.94; FCM-2: r=0.88) and precise (CV: FCM-1=2.5; FCM-2=2.7%). Overall, CASA-2 and FCM-1 feature reliable methods for quantification of European eel sperm, but FCM-1 has a clear advantage featuring highest precision and accuracy. Together, these results provide a useful basis for gamete management in fertilization protocols.
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Thanks:
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This work was conducted as part of the project 'Reproduction of European Eel: towards a Self-sustained Aquaculture' (PRO-EEL) supported financially by the European Commission's 7th Framework Programme under the Theme 2 ...[+]
This work was conducted as part of the project 'Reproduction of European Eel: towards a Self-sustained Aquaculture' (PRO-EEL) supported financially by the European Commission's 7th Framework Programme under the Theme 2 'Food, Agriculture and Fisheries, and Biotechnology', Grant Agreement no245257. Juan F. Asturiano and Luz Perez received a grant to stay in Denmark from Programa de Apoyo a la Investigacion y Desarrollo (PAID-00-11) of the Universitat Politecnica de Valencia. Victor Gallego has a predoctoral grant from Spanish Ministry of Science and Innovation (MICINN). Fish were raised at a commercial eel farm in Jutland, Denmark (Stensgard Eel Farm A/S). We want to thank Peter Lauesen, Billund Aquaculture Service, Christian Graver, Danish Eel Producers Association and Maria K. Johnsen, Technical University of Denmark, for help and assistance in experiments. All fish were handled in accordance with the European Union regulations concerning the protection of experimental animals (Dir 86/609/EEC).
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