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Evaluation of Methods to Determine Sperm Density for the European eel, Anguilla anguilla

Authors

  • SR Sørensen,

    Corresponding author
    1. National Institute of Aquatic Resources, Technical University of Denmark, Charlottenlund, Denmark
    • Author's address (for correspondence): SR Sørensen, National Institute of Aquatic Resources, Technical University of Denmark, Kavalergården 6, 2920 Charlottenlund, Denmark. E-mail: srs@aqua.dtu.dk

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  • V Gallego,

    1. Grupo de Acuicultura y Biodiversidad, Instituto de Ciencia y Tecnología Animal, Universitat Politècnica de València, Valencia, Spain
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  • L Pérez,

    1. Grupo de Acuicultura y Biodiversidad, Instituto de Ciencia y Tecnología Animal, Universitat Politècnica de València, Valencia, Spain
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  • IAE Butts,

    1. National Institute of Aquatic Resources, Technical University of Denmark, Charlottenlund, Denmark
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  • J Tomkiewicz,

    1. National Institute of Aquatic Resources, Technical University of Denmark, Charlottenlund, Denmark
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  • JF Asturiano

    1. Grupo de Acuicultura y Biodiversidad, Instituto de Ciencia y Tecnología Animal, Universitat Politècnica de València, Valencia, Spain
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  • The work presented in this manuscript was carried out at: National Institute of Aquatic Resources, Technical University of Denmark and Grupo de Acuicultura y Biodiversidad. Instituto de Ciencia y Tecnología Animal, Universitat Politècnica de València, Spain.

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 6000 × g (= 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 (= 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: = 0.94; FCM-2: = 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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