Evaluation of methods to determine sperm density for the European eel, Anguilla anguilla
Sune R. Sørensen, Victor Gallego, Luz Pérez, Ian
A.E. Butts, J. Tomkiewicz, J.F. Asturiano
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 hemocytometer
as benchmark. Initially, relationships between spermatocrit, hemocytometer
counts, and sperm motility were analyzed, as well as the effect of sperm
dilution on hemocytometer 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 hemocytometer sperm density estimates, whereas motility associated
positively with hemocytometer counts, but not with spermatocrit. Results from
all techniques, spermatocrit, CASA and FCM, showed significant positive
correlations with hemocytometer counts. The best correlation between spermatocrit
and hemocytometer counts was obtained at 6000 × g (r = 0.68). Out of two CASA
variants, one or three photographic fields (CASA-1 and CASA-2), CASA-2 showed a
very high accuracy to hemocytometer 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 hemocytometer 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 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.
