Motility and fertilizing
capacity of frozen/thawed common carp (Cyprinus carpio L.) sperm
using dimethyl-acetamide as the main cryoprotectant
D. Warnecke, H.-J. Pluta-2003
Aquaculture 215(1-4): 167-185
Abstract:
In the present study, motility and fertilizing
capacity of fresh and their corresponding frozen/thawed common carp (Cyprinus
carpio L.) sperm were investigated in order to evaluate several
dimethyl-acetamide (DMA)-containing cryo-diluents. Sperm motility was
analyzed objectively through computer-assisted sperm motion analysis (CASA).
In five consecutive test series, the effects on post-thaw motility of 11
different cryo-diluents added in a ratio of 1:6 were assessed. The
cryo-diluent composition varied by two different extenders, sugar addition
(sucrose or trehalose; 100–300 mM), and DMA concentrations between 10% and
25%. Three freezing profiles were applied, which differed in freezing speed
(averaged rate until -196 °C: 3, 6, and 10 °C min-1) and method
(freezer-controlled vs. liquid nitrogen (LN2) vapor). For
thawing, straws (0.25 ml) were immersed for 3 s in a water bath at 40 °C.
Optimal post-thaw motility results were obtained when using Cryo3 (modified
Kurokura's extender 2 (MK2)/200 mM sucrose/15% DMA) and Cryo10 (MK2/200 mM
trehalose/20% DMA) in combination with profile III (10 °C min-1,
above LN2). Maximum initial (15–20 s post-activation) motility
rates exhibited 40±6% and reached about half the values of the
corresponding fresh sperm. Initial averaged path velocity (VAP) was reduced
from 70–90 µm s-1 with fresh sperm to maximum values of
60–65 µm s-1 with frozen/thawed sperm. In a final test series,
the fertilizing capacity of sperm frozen with Cryo3 and Cryo10 was
investigated. Calculated from the total number of eggs, hatching rate was 80±2%
and percentage of swim-up-stage larvae was 78±2% when sperm were frozen for
6 days in Cryo10. These results were nearly identical with the control
values (fresh sperm). Sperm frozen with Cryo3 and stored for 349 days in LN2
still produced 38±5% of healthy larvae. Five minutes equilibration time of
fresh sperm with Cryo10 resulted in a steep decrease of motile cells to
levels of frozen/thawed sperm, but 68±7% of the embryos developed normally.
Using DMA as internal cryoprotectant in combination with the MK2/sugar
extender proved to be very suitable for cryopreservation of common carp
sperm, especially with regard to fertilization and hatching success.
(FG
Ökotoxikologie and Biochemie, Institut für Biologie, Freie Universität
Berlin, Ehrenbergstr. 26-28,
D-14195, Berlin, Germany,
Tel.: +49-30-838-54324; fax: +49-30-838-54585; email: dietmarw@zedat.fu-berlin.de)