Motility and fertilizing capacity of frozen/thawed common carp ( Cyprinus carpio L.) sperm using dimethyl-acetamide as the main cryoprotectant

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 (LN 2) 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 LN 2). 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 LN 2 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.