Sperm cryopreservation protocols for the large-scale fertilization of Japanese eel using a combination of large-volume straws and low sperm dilution ratio

The aim of this study was to develop sperm cryopreservation protocols for large-scale fertilization in Japanese eel (Anguilla japonica), by testing the feasibility of combining large-volume straws and a low sperm dilution ratio. Experiments were conducted using both 0.25-ml straws and 2.5- and 5-ml straws (macrotubes) for sperm cryopreservation. Cooling rates in macrotubes were measured at three internal positions and at different heights above the surface of liquid nitrogen (LN). There was no significant difference in the cooling rate among internal positions in the straws. The cooling rate could be adjusted to the appropriate range for Japanese eel sperm (6.3 °C-8.6 °C min−1) by positioning the 2.5- and 5.0-ml straw 0.5-5.0 cm above the surface of LN. We succeeded in scaling up the storage volume from 0.25 to 4.5 ml (18-fold) without decreasing the motility using a 5.0-ml straw. The number of storable motile spermatozoa was the highest at a range of dilution ratios (sperm to extender, v/v) from 1:1 to 1:5. Although frozen-thawed sperm showed a significant decrease in the proportion of motile sperm to egg compared with than fresh sperm, there was no significant difference in the fertilization rate, and fertilized eggs with cryopreserved sperm did not affect the hatching rate or survival rate at 7 days after hatching. Using a segmented linear analysis, we found that 10,180 ± 3077 motile spermatozoa per egg are required to maximize fertilization success. We estimated that spermatozoa stored in a 2.5- and 5.0-ml straw can fertilize approximately 184,185 and 331,532 eggs, respectively. Furthermore, our investigation of the effect of long-term storage in LN on the sperm quality showed that the storage period does not affect the post-thaw motility rate even after 2 years of storage. Our results demonstrate that cryopreservation techniques for Japanese eel sperm using 2.5- and 5.0-ml straws could be used for large-scale fertilization programs.