Large-scale cryopreservation of Japanese pearl oyster Pinctada fucata martensii sperm

Abstract

To develop methods for large-scale cryopreservation of Japanese pearl oyster spermatozoa, post-thaw motility and fertility of spermatozoa cryopreserved in straw and in a flat-bottomed aluminum cup were compared. Spermatozoa diluted 1:19 with an extender comprising 10% methanol, 72% seawater, and 18% fetal bovine serum were placed in the straw (0.25, 1.0, and 2.0 mL) and in the cup (10 mL). The vessels were then cooled in LN vapor to - 50°C at a cooling rate of -17.6 to - 20.6°C/min, and immersed in LN. The percentages of motility of spermatozoa cryopreserved in 0.25 mL, 1.0 mL, 2 mL straws, and the cup were 35.2%, 31.1%, 23.2%, and 31.8 %, respectively, of the pre-cryopreserved spermatozoa. When 100 μL of semen (pre-cryopreserved or cryopreserved in the 3 kinds of straw) was introduced to 2.5 million eggs, the percentages of fertility were invariably high, but not significantly different from one another. When fresh spermatozoa with low motility were cryopreserved, thawed, and then introduced to eggs, the percentage of fertility increased in a time-dependent fashion, until 20 min after the commencement of insemination. There were no harmful effects on growth and feed intake ability of pearl oyster larvae when the contact time of gametes at fertilization had been prolonged from 5 min to 60 min.