Development of an Artificial Fertilization Method for the Japanese Pearl Oyster Pinctada fucata martensii by Using a Microplate

Abstract

Japanese pearl oysters are widely cultivated by artificial fertilization using gametes obtained from the ovary and testis. Using the gametes from the Japanese pearl oyster, we developed a method for conducting small-scale artificial fertilization tests in a 24-well microplate. Following induction of germinal vesicle breakdown (GVBD), we inseminated 20,000 eggs in 2.0 ml seawater in a single well of a microplate. The highest fertilization rates were obtained using a testicular semen volume of 1.25μl when the concentration of NH₃ in seawater was 0.75 mM during insemination. Changes in contact time (the time from commencement of insemination to rinsing the eggs) from 3 to 60 min did not affect the fertilization rate. However, a contact time of >12 min was necessary to ensure consistency in contact time among the 24 wells.