Effects of cryopreservation on sperm structure in Japanese pearl oysterPinctada fucata martensii

Abstract

To clarify factors reducing the motility and fertility of cryopreserved spermatozoa of the Japanese pearl oyster Pinctada fucata martensii, the structure of spermatozoa before and after cryopreservation was observed by scanning electron microscopy. Testicular spermatozoa were diluted with cryopreservation diluent (10% methanol + 18% fetal bovine serum + 72% sea water), and dispensed into 0.25-mL straws. The straws were cooled at a rate of approximately -20°C/min to -50°C, and subsequently immersed in liquid nitrogen. Percentage motility of spermatozoa before cryopreservation was 69.9 Ϯ 4.2%, and that of cryopreserved spermatozoa was 24.0 Ϯ 1.8%, respectively. In cryopreserved spermatozoa, the percentage that lacked or had a deformed flagellum was 56.6 Ϯ 3.9%, while in fresh spermatozoa this was 8.7 Ϯ 2.0%. In cryopreserved spermatozoa, the percentage of deformed acrosomes was 76.6 Ϯ 5.2%, while in fresh spermatozoa this was only 0.9 Ϯ 0.3%. Cryopreserved spermatozoa with a normal acrosome and flagellum were only 15.4 Ϯ 3.5% of those in fresh spermatozoa. These results indicate that lesion of the flagellum and deformation of the acrosome occurred through the cryopreservation procedure, and both types of damage lead to loss of the motility and fertility in thawed spermatozoa.

Motility and fertility of cryopreserved spermatozoa of the Japanese sea cucumber Apostichopus japonicus

“…They found that more than 10 times the volume of post‐thaw semen was necessary to obtain similar fertilization success as with fresh semen. They showed that the difference in the fertility of sperm before and after cryopreservation originated from 56.6% of the post‐thaw sperm with damaged flagella (that of fresh sperm was 8.7%) and 76.6% of post‐thaw sperm had damaged acrosomes (similarly, 0.9%) (Narita, Kawamoto, Isowa, Aoki, Hayashi, Ohta, et al, ). In this study, we developed a sperm cryopreservation method based on post‐thaw motility, but it will be important to examine the integrity of the acrosomes to elucidate the post‐thaw fertility of the sperm.…”