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

Abstract

We developed both a cryopreservation method for Japanese sea cucumber spermatozoa and an artificial fertilization method using post-thaw spermatozoa. Twenty per cent dimethyl sulfoxide (DMSO), 16% foetal bovine serum, and 64% artificial seawater were suitable cryodiluent, and the diluent was pre-cooled to 0°C. Semen was diluted with the solution and enclosed in a 250 μl straw, cooled to −50°C at 10.4 ± 0.4°C/ min, and immediately immersed in liquid nitrogen. Although this method showed the highest post-thaw motility in all the conditions we examined, its post-thaw motility was still less than approximately 15%. Artificial fertilization was carried out by adding post-thaw semen with a cryodiluent to the oocytes. The fertilization rate of 200 oocytes/ml seawater increased with the amount of post-thaw semen from 1 to 5 μl but showed a significant decrease at 25 μl. This decrease was considered to be due to DMSO in the cryodiluent, because the fertilization rate of the fresh semen decreased sharply when the DMSO concentration around the oocytes was 1.0% or more. Further improvement in increasing post-thaw motility and lowering the cryoprotectant concentration is necessary for commercial-scale artificial fertilization.

Optimizing cryopreservation of sea cucumber (Apostichopus japonicus) sperm using a programmable freezer and computer-assisted sperm analysis

“…Sperm were activated by a 1:1000 dilution with activator. The composition of the activator was filter-sterilized (the filter mesh size: 0.45mm) NSW containing 5 wt% albumin (EVERY GREEN, Zhejiang, China) (Paredes et al, 2013;Mizuno et al, 2019). After the sperm were activated for about 30 s, we added 10 mL of the mixed solution to the sperm counting plate and placed it in the CASA system (ML-500JZ, Nanning Songjintianlun Biotechnology Co., Ltd, Nanning, China) to quantify sperm motility.…”

“…Of the exiting studies, mollusks (mainly oysters) and echinoids (mainly sea urchins) together account for 72% of the research subjects (Liu et al, 2015;Paredes, 2015;Paredes, 2016;Paredes et al, 2019). At present, only two studies of cryopreservation of sea cucumber sperm have been published (Shao et al, 2006;Mizuno et al, 2019). Shao et al (2006) reported that the optimal freezing rate, sperm motility, and fertilization rate after thawing were 35°C/ min, 76.7 ± 2.9%, and 60.2 ± 5.4% respectively, whereas Mizuno et al (2019) reported 10.4°C/min, 19.3 ± 1.1%, and 89.8 ± 1.7%, respectively.…”

“…At present, only two studies of cryopreservation of sea cucumber sperm have been published (Shao et al, 2006;Mizuno et al, 2019). Shao et al (2006) reported that the optimal freezing rate, sperm motility, and fertilization rate after thawing were 35°C/ min, 76.7 ± 2.9%, and 60.2 ± 5.4% respectively, whereas Mizuno et al (2019) reported 10.4°C/min, 19.3 ± 1.1%, and 89.8 ± 1.7%, respectively.…”

“…To date, most studies of cryopreservation of echinoderm sperm have controlled the freezing rate by adjusting the distance of the sample from the liquid nitrogen surface, and assessment of sperm motility has been conducted visually using an optical microscope. These methods have too many subjective factors and potential procedural errors to accurately assess the quality of frozen sperm (Dunn and Mclachlan, 1973;Asahina and Takahashi, 1978;Adams et al, 2004;Shao et al, 2006;Jalali and Crawford, 2012;Fabbrocini et al, 2014;Paredes, 2015;Paredes, 2016;Mizuno et al, 2019;Paredes et al, 2019).…”

Optimización de un protocolo para la criopreservación del espermatóforo y masa espermática del langostino blanco Litopenaeus vannamei (Boone, 1931)

“…El suero fetal bovino (FBS) es convencionalmente incluido en las soluciones de criopreservación debido a que contiene una mezcla de factores de crecimiento y sustancias indefinidas (Tae et al, 2017) que potencian la supervivencia de los espermatozoides durante del proceso de criopreservación y descongelamiento (Yong et al, 2014). Se ha reportado su uso como agente crioprotector a concentraciones entre 10 a 20% lo cual se considera recomendable, dado que una mayor concentración presenta efectos negativos sobre la movilidad espermática (Mizuno et al, 2018). En este trabajo se utilizó FBS al 10% junto a sucrosa a 0.2M y MgCl 2 a concentración final 15% (Solución 6) obteniendo un porcentaje de viabilidad de 80.0 ± 1.7 (espermatóforos) y 65.6 ± 20.4 (masa espermática), estadísticamente semejante a los valores obtenidos con las soluciones seleccionadas.…”