Cryopreservation of great scallop (Pecten maximus) sperm: effect of extender, cryoprotectant and cooling rate on sperm survival

Abstract

-To develop selection programs for the great scallop, artificial reproduction of the species needs to be improved by gamete cryobanking. Here, a set of four experiments was designed in order to define the basic elements of a cryopreservation protocol for scallop sperm, including extender composition (experiment 1), cryoprotectant selection (2), cooling rate (3), and assessment of the effects of sperm cryopreservation on sperm motility and fertilization capacity (4). Sperm was collected after serotonin injection (100 µl of a 10-mM solution) and frozen in 500-µl straws. For the first three experiments, the percentage of motile fresh sperm (80 ± 4%, mean ± SEM) was significantly higher than that observed for thawed sperm (11 ± 2%). During the first experiment, no significant difference of the percentage of motile thawed sperm was observed among the three saline extenders tested: seawater, calcium-free Hanks' balanced salt solution (Ca-free HBSS) and DCSB4 solution. However, a complementary experiment (2) showed that a significantly higher percentage of motile thawed sperm was recorded using DCSB4 than with Ca-free HBSS as an extender. During the third experiment, sperm motility was higher when using polyethylene glycol (10% PEG) as a cryoprotectant, than when using dimethyl sulphoxide, DMSO (10 and 20%), methanol (10%) and ethylene glycol (10% EG). A higher D-larval rate was obtained during the fourth experiment, using fresh sperm than thawed sperm, and using a 500:1 sperm-to-egg ratio compared with a 50:1 ratio. There is some evidence of inter-individual variations in sperm tolerance to cryopreservation. In conclusion, the highest survival of great scallop sperm after thawing was recorded using the following conditions: DCSB4 extender (1:3 vol/vol sperm to extender dilution), PEG cryoprotectant (10%) and straws maintained at 5.5 cm above liquid nitrogen.

Optimizing sperm cryopreservation in great scallop (Pecten maximus)

“…A basic cryopreservation protocol previously published for great scallop sperm (Suquet et al 2014) led to limited postthawing survival of 25 ± 2% (calculated as a percentage of the percentage of motile sperm from a fresh control), whereas the modifications adopted in the conclusion of the present study improve post-thawing sperm survival to 67 ± 5%. The present improvement was mainly due to the optimisation of CPA concentration and sperm dilution rate in the extender.…”

“…The great scallops were then transferred to the Argenton experimental hatchery (Ifremer) and maintained in a 100-L race-way (17 • C, salinity 34‰) where they were fed daily on a mixture of microalgae (Isochrysis galbana and Chaetoceros calcitrans, 10 9 cells of each species/day/animal) for one month. The sperm collection method and laboratory analysis are only given in brief below as these were previously described in detail in Suquet et al (2014).…”

“…Recently, a basic cryopreservation protocol was defined for great scallop sperm, using a saline solution (DCSB4) as an extender and polyethylene glycol (PEG) as a CPA. However, the survival rate of the thawed sperm remained limited (Suquet et al 2014). The aims of the present paper are to improve post thawing sperm motility by (1) studying the effects of PEG concentration;…”

Sperm cryopreservation of the noble Scallop Chlamys nobilis by a programmable freezing method: Effect of cryoprotectant

“…These studies concerned oyster species (Boulais et al, ; Suquet et al, ; Suquet, Rimond, et al, ), pearl oyster species (Demoy‐Schneider, Levêque, Schmitt, Le Pennec, & Cosson, ; Demoy‐Schneider, Schmitt, Le Pennec, Suquet, & Cosson, ; Zheng et al, ) and so on. However, studies for scallop species were limited to Pecten maximus (Suquet, Arsenault‐Pernet, Ratiskol, & Mingant, ; Suquet, Gourtay, et al, ; Suquet, Malo, et al, ; Suquet, Quéré, et al, ), Argopecten purpuratus (Lamarck, 1819) (Dupré, Covarrubias, Goldstein, Guerrero, & Rojas, ; Espinoza, Valdivia, & Dupre, ), Chlamys farreri (Li, Li & Xue, 2000; Xue, ) and Patinopecten yessoensis (Yang et al, ; Yang, Yang, Liu, & Zhou, , ). Because of the difference in species, spermatozoa cryopreservation methods and related parameters in different species are different.…”

“…These studies concerned oyster species (Boulais et al, 2018;Suquet et al, 2012;Suquet, Rimond, et al, 2013), pearl oyster species (Demoy-Schneider, Levêque, Schmitt, Le Pennec, & Cosson, 2012;Zheng et al, 2018) and so on. However, studies for scallop species were limited to Pecten maximus (Suquet, Arsenault-Pernet, Ratiskol, & Mingant, 2014;Suquet, Quéré, et al, 2013), Argopecten purpuratus (Lamarck, 1819) (Dupré, Covarrubias, Goldstein, Guerrero, & Rojas, 2016;Espinoza, Valdivia, & Dupre, 2010), Chlamys farreri (Li, Li & Xue, 2000;Xue, 1994) and Patinopecten yessoensis (Yang et al, 1999;Yang, Yang, Liu, & Zhou, 2007.…”