Development of a novel non-programmable cryopreservation method capable of accurate cooling rate manipulation

Arita, Kahori; Takamatsu, Shinya; Isowa, Kiyoshi; Aoki, Hideo; Ohta, Hiromi

doi:10.1016/j.aquaculture.2017.11.008

Cited by 3 publications

(1 citation statement)

References 15 publications

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“…In addition, when the height was fixed at 15 cm, the reached temperatures of −30°C or −70°C showed lower post‐thaw motility compared than those between −40°C and −60°C. The cooling rate of the sample and the temperature just before immersing in the LN regulate the dehydration of intracellular water, and greatly affect the survival of the cell during cryopreservation (Arita, Takamatsu, Isowa, Aoki, & Ohta, ; Denniston, Michelet, & Godke, ). If the temperature is fixed, the fast cooling rate will decrease dehydration of the cell and the slow cooling rate will increase it.…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2018

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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.

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Section: Discussionmentioning

confidence: 99%

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

et al. 2018

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Non-programmable cryopreservation of sperm from industrial-farmed Murray cod (Maccullochella p peelii)

et al. 2021

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Optimization of Sperm Cryopreservation Formulation in Portunus trituberculatus

Chang

Zhu

et al. 2023

IJMS

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Portunus trituberculatus is a very important marine economic species, and its aquaculture industry has been developing rapidly. However, the phenomenon of marine wild capture of P. trituberculatus and germplasm degradation has become increasingly serious. It is necessary to develop the artificial farming industry and carry out germplasm resource protection, for which sperm cryopreservation technology is an effective method. This research compared three methods (mesh-rubbing, trypsin digestion, and mechanical grinding) for acquiring free sperm, and the best method was mesh-rubbing. Then, the optimal cryopreservation conditions were selected, and the optimal formulation was sterile calcium-free artificial seawater, the optimal cryoprotectant was 20% glycerol, and the best equilibrium time was 15 min at 4 °C. The optimal cooling program was suspending the straws at 3.5 cm on the liquid nitrogen surface for 5 min and then storing them in liquid nitrogen. Finally, the sperm were thawed at 42 °C. However, the expression of sperm-related genes and the total enzymatic activities of frozen sperm were significantly decreased (p < 0.05), which showed that sperm cryopreservation damaged the sperm. Our study improves the sperm cryopreservation technology and the yield of aquaculture in P. trituberculatus. Additionally, the study provides a certain technical basis for the establishment of a sperm cryopreservation library of crustaceans.

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Development of a novel non-programmable cryopreservation method capable of accurate cooling rate manipulation

Cited by 3 publications

References 15 publications

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

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

Non-programmable cryopreservation of sperm from industrial-farmed Murray cod (Maccullochella p peelii)

Optimization of Sperm Cryopreservation Formulation in Portunus trituberculatus

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