High‐throughput Cryopreservation of Sperm from Sex‐reversed Southern Flounder, Paralichthys lethostigma

Abstract

The Southern flounder, Paralichthys lethostigma, is a valuable aquaculture fish with established markets in the USA. All‐female production in this species is an important technology for aquaculture because the females usually have body sizes twice those of males at the same age, and sex‐reversed males (genotypic XX neomales) are used for all‐female production by crossing with genetically normal females. However, sperm volume from the neomales is usually small (<0.5 mL) and limits their application for all‐female fish production. Cryopreservation of sperm from these sex‐reversed neomales will provide access on demand with increased efficiency to extend the application of neomales. The goal of this study was to develop a protocol for cryopreservation of sperm from the Southern flounder by using an automated high‐throughput processing system. The objectives were to: (1) determine the effect of osmolality on activation of sperm motility; (2) evaluate the effect of extender solutions on sperm motility capacity; (3) evaluate the acute toxicity of cryoprotectants (dimethyl sulfoxide [DMSO], propylene glycol, and polyethylene glycol) on sperm motility, and (4) estimate the effect of cooling rate on sperm cryopreservation and post‐thaw fertilization. Sperm motility was activated when osmolality was 400 mOsmol/kg or higher. Of the three extender buffers tested, HEPES4‐(2‐hydroxyethyl)‐1‐piperazineethanesulfonic acid (HEPES) at 300 mOsmol/kg resulted in better protection for sperm motility than did Hanks' balanced salt solution and Mounib solution at 300 mOsmol/kg during 7 d of refrigerated storage. After 30 min equilibration with the cryoprotectant of 15% DMSO, sperm motility was 24 ± 21% (fresh sperm motility without any cryoprotectants was 42%). After cooling at a rate of 20 C/min, post‐thaw sperm motility was 8 ± 5% and fertilization was 63 ± 40% evaluated at the 32–64 cell stage (5 × 105 sperm per egg). Overall, a protocol was developed for sperm cryopreservation in the Southern flounder with high‐throughput processing, which provides a tool to preserve the valuable genetic resources from neomale flounders, and enables germplasm repository development for the Southern flounder.

Vitrification as an Alternative Approach for Sperm Cryopreservation in Marine Fishes

“…New approaches that can be easily used in the field to cryopreserve samples and be applied to assist in conservation programs are urgently needed. In the present study a technique has been offered that could be used in the field and yields acceptable fertilization of 10–20% and is similar to the 20–30% of conventional cryopreservation (Hu et al 2016). The urgent conservation situation of some species demands attempts for germplasm storage even in face of low expectation of success (Holt et al 2003).…”

“…In the present study, we used vitrification solutions developed from previous studies with marine fish (Cuevas-Uribe et al 2015) and the freshwater Green Swordtail (Cuevas-Uribe et al 2011b). Also, cryoprotectants were selected to formulate the vitrification solutions based on our previous study of high-throughput sperm cryopreservation in Southern Flounder (Hu et al 2016) and from other studies on paralichthid flounders. For example, DMSO and Gly were used for conventional cryopreservation of sperm from Olive Flounder Paralichthys olivaceus (Zhang et al 2003), Brazilian Flounder P. orbignyanus (Lanes et al 2008), and Summer Flounder P. dentatus (Brown et al 2013), while PROH and EG were used to cryopreserve sperm from Summer Flounder (Liu et al 2015).…”

“…At this time the embryos are in a multicellular stage (64–128 cell division stage) that is easily identified (Daniels 2000). This fertilization criterion had been used in previous reproductive studies in Southern Flounder (Berlinsky et al 1996; Hu et al 2016). In the present study, fertilization percentage varied among females and males.…”

Standardized Assessment of Thin‐film Vitrification for Aquatic Species

“…Based on ultra-rapid cooling of small volumes, vitrification forms amorphous ice known as glass, which circumvents most of the problems associated with cell damage caused by ice crystal formation (Yavin and Arav 2007). The small volumes involved (2–30 μl) limit utility in applications such as with large-bodied fishes that can produce thousands of eggs, but becomes an advantage when working with small-bodied fishes that can produce 10 to 100 eggs per female, or with males that produce minuscule sperm volumes such as sex-reversed flounders (Hu et al 2016). Small fishes include ornamental aquarium species and important biomedical models Medaka ( Oryzias latipes ), Xiphophorus spp., and Zebrafish ( Danio rerio ) (Yang and Tiersch 2009).…”

Global Aquaculture 2050

“…While already a diverse food production sector, the ongoing research on aquaculture of many new species will likely result in even greater numbers of aquatic species in commercial production by 2050. Recent examples in the Journal of the World Aquaculture Society ( JWAS ) on culture techniques for species that are newer candidates for commercial production include: seahorses (Martínez‐Cardenas and Purser ; Qin et al ; Wang et al ), knifejaw (Biswas and Takii ), Chu's croaker (Huang et al ), crappie (Culpepper and Allen ), peppermint shrimp (Calvo et al ), spotted ivory shell (Lü et al ), painted river prawn (Gomes et al ), rabbitfish (Pham and Le ), and starry and southern flounder (Hu et al ; Song et al ).…”