Vitrification as an Alternative Approach for Sperm Cryopreservation in Marine Fishes

Abstract

The Southern Flounder Paralichthys lethostigma is a high-value species and a promising aquaculture candidate. Because sperm volume can be limited in this species (<500 μL), new sperm cryopreservation methods need to be evaluated. Vitrification is an alternative to conventional slow-rate freezing, whereby small volumes are cryopreserved at high cooling rates (>1,000°C/min). The goal of this work was to develop a standardized approach for vitrification of Southern Flounder sperm. The specific objectives were to (1) evaluate thawing methods and vitrification solutions, (2) evaluate the postthaw membrane integrity of sperm vitrified in different cryoprotectant solutions, (3) examine the relationship between membrane integrity and motility, and (4) evaluate the ability of vitrified sperm to fertilize eggs. From the vitrification solutions tested, the highest postthaw motility (28 ± 9% [mean ± SD]) and membrane integrity (11 ± 4%) was observed for 20% ethylene glycol plus 20% glycerol. There was no significant difference in postthaw motility of sperm thawed at 21°C or at 37°C. Fertilization from vitrified sperm in one trial yielded the same fertilization rate (50 ± 20%) as the fresh sperm control, while the sperm from the other two males yielded 3%. This is the first report of fertilization by vitrified sperm in a marine fish. Vitrification can be simple, fast, inexpensive, performed in the field, and, at least for small fishes, offers an alternative to conventional cryopreservation. Because of the minute volumes needed for ultrarapid cooling, vitrification is not presently suited as a production method for large fishes. Vitrification can be used to reconstitute lines from valuable culture species and biomedical models, conserve mutants for development of novel lines for ornamental aquaculture, and transport frozen sperm from the field to the repository to expand genetic resources.

A 3D Printed Vitrification Device for Storage in Cryopreservation Vials

“…Due to its lower viscosity than most vitrification solutions, water was used in this study to represent a conservative model for film retention and, also, to facilitate standard comparisons across other studies [ 20 ]. However, cryoprotectants (e.g., dimethyl sulfoxide, ethylene glycol, and glycerol) [ 2 ] with higher viscosities will be used for vitrification in actual applications [ 30 ] and, thus, likely increase the film retention compared with the observations herein.…”

“…This provided ratios of 1–2 × 10 7 sperm/vial, 1 to 2 males/vial (based on the 1 × 10 8 cells/mL cooling concentration), and 300–600 eggs/vial. Upon collection, sperm can be diluted to 1 × 10 9 cells/mL for vitrification [ 2 ], with a diluted volume of 1–4 μL. It is necessary to point out that the sperm concentration for vitrification is higher than those used in equilibrium freezing [ 36 , 37 ].…”

“…This approach is ideal for processing large numbers of samples in batches, but specialized equipment can cost tens of thousands of dollars (e.g., >US$20,000 for a computer-programmed freezer). An alternative and relatively new method for sperm cryopreservation is vitrification, by which the liquid is cooled at >1000 °C/min (‘rapid cooling’) to transform into an amorphous solid (glass) phase without the formation of crystalline ice [ 2 , 3 ]. The rapid cooling can be obtained simply by plunging a thin film (e.g., several μL loaded on loops) or droplets (e.g., on plates or strips) of the sample into liquid nitrogen.…”

A brief review of the methodology and cryoprotectants in selected fish and mammalian species

“…As shown in Figure 1, cryoprotectants can be divided into two permeable and non‐permeable groups. Those in the first group can permeate the cell membrane; these agents are generally low molecular weight (MW: 400 Da), non‐electrolytic and highly soluble in water (Cuevas‐Uribe & Tiersch, 2011). The most common permeable agents used for semen cryopreservation are dimethyl sulfoxide (DMSO), methanol (MET), glycol (Gly), dimethylacetamide (DMA) and ethylene glycol (EG).…”

“…The second group of cryoprotectants consists of non‐permeable agents of high MW, which may be monosaccharide (glucose) or disaccharide (sucrose) sugars, or macromolecules such as lipoproteins or proteins derived from milk, egg yolk, vegetable oils or other sources. These macromolecules have two main functions: they increase the osmolality of the extracellular space, thus causing dehydration of the cells during freezing, and prevent excessive osmotic expansion during thawing (Cuevas‐Uribe & Tiersch, 2011).…”