Abstract
The Southern Flounder (Paralichthys lethostigma) is a commercially valuable fish species with significant aquaculture potential. Given the limited sperm volume typically obtainable from this species (less than 500 μL), there is a pressing need to explore and refine innovative sperm cryopreservation techniques. Vitrification, an alternative to traditional slow-rate freezing, offers a promising solution by cryopreserving minute volumes at exceptionally rapid cooling rates (exceeding 1,000°C/min). This research aimed to establish a standardized vitrification protocol for Southern Flounder sperm. Our specific objectives were to: (1) assess different thawing methods and vitrification solutions, (2) evaluate post-thaw sperm membrane integrity using various cryoprotectant solutions, (3) investigate the correlation between membrane integrity and motility, and (4) determine the fertilization capacity of vitrified sperm. Among the vitrification solutions tested, a combination of 20% ethylene glycol and 20% glycerol yielded the most favorable post-thaw results, exhibiting the highest motility (28 ± 9% [mean ± SD]) and membrane integrity (11 ± 4%). No significant difference in post-thaw sperm motility was observed between thawing at 21°C and 37°C. Fertilization trials using vitrified sperm from one male resulted in a fertilization rate (50 ± 20%) comparable to that of fresh sperm controls. However, sperm from the other two males showed significantly lower fertilization rates (3%). This study marks the first successful instance of fertilization using vitrified sperm in a marine fish species. Vitrification, characterized by its simplicity, speed, low cost, and feasibility in field settings, presents a viable alternative to conventional cryopreservation, particularly for small fish species. While not currently suitable for large-scale production in large fish due to the minute volumes required for ultrarapid cooling, vitrification holds significant potential for reconstituting genetic lines of valuable aquaculture species and biomedical models, conserving mutant strains for ornamental aquaculture development, and facilitating the transport of frozen sperm from field locations to repositories for broader genetic resource management.
Cryopreservation technology has become an indispensable tool in aquatic species management, significantly enhancing hatchery and aquaculture operations. It provides flexibility in managing spawning cycles of females, enhances control over breeding programs, and enables the long-term storage of valuable genetic material (Tiersch et al. 2007; Martinez-Paramo et al., in press). Furthermore, growing concerns about the conservation of native fish populations have spurred the exploration of sperm cryopreservation as a crucial method for preserving genetic diversity and facilitating gene transfer between wild and hatchery populations (Tiersch 2011a). However, cryopreservation efforts for threatened and endangered fish species often face challenges, including limited access to captive broodstock and a scarcity of cryopreservation expertise (Tiersch et al. 2004). Therefore, the development of user-friendly and accessible cryopreservation methods suitable for field application is paramount. Vitrification emerges as a compelling alternative cryopreservation approach, transforming a liquid into a non-crystalline solid state known as “glass.” This amorphous solid retains the random molecular arrangement characteristic of a liquid (Fahy and Wowk 2015). Achieving this glassy state typically necessitates the use of high concentrations of cryoprotectants (40–50%, v:v) and rapid cooling and warming rates (exceeding 1,000°C/min) (Fahy and Rall 2007; Fahy and Wowk 2015).
While conventional cryopreservation remains a well-established method for long-term genetic material storage, vitrification offers an attractive alternative. It has been successfully employed in mammals for the cryopreservation of spermatozoa, embryos, oocytes, stem cells, and even organs (Tucker and Liebermann 2007). Vitrification presents a novel strategy for expanding genetic resource management, safeguarding valuable stocks, reconstituting genetic lines, and enabling the efficient transport of frozen sperm from field locations to centralized repositories. The earliest attempts at fish vitrification date back to 1938, when Basile Luyet experimented with vitrifying juvenile Goldfish (Carassius auratus, 40 mm SL) by immersing them in liquid air (−194°C) (Luyet 1938). Subsequent efforts to vitrify fish embryos have met with limited success (reviewed in Cuevas-Uribe and Tiersch 2011a).
For large-bodied fish aquaculture production, vitrification may offer minimal advantages over conventional cryopreservation due to the small volumes (<30 μL) typically used. However, its simplicity, speed, and suitability for field applications without the need for specialized equipment make it a valuable tool for genetic resource preservation and strain or line reconstitution (Moore and Bonilla 2006; Saragusty and Arav 2011; Magnotti et al., in press). Vitrification is particularly well-suited for three key areas: biomedical research fish models, genetically improved lines, and endangered species. Certain fish species, such as Zebrafish (Danio rerio, <5 μL) (Jing et al. 2009) and Green Swordtail (Xiphophorus hellerii, <9 μL) (Huang et al. 2004), naturally produce small sperm volumes. Even in larger-bodied species, obtaining substantial sperm volumes can be challenging, as exemplified by the endangered Apache Trout (Oncorhynchus apache, <500 μL) (David et al. 2011). Furthermore, selective breeding programs in fish can sometimes lead to undesirable genetic correlations, such as reduced reproductive performance. For instance, Atlantic Salmon (Salmo salar) broodstock selected for rapid growth and late maturation produced limited sperm volumes (<100 μL) (Zohar 1996) compared to their wild counterparts (>10 mL) (Kazakov 1981). Similarly, sex-reversed males of Dusky Grouper (Epinephelus marginatus) exhibit low sperm volumes (<400 μL) (Cabrita et al. 2009). Vitrification perfectly addresses the need to conserve germplasm from these critical fish populations.
Sperm vitrification has already been successfully implemented in freshwater fish species, with offspring produced from vitrified sperm samples of Russian Sturgeon (Acipenser gueldenstaedtii) (Andreev et al. 2009), Channel Catfish (Ictalurus punctatus) (Cuevas-Uribe et al. 2011a), Green Swordtail (Cuevas-Uribe et al. 2011b), Rainbow Trout (O. mykiss) (Figueroa et al. 2013), Atlantic Salmon (Figueroa et al. 2015), and Eurasian Perch (Perca fluviatilis) (Kasa et al., in press). Our recent investigations have evaluated sperm vitrification in three marine species—Spotted Seatrout (Cynoscion nebulosus), Red Drum (Sciaenops ocellatus), and Red Snapper (Lutjanus campechanus)—using sperm motility and membrane integrity as key indicators of gamete quality (Cuevas-Uribe et al. 2015). More recently, sperm motility and head morphometry were assessed following vitrification of European Eel (Anguilla anguilla) sperm (Kasa et al., in press). However, these prior studies did not attempt fertilization or offspring production (Magnotti et al., in press).
The Southern Flounder (Paralichthys lethostigma) is a high-value species and a promising candidate for aquaculture diversification. Given that female Southern Flounder grow two to three times larger than males, all-female culture is highly desirable for commercial aquaculture operations (Morgan et al. 2006). Neomales with an XX genotype have been produced through sex-reversal of gynogenetic offspring. However, the production of these neomales is a complex and labor-intensive process, due to the time required for progeny testing and the low survival rates of gynogens (<2%) (Morgan et al. 2006). Furthermore, males produce very small sperm volumes (<500 μL) (Daniels 2000). Vitrification could provide a valuable tool for safeguarding this investment and reconstituting these valuable sex-reversed male lines, as previously demonstrated for Rainbow Trout neomales (Figueroa et al. 2013).
The primary objective of this research was to develop a standardized vitrification method for Southern Flounder sperm. The specific aims of this study were to: (1) evaluate thawing methods and vitrification solutions, (2) assess post-thaw membrane integrity of sperm vitrified in various cryoprotectant solutions, (3) examine the relationship between membrane integrity and motility, and (4) evaluate the fertilization potential of vitrified sperm. Here, we present the first documented instance of successful fertilization using vitrified sperm in a marine fish species. Vitrification is well-suited for field applications and offers a novel approach for conservation biology.
Southern Flounder
Methods
Sperm Collection
Adult Southern Flounder broodstock, maintained at the North Carolina State University (NCSU) Lake Wheeler Field Laboratory Facilities in Raleigh, North Carolina, were induced to spawn through controlled manipulation of photoperiod and temperature (Daniels and Watanabe 2002; Watanabe et al. 2006) during April 2009 and March–April and August 2010. The fish were cultured in an artificial seawater system (33 g/L) (Crystal Sea Marinemix, Marine Enterprises International, Baltimore, Maryland) with a 9 h light : 15 h dark photoperiod at 16°C and were fed BioBrood pellets (Bio-Oregon, Longview, Washington) every other day to satiation.
The fish used in this study were 3-year-old, F3, sex-reversed males (XX neomales), with an average weight of 0.41 ± 0.23 kg (mean ± SD). Males were anesthetized using tricaine methanesulfonate (40 mg/L) (MS-222; Argent Chemical Laboratories, Redmond, Washington) and assessed for spermiation by applying gentle pressure to the gonadal area. Spermiating males were carefully dried with towels, and sperm was aspirated using 1-mL pipette tips by applying slight abdominal pressure. Precautions were taken to prevent contamination of sperm samples with urine, feces, or water. Feed was withheld for 2 days prior to sperm collection to minimize fecal release during handling. Sperm samples, along with a seawater sample from the culture system, were secured in ZipLoc plastic bags and shipped overnight to the Aquaculture Research Station (ARS) of the Louisiana State University Agricultural Center in a styrofoam box containing two foam refrigerant blocks frozen to −20°C. A cardboard divider was placed above the refrigerant blocks to prevent direct contact with the samples (Tiersch 2011b). Consequently, all vitrification experiments were conducted on samples 24–48 h post-collection.
Motility Assessment and Sperm Preparation
Upon arrival at the ARS, sperm samples were gently inverted to ensure mixing, and sperm motility was assessed using darkfield microscopy (Optiphot-2, Nikon, Garden City, New York) at 200× magnification. A 1-μL sperm suspension was placed on a glass slide and activated by mixing with 20 μL of artificial seawater (995 mOsmol/kg). Motility was estimated by observing three to five different fields within 20 seconds of activation and expressed as the percentage of sperm exhibiting progressive forward movement. Sperm cells exhibiting only vibratory motion were not considered motile.
Sperm concentration was estimated spectrophotometrically by measuring the absorbance of 2-μL aliquots at 601 nm using a microspectrophotometer (Nanodrop 1000, Thermo Scientific, Wilmington, Delaware). Absorbance values were used in the following equation, derived from a standard curve relating absorbance readings to sperm concentration determined by hemocytometer counts (r2 = 0.987) (Cuevas-Uribe and Tiersch 2011b):
Sperm concentration (cells/mL) = absorbance × 9.77 × 108 − 7.68 × 107
Samples were diluted to a concentration of 2 × 109 cells/mL using sperm motility-inhibiting saline solution (SMIS) (Lahnsteiner 2000). SMIS composition was: 600 mg NaCl, 315 mg KCl, 15 mg CaCl2·2 H2O, 20 mg MgSO4·7H2O, and 470 mg HEPES in 100 mL ultrapure water (pH 7.8), with 1.5 g bovine serum albumin and 0.5 g sucrose, resulting in an osmolality of 324 mOsmol/kg. Osmolality was measured using a vapor pressure osmometer (Model 5520, Wescor, Logan, Utah). Diluted samples were kept on ice until use in vitrification experiments.
Sperm Vitrification Procedure
As described above, sperm were diluted to 2 × 109 cells/mL in SMIS. Cryoprotectant solutions were prepared at double strength in SMIS. For vitrification, sperm samples were mixed with double-strength cryoprotectants at a 1:1 (v/v) ratio. Within 15 seconds of cryoprotectant addition, samples were immediately loaded into 10-μL polystyrene loops (Nunc, Roskilde, Denmark) without equilibration and rapidly immersed individually in liquid nitrogen within approximately 1 minute (∼50 s). Loops were stored in goblets (three per goblet) in liquid nitrogen. After a minimum of 12 hours of liquid nitrogen storage, vitrified loop samples were thawed directly onto a microscope slide containing a 30-μL droplet of seawater (∼1,000 mOsmol/kg) at room temperature (24°C) or other specified temperatures. Thawed sperm motility was assessed within 30 seconds.
Experiment 1: Effect of Thawing Temperatures
The cryoprotectants evaluated were dimethyl sulfoxide (DMSO; OmniSolv, France), ethylene glycol (EG; Mallinckrodt Baker, Paris, Kentucky), 1,2-propanediol (PROH; Sigma-Aldrich, St. Louis, Missouri), and glycerol (Gly; Mallinckrodt Baker). Six vitrification solutions were tested: (1) 20% DMSO + 20% EG, (2) 20% DMSO + 20% PROH, (3) 20% DMSO + 20% Gly, (4) 20% EG + 20% PROH, (5) 20% EG + 20% Gly, and (6) 20% PROH + 20% Gly. Double-strength cryoprotectant solutions were prepared in SMIS and diluted at 4°C with sperm suspension at a 1:1 ratio (final sperm concentration, 1 × 109 cells/mL). Samples were immediately loaded (within 15 seconds) into 10-μL polystyrene loops without equilibration and submerged in liquid nitrogen within 1 minute of vitrification solution addition.
Glass formation was visually assessed by examining the appearance of the vitrified sample. A milky appearance indicated ice crystal formation (Ali and Shelton 1993). Loops were thawed directly onto microscope slides containing 30-μL seawater droplets at two temperatures (21°C and 37°C). Motility was assessed immediately after thawing. Sperm from three males were used, with one replicate per male for each treatment.
Experiment 2: Evaluation of Vitrification Solutions
Sperm samples from three males were used to evaluate three vitrification solutions: (1) 20% EG + 20% Gly, (2) 10% DMSO + 30% EG + 0.25 M trehalose dehydrate (Tre; Acros Organics, Fair Lawn, New Jersey), and (3) 15% DMSO + 15% EG + 10% Gly + 1% X-1000 (21st Century Medicine, Fontana, California) + 1% Z-1000 (DEGXZ) (21st Century Medicine). The general vitrification procedure was followed. Loops were thawed directly onto microscope slides containing 30 μL of seawater at room temperature (24°C). Motility was assessed immediately after thawing. All trials were replicated at least twice for each male.
Experiment 3: Effect of Vitrification Solutions on Membrane Integrity
Sperm samples from three males were vitrified using three solutions: (1) 20% DMSO + 20% EG, (2) 20% DMSO + 20% Gly, and (3) 20% EG + 20% Gly. Samples were stored in liquid nitrogen for 13 days before flow cytometry analysis. To thaw, each loop was warmed in 495 μL of SMIS at room temperature (24°C) to achieve a sperm concentration of approximately 5 × 106 cells/mL. Plasma membrane integrity was assessed using the SYBR 14–propidium iodide (PI) staining method (live–dead sperm viability kit, Molecular Probes, Eugene, Oregon) (Daly and Tiersch 2011). Fresh and thawed sperm were filtered through 35-μm nylon mesh, and duplicate 250-μL aliquots were stained with 100 nM SYBR-14 and 12 μM PI. Samples were incubated in the dark for 10 minutes at room temperature before analysis. Flow cytometry was performed using a C6 Accuri Cytometer (Ann Arbor, Michigan) with a 488-nm, 50-mW solid-state laser. Instrument performance was validated using fluorescent validation beads (Spherotech, Accuri Cytometers) to ensure CV (100·SD/mean) values were <3.0% for fluorescence detectors (FL1, FL2, FL3, and FL4). Each microcentrifuge tube was gently flicked three times before analysis to ensure cell suspension. 10 μL of sample were analyzed at a flow rate of 35 μL/min using Cflow software (version 1.0.202.1, Accuri Cytometers). Green fluorescence (SYBR 14) was detected with a 530 ± 15-nm bandpass filter (FL1), and red fluorescence (PI) was detected with a >670-nm longpass filter (FL3). Events were visualized on forward-scatter (FSC) versus side-scatter (SSC) plots. A gate was drawn around the sperm population to exclude non-sperm events. Gated events were viewed on FL1 versus FL3 scatter plots. Fluorescence compensation, based on median fluorescence values, was applied to reduce spectral overlap. Sperm stained with SYBR 14 alone were considered membrane-intact, while those stained with both SYBR 14 and PI or PI alone were considered membrane-compromised.
Experiment 4: Fertilization Trials
Females (body weight, 1.09 ± 0.28 kg [mean ± SD]) were injected intramuscularly with 0.5 mL/kg Ovaprim (10 μg/kg salmon gonadotropin releasing hormone analog + 10 μg/kg Domperidone; Syndel International, Vancouver, British Columbia) at NCSU Lake Wheeler Field Laboratory Facilities. Hormones were administered in two injections: 10% of the total dosage initially, followed by the remaining 90% after 24 hours. Eggs were collected approximately 48 hours after the second injection. 0.1 mL egg aliquots (129 ± 35 eggs) were placed in 60-mL plastic cups and held (<1 hour) for fertilization trials.
Based on Experiment 1 results, sperm from three males were vitrified at a final concentration of 5 × 108 cells/mL using 20% EG + 20% Gly in 10-μL polystyrene loops at the ARS and shipped overnight to NCSU for fertilization trials in April 2009. In addition to the vitrification solution, sperm from the same three males were vitrified without cryoprotectants (cryoprotectant-free vitrification) at a final concentration of 1 × 109 cells/mL. For artificial fertilization, three loops of vitrified samples from each male were thawed in 15-mL conical tubes (Corning Inc., Corning, New York) containing 5 mL seawater at ∼20°C. Loops were gently agitated for <10 s, and suspensions were mixed with egg aliquots. Estimated sperm-to-egg ratio was 1 × 105 sperm per egg. Control egg aliquots were mixed with 30 μL of pooled fresh sperm from at least two males, collected on the same day and refrigerated until use. Eggs from three females were used in 2009 fertilization trials. Eggs were incubated at ∼20°C. Fertilization rate was assessed by examining embryo development to the 64–128 cell division stage (3–5 hours post-fertilization) using a dissecting microscope.
In March 2011, sperm from three males were vitrified at a final concentration of 1 × 109 cells/mL using 20% EG + 20% Gly in 10-μL polystyrene loops at the ARS and shipped overnight to NCSU for fertilization trials in February–March 2011. Eggs from eight females were used in 2011 trials. Estimated sperm-to-egg ratio was 3 × 105 thawed sperm per egg.
Statistical Analysis
Data were analyzed using SAS software (Statistical Analysis System, version 9.1; SAS institute, Cary, North Carolina). Mixed ANOVA procedures were used for all interactions. For the thawing experiment, fixed treatments were temperature and vitrification solution, with post-thaw motility as the dependent variable. Membrane integrity data were analyzed using mixed ANOVA with cryoprotectants as fixed treatments and membrane intact as the dependent variable. Percentage data were arcsine-square-root transformed for normalization before analysis. Post hoc Tukey’s test was used to identify significant differences, with significance set at P < 0.05.
Results
Experiment 1: Thawing Temperatures
Fresh sperm motility prior to vitrification was 50 ± 10% (mean ± SD). No significant differences (P = 0.697) in post-thaw motility were observed between sperm thawed at 21°C or 37°C across all treatments (Table 1). The highest post-thaw motility (35%) was achieved with 20% EG + 20% Gly and 20% DMSO + 20% Gly, which were not significantly different (P = 0.606). Motility in the 20% PROH + 20% Gly solution (14 ± 10%) was not significantly different from 20% DMSO + 20% Gly (21 ± 9%; P = 0.059), but was significantly different from 20% EG + 20% Gly (22 ± 7%; P = 0.018). Motilities in 20% DMSO + 20% EG (2 ± 2%), 20% DMSO + 20% PROH (2 ± 1%), and 20% EG + 20% PROH (3 ± 3%) were not significantly different (P > 0.446). Complete glass formation was observed in all solutions except 20% DMSO + 20% Gly, which exhibited approximately 80–90% glass formation (<20% milky appearance).
Table 1. Percent sperm motility (mean ± SD) of Southern Flounder after thawing at different temperatures and the osmolality for different vitrification solutions. DMSO: dimethyl sulfoxide; EG: ethylene glycol; PROH: propanediol; Gly: glycerol; Tre: trehalose; X: X-1000; Z: Z-1000. Mean values with different letters within a row are significantly different (P < 0.05).
| Vitrification solution | Thawing temperature | Osmolality (mOsmol/kg) |
|---|---|---|
| 21°C | 24°C | |
| 20% DMSO + 20% EG | 2 ± 2 z | – |
| 20% DMSO + 20% PROH | 2 ± 2 z | – |
| 20% DMSO + 20% Gly | 20 ± 12 z | – |
| 20% EG + 20% PROH | 3 ± 4 z | – |
| 20% PROH + 20% Gly | 15 ± 12 z | – |
| 20% EG + 20% Gly | 19 ± 5 z | 28 ± 9 y |
| 10% DMSO + 30% EG + 0.25 M Tre | 7 ± 3 | – |
| 15% DMSO + 15% EG + 10%Gly + 1% X + 1% Z | 14 ± 10 | – |
Experiment 2: Vitrification Solutions
Fresh sperm motility before vitrification was 60 ± 10%. The highest post-thaw motility (40%) was observed with 20% EG + 20% Gly, followed by 30% with 15% DMSO + 15% EG + 10% Gly + 1% X-1000 + 1% Z-1000 (DEGXZ). These two treatments showed significantly different motility (P = 0.039) (Table 1). Motility in 10% DMSO + 30% EG + 0.25 M Tre (7 ± 3%) was not significantly different from DEGXZ (14 ± 10%; P = 0.114) but was significantly lower than 20% EG + 20% Gly (28 ± 9%; P = 0.008).
Experiment 3: Membrane Integrity
Fresh sperm motility (57 ± 9%) showed a positive correlation (r = 0.80) with membrane-intact cells (89 ± 1%), although a significant difference was observed between them (P < 0.001) (Table 2). No correlation between thawed sperm motility and membrane integrity was found across all treatments (Table 2). The highest percentage of membrane-intact sperm post-vitrification (17%) was achieved with 20% EG + 20% Gly, but this was not significantly different in motility (P = 0.252). The 20% EG + 20% Gly solution was not significantly different in motility (P = 0.076) from 20% DMSO + 20% Gly, but showed significantly higher membrane integrity (P = 0.037). The 20% DMSO + 20% EG solution had significantly lower membrane integrity compared to both 20% EG + 20% Gly (P = 0.004) and 20% DMSO + 20% Gly (P = 0.045).
Table 2. Post-thaw motility (percent [mean ± SD]), membrane-intact cells (percent intact [mean ± SD]), and their correlation of Southern Flounder sperm vitrified with different vitrification solutions. DMSO: dimethyl sulfoxide; EG: ethylene glycol; Gly: glycerol. Motility and membrane integrity by fresh sperm are included for comparison. Mean values with different letters within rows are significantly different (P < 0.05).
| Treatment | % Motility | % Intact | Correlation coefficient (r) |
|---|---|---|---|
| Fresh | 57 ± 9 z | 89 ± 1 y | 0.80 |
| 20% DMSO + 20% EG | 0 ± 1 z | 2 ± 1 y | -0.26 |
| 20% DMSO + 20% Gly | 7 ± 6 z | 6 ± 4 z | -0.33 |
| 20% EG + 20% Gly | 13 ± 6 z | 11 ± 4 z | 0.18 |
Experiment 4: Fertilization Trials
In 2009, only one of three females yielded viable eggs (>20% fertilization in control). Vitrified sperm from one male resulted in fertilization rates comparable to fresh sperm controls (Table 3), while sperm from the other two males yielded low fertilization (<5%). Cryoprotectant-free vitrification did not result in fertilization.
Table 3. Percent egg fertilization (mean ± SD) achieved by Southern Flounder sperm vitrified with 20% ethylene glycol (EG) + 20% glycerol (Gly) in 10-μL polystyrene loops. Fertilization capability was assessed at the 64–128 cell division stage. The same males were used for females 1, 2, and 3 in 2011. Fertilization rates by fresh sperm are included for comparison of egg quality.
| Female | Year | Male | Average | Control |
|---|---|---|---|---|
| 1 | 2 | 3 | ||
| 1a | 2009 | 50 ± 20 | 3 ± 2 | 3 ± 1 |
| 1b | 2011 | 12 | 23 | 6 |
| 2b | 2011 | 8 | 7 | 8 |
| 3b | 2011 | 13 | 13 ± 0 | – |
| 4c | 2011 | 1 ± 0 | 0 ± 0 | 9 ± 2 |
a Mean ± SD of three replicates of egg batches.
b No replicates for individual males.
c Mean ± SD of two replicates of egg batches.
In 2011, data from four of eight females with control fertilization rates >9% were used. The same males were used for females 1, 2, and 3 (Table 3). Due to logistical issues, fertilization trials were not performed for males 1 and 3 with female 3. Male-to-male variation and variation within egg batches were observed in fertilization trials. For example, male 2 yielded 23% fertilization with female 1, while male 3 yielded 6% with the same female. For female 2, fertilization with vitrified sperm achieved rates comparable to fresh sperm controls. Fertilization rates for female 4 were low (<10% vs. 20% for control) (Table 3).
Discussion
Vitrification presents a promising alternative to conventional cryopreservation, offering a novel approach for genetic material conservation (Tucker and Liebermann 2007). Recent studies have explored vitrification for sperm cryopreservation in marine fish (Cuevas-Uribe et al. 2015; Kasa et al., in press). Vitrified sperm from Spotted Seatrout, Red Snapper, and Red Drum exhibited high motility (up to 60%) and membrane integrity (up to 23%). Another study on European Eel sperm vitrified using Cryotop devices (Kitazato BioPharma, Shizuoka, Japan) reported 5% motility (Kasa et al., in press). However, these studies did not assess fertilization or offspring production. Fertilization capacity is a critical assessment criterion for cryopreserved sperm quality. This study aimed to evaluate the fertilization ability of vitrified Southern Flounder sperm.
The primary challenge in vitrification lies in formulating suitable vitrification solutions and developing equilibration and dilution protocols that minimize osmotic and toxic damage (Rall 1991). In this study, we utilized vitrification solutions adapted from previous research on marine fish (Cuevas-Uribe et al. 2015) and Green Swordtail (Cuevas-Uribe et al. 2011b). Cryoprotectants were selected based on our prior high-throughput sperm cryopreservation study in Southern Flounder (Hu et al. 2016) and other studies on paralichthid flounders. For instance, DMSO and Gly were used for conventional cryopreservation of 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) sperm, while PROH and EG were used for Summer Flounder sperm cryopreservation (Liu et al. 2015). These prior studies used relatively low cryoprotectant concentrations (<20%). The high cryoprotectant concentrations required for vitrification (>40%) approach cellular toxicity limits (Mazur et al. 2008). To mitigate individual cryoprotectant toxicity, we used mixtures to combine beneficial properties such as permeability and glass formation (Weiss et al. 2010).
In Experiment 1, we tested eight vitrification solutions using four cryoprotectants. Solutions containing Gly yielded the highest post-thaw motilities. Osmolality of vitrification solutions did not correlate with post-thaw motility, contrasting with mammalian studies suggesting lower molarity to reduce toxicity (Ali and Shelton 2007). The combination of Gly and EG resulted in the highest post-thaw motility. A previous Summer Flounder study using 20% EG for conventional cryopreservation reported 70% post-thaw motility (Liu et al. 2015). A Red Snapper vitrification study using 20% EG + 20% Gly reported 23% average post-thaw motility (Cuevas-Uribe et al. 2015), similar to our 26% result. In Green Swordtail vitrification using the same solutions, post-thaw motility was approximately 10% (Cuevas-Uribe et al. 2011b).
Thawing temperatures (21°C and 37°C) did not significantly affect motility, consistent with Green Swordtail studies showing no motility difference between 24°C and 37°C thawing (Cuevas-Uribe et al. 2011b). The small vitrification solution volumes ensured sufficient warming rates at both temperatures to prevent ice crystal formation (devitrification). Katkov et al. (2003) estimated warming rates for similar loops at 37°C to be as high as 200,000°C/min. This aligns with Mazur and Seki (2011), who found rapid warming rates (118,000°C/min) more critical than cooling rates (95–69,250°C/min) for vitrified mammalian oocyte and embryo survival.
In Experiment 2, we evaluated two vitrification solutions previously used for marine fish with high motility (>20%) (Red Snapper, Spotted Seatrout, Red Drum) (Cuevas-Uribe et al. 2015). Trehalose or proprietary polymers X-1000 and Z-1000 were added to enhance glass formation. Trehalose can protect marine fish during conventional cryopreservation, as seen in Orange-spotted Grouper (Epinephelus coioides) (Peatpisut and Bart 2010) and Longtooth Grouper (E. bruneus) (Miyaki et al. 2005). However, trehalose addition in our study did not improve Southern Flounder sperm survival after vitrification, similar to low post-thaw motility (~4%) observed in Green Swordtail using the same solution (Cuevas-Uribe et al. 2011b).
We also tested commercial polymers X-1000 and Z-1000 (DEGXZ). These polymers can reduce cryoprotectant concentrations needed for vitrification and act as “ice blockers” (Wowk and Fahy 2002). DEGXZ has yielded high post-thaw motility in marine fish (Cuevas-Uribe et al. 2015). However, our results showed only ~14% post-thaw motility with DEGXZ in Southern Flounder.
The highest post-thaw motilities in Experiments 1 and 2 were achieved with 20% EG + 20% Gly. These cryoprotectants have low toxicity (Shaw and Jones 2003, although Gly is a less effective glass former than EG, and EG permeates cells faster (Shaw and Jones 2003). The combination of Gly and EG is commonly used for vitrification (Ali and Shelton 2007). This mixture of a less effective glass former and a fast-permeating cryoprotectant proved advantageous for Southern Flounder, yielding 20–30% post-thaw motility.
Sperm motility alone is not always a reliable fertilization predictor (Kopeika and Kopeika 2008). High cryoprotectant concentrations (>40%) and osmotic pressures (>4,500 mOsmol/kg) in vitrification solutions can damage sperm through chemical toxicity or osmotic effects, affecting plasma membrane integrity. Compromised plasma membrane integrity can reduce sperm viability and fertilization capacity (Silva and Gadella 2006).
Membrane-intact cells were defined as “viable sperm.” In Channel Catfish sperm vitrification, membrane-intact cell percentage increased with improved glass formation (Cuevas-Uribe et al. 2011a). In Green Swordtail sperm vitrification, membrane integrity was evaluated before and after vitrification. Before vitrification, ~70% cells were membrane-intact, dropping to ~10% post-vitrification (Cuevas-Uribe et al. 2011b). In our study, the highest membrane-intact cell percentage was with 20% EG + 20% Gly, corresponding to the highest post-thaw motility treatment, which did not contain DMSO.
Post-thaw sperm motilities in the membrane integrity experiment (Experiment 3) were lower than in previous experiments, possibly due to male-to-male variation or incomplete recrudescence. Sperm for Experiment 2 were collected in March, while those for Experiment 3 were collected five months later. Although males were held in controlled recirculating systems, at least 5 months are needed for recrudescence to restore energy and storage depots like lipids (Watanabe et al. 2006).
The 20% EG + 20% Gly solution, exhibiting the highest post-thaw motility and membrane integrity, was used for fertilization trials. Fertilization was assessed by examining incubated eggs for early embryonic cleavage (64–128 cell stage, 3–6 hours post-fertilization) (Daniels 2000), a criterion used in previous Southern Flounder reproductive studies (Berlinsky et al. 1996; Hu et al. 2016). Fertilization percentages varied among females and males. Vitrified sperm fertilization rates reached up to 70% (fresh sperm control, 50%). Average fertilization with vitrified sperm ranged from 10% to 20%. Low fertilization rates could be due to loop-to-loop variability or egg batch differences. Berlinsky et al. (1996) noted significant fertilization rate variation between females and spawns in Southern Flounder, a serial spawner with multiple egg batches during the spawning season (Watanabe and Daniels 2010). Each batch may contain oocytes at different stages (Berlinsky et al. 1996), and not all buoyant eggs are fertilized (Daniels et al. 2010). Batch variance also occurs, with initial spawns having low fertility (<10%), improving (>50%) within a week and remaining high for a month before declining (Daniels and Watanabe 2002).
Sperm vitrified without cryoprotectants (cryoprotectant-free vitrification) did not result in fertilization, contrasting with Rainbow Trout studies reporting ~80% motility post-cryoprotectant-free vitrification (Merino et al. 2011, although fertilization was not assessed, and motility assessment details were limited. Another cryoprotectant-free vitrification attempt in Red Drum showed no motility (Cuevas-Uribe et al. 2015). Motility estimation should only include actively forward-swimming sperm (Tiersch 2011c). The Rainbow Trout study reported ~50% mitochondrial membrane potentials with bovine serum albumin. Our study used SMIS extender containing bovine serum albumin, and no motility was observed. Cryoprotectant-free vitrification has been successful in mammals, with human cryoprotectant-free vitrification achieving fertilization rates comparable to slow cooling (Isachenko et al. 2004). In fish, cryoprotectant-free vitrification has shown limited success in Channel Catfish (<2% fertilization in 2 of 16 trials) (Cuevas-Uribe et al. 2011a) and Persian Sturgeon (Acipenser persicus) (6% motility) (Abed-Elmdoust et al. 2015). Further research is needed to evaluate cryoprotectant-free sperm vitrification fertilization capacity in other fish species.
With increasing threats to marine fish from anthropogenic disturbances and climate change, urgent conservation action is critical. Cryopreservation can contribute to conservation programs, but conventional techniques require specialized equipment unsuitable for field use. New field-deployable cryopreservation methods are urgently needed. This study presents a field-applicable vitrification technique achieving acceptable fertilization rates (10–20%), comparable to conventional cryopreservation (20–30%) (Hu et al. 2016). In urgent conservation situations, germplasm storage attempts are warranted even with low success expectations (Holt et al. 2003). Sperm vitrification for genetic diversity protection offers novel approaches for conservation biology, biomedical models, wild fisheries, and aquaculture species.
Acknowledgments
This work was supported by the National Institutes of Health, Office of Research Infrastructure Programs (R24-RR023998 and R24-OD011120), with additional support from the U.S. Department of Agriculture, National Institute of Food and Agriculture (Hatch project LAB94231). We thank P. Turner and R. Sanderson for early project assistance, N. Novelo for manuscript review, and J. Daly for technical assistance. This report was approved for publication by the Director of the Louisiana Agricultural Experiment Station as number 2016-241-30653.
Contributor Information
Rafael Cuevas-Uribe, Department of Fisheries Biology, Humboldt State University, One Harpst Street, Arcata, California 95521, USA.
E. Hu, Center for Aquaculture Technologies, Inc., 8395 Camino Santa Fe, Suite E, San Diego, California 92121, USA
Harry Daniels, Department of Applied Ecology, North Carolina State University, Raleigh, North Carolina 27695, USA.
Adriane O. Gill, Department of Applied Ecology, North Carolina State University, Raleigh, North Carolina 27695, USA
Terrence R. Tiersch, Aquatic Germplasm and Genetic Resources Center, School of Renewable Natural Resources, Louisiana State University Agricultural Center, Baton Rouge, Louisiana 70820, USA
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