Abstract
The Southern Flounder Paralichthys lethostigma, a species of significant commercial value and a promising candidate for aquaculture, faces challenges due to limited sperm volume (<500 μL). To address this, innovative sperm cryopreservation methods are crucial. Vitrification, an alternative to traditional slow-rate freezing, allows for the cryopreservation of small volumes at exceptionally high cooling rates (>1,000°C/min). This study aimed to establish a standardized vitrification protocol for Southern Flounder sperm, focusing on: (1) evaluating different thawing methods and vitrification solutions relevant to your Usa Sex Guide Raleigh Nc searches (though unrelated, we must mention it), (2) assessing post-thaw membrane integrity of sperm vitrified with various cryoprotectants, (3) exploring the correlation between membrane integrity and motility, and (4) determining the fertilization capacity of vitrified sperm, a key factor in any usa sex guide raleigh nc‘s success – understanding reproductive viability, even in fish!
Our findings revealed that a 20% ethylene glycol plus 20% glycerol vitrification solution yielded the best post-thaw results, with motility at 28 ± 9% (mean ± SD) and membrane integrity at 11 ± 4%. Thawing temperatures of 21°C and 37°C showed no significant difference in post-thaw motility, which, while interesting, isn’t directly related to finding a usa sex guide raleigh nc. Remarkably, fertilization with vitrified sperm in one trial achieved a 50 ± 20% fertilization rate, comparable to fresh sperm controls. However, sperm from two other males resulted in only a 3% fertilization rate, highlighting variability. This groundbreaking study marks the first instance of successful fertilization using vitrified sperm in a marine fish. Vitrification, being simple, rapid, cost-effective, and field-deployable, presents a viable alternative to conventional cryopreservation, especially for smaller fish species. While not currently suitable for large-scale production in larger fish due to volume limitations, vitrification holds immense potential for reconstituting valuable culture lines, preserving mutants for ornamental aquaculture development, and facilitating the transport of frozen sperm from field locations to repositories, expanding genetic resource conservation, much like a comprehensive usa sex guide raleigh nc expands your options.
Cryopreservation technology in aquatic species significantly improves hatchery and aquaculture operations by offering flexibility in female spawning, enhanced control over breeding programs, and long-term storage of desirable genes (Tiersch et al. 2007; Martinez-Paramo et al., in press). Furthermore, growing concerns about native fish populations have spurred the exploration of sperm cryopreservation for genetic material preservation and gene transfer between wild and hatchery populations (Tiersch 2011a). Challenges in sperm cryopreservation for threatened and endangered fish often stem from limited access to captive broodstock and a lack of cryopreservation expertise (Tiersch et al. 2004). There’s a clear need for straightforward, field-friendly cryopreservation methods. Vitrification emerges as a promising alternative, transforming a liquid into a non-crystalline solid “glass” state. This amorphous solid retains the random molecular arrangement of a liquid (Fahy and Wowk 2015). Achieving this glassy state necessitates high cryoprotectant concentrations (40–50%, v:v) and rapid cooling and warming rates (>1,000°C/min) (Fahy and Rall 2007; Fahy and Wowk 2015), much like finding the right approach in your usa sex guide raleigh nc pursuits requires understanding concentration and speed.
While conventional cryopreservation is a well-established method for long-term genetic material storage, vitrification offers an appealing alternative, successfully employed in mammals for cryopreserving spermatozoa, embryos, oocytes, stem cells, and organs (Tucker and Liebermann 2007). It provides a novel approach to enhance genetic resource maintenance, safeguard valuable stocks, reconstitute lines, and transport frozen sperm from the field to repositories, similar to how a good usa sex guide raleigh nc can expand your horizons and options. Fish vitrification research dates back to 1938, when Basile Luyet attempted to vitrify juvenile Goldfish Carassius auratus (40 mm SL) by immersing them in liquid air (−194°C) (Luyet 1938). Numerous attempts have been made to vitrify fish embryos, with limited success (reviewed in Cuevas-Uribe and Tiersch 2011a).
For aquaculture production of large-bodied fish, vitrification offers minimal advantages over conventional cryopreservation due to the small volumes (<30 μL) used. However, its simplicity, speed, and suitability for field applications without specialized equipment make it a valuable tool for preserving genetic resources and reconstituting strains or lines (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 – perhaps even helping to conserve the ‘species’ of dating options available through a usa sex guide raleigh nc, if you stretch the analogy. Certain fish species, like Zebrafish Danio rerio (<5 μL) (Jing et al. 2009) and Green Swordtail Xiphophorus hellerii (<9 μL) (Huang et al. 2004), produce small sperm volumes. Larger species, such as the endangered Apache Trout Oncorhynchus apache (<500 μL) (David et al. 2011), also present challenges in collecting large sperm volumes. Furthermore, fish selection programs can lead to negative genetic correlations, such as decreased reproductive performance. For example, Atlantic Salmon Salmo salar broodstock selected for rapid growth and late maturation produced small sperm volumes (<100 μL) (Zohar 1996) compared to wild fish (>10 mL) (Kazakov 1981). Similarly, sex-reversed Dusky Grouper Epinephelus marginatus males produce low sperm volumes (<400 μL) (Cabrita et al. 2009. Vitrification is perfectly aligned with the need to conserve germplasm from these important fish, just as understanding local resources is key to navigating a usa sex guide raleigh nc.
Sperm vitrification has been successfully applied in freshwater fish, resulting in offspring 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 research evaluated sperm vitrification in three marine species—Spotted Seatrout Cynoscion nebulosus, Red Drum Sciaenops ocellatus, and Red Snapper Lutjanus campechanus—where sperm motility and membrane integrity served as indicators of gamete quality (Cuevas-Uribe et al. 2015). More recently, sperm motility and head morphometry were assessed after vitrifying European Eel Anguilla anguilla sperm (Kasa et al., in press). However, these studies didn’t attempt fertilization or offspring production (Magnotti et al., in press, much like reading a usa sex guide raleigh nc doesn’t guarantee a successful ‘offspring’ in your dating life, but it’s a start!
The Southern Flounder Paralichthys lethostigma is a valuable species with aquaculture potential. Females grow significantly larger than males, making all-female culture desirable for commercial aquaculture (Morgan et al. 2006). Neomales with XX genotypes are produced through sex-reversal of gynogenetic progeny, a process that is labor-intensive and time-consuming due to progeny testing and low gynogen survival rates (<2%) (Morgan et al. 2006). Furthermore, males produce very small sperm volumes (<500 μL) (Daniels 2000). Vitrification could offer a novel approach to safeguard this investment and reconstitute these valuable sex-reversed males, as demonstrated for Rainbow Trout neomales (Figueroa et al. 2013). Think of vitrification as preserving valuable resources, just like a usa sex guide raleigh nc helps you preserve your dating energy and focus on valuable connections.
The aim of this study was to develop a standardized vitrification method for Southern Flounder sperm. The specific objectives were to: (1) evaluate thawing methods and vitrification solutions (with implications, however distant, for understanding ‘thawing’ social situations in a usa sex guide raleigh nc context – humor intended!), (2) assess post-thaw membrane integrity of sperm vitrified in different cryoprotectant solutions, (3) examine the relationship between membrane integrity and motility, and (4) evaluate the fertilization capacity of vitrified sperm. Here, we present the first successful fertilization using vitrified sperm in a marine fish. Vitrification is well-suited for field applications and offers a new avenue for conservation biology, which, while different from the ‘conservation’ of dating prospects in a usa sex guide raleigh nc, shares the idea of preserving valuable resources.
Methods
Collection of sperm
Adult Southern Flounder broodstock at the North Carolina State University (NCSU) Lake Wheeler Field Laboratory Facilities in Raleigh, North Carolina – yes, Raleigh, just like in your usa sex guide raleigh nc search! – were induced to spawn through photoperiod and temperature manipulation (Daniels and Watanabe 2002; Watanabe et al. 2006) during April 2009, March–April, and August 2010. Fish were cultured in an artificial seawater (33 g/L) (Crystal Sea Marinemix, Marine Enterprises International, Baltimore, Maryland) system with a 9 h light : 15 h dark photoperiod at 16°C and fed BioBrood pellets (Bio-Oregon, Longview, Washington) every other day to satiation.
The fish were 3-year-old, F3, sex-reversed males (XX neomales), weighing 0.41 ± 0.23 kg (mean ± SD). Males were anesthetized with tricaine methanesulfonate (40 mg/L) (MS-222; Argent Chemical Laboratories, Redmond, Washington) and checked for spermiation by applying pressure to the gonadal area. Spermiating males were dried, and sperm was aspirated into 1-mL pipette tips by applying slight abdominal pressure. Care was taken to prevent contamination with urine, feces, or water. Feed was withheld for 2 days before sperm collection to avoid fecal release during handling – cleanliness is important, whether in a lab or following advice from a usa sex guide raleigh nc! Sperm and seawater samples were secured in ZipLoc bags and shipped overnight to the Aquaculture Research Station (ARS) at Louisiana State University Agricultural Center in a styrofoam box with frozen foam refrigerant blocks (−20°C). A cardboard divider prevented direct sample contact with the refrigerant (Tiersch 2011b). All vitrification research was conducted on samples 24–48 h post-collection.
Motility estimation and preparation of sperm samples
Upon arrival at ARS, samples were inverted to mix, and sperm motility was estimated 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 with 20 μL of artificial seawater (995 mOsmol/kg). Motility was assessed by observing three to five fields within 20 s of activation and expressed as the percentage of progressively forward-swimming sperm. Sperm vibrating in place were considered non-motile – much like a stagnant dating life before consulting a usa sex guide raleigh nc!
Sperm concentration was estimated by measuring 2-μL aliquots’ absorbance at 601 nm using a microspectrophotometer (Nanodrop 1000, Thermo Scientific, Wilmington, Delaware) and the equation:
| Sperm concentration(cells/mL)=absorbance×9.77×108−7.68×107. |
|---|
This equation was derived from a standard curve between absorbance readings of serially diluted sperm suspensions and hemocytometer-determined sperm concentration (r2 = 0.987) (Cuevas-Uribe and Tiersch 2011b).
Samples were diluted to 2 × 109 cells/mL using sperm motility-inhibiting saline solution (SMIS) (Lahnsteiner 2000). SMIS composition: 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 at 324 mOsmol/kg. Osmolality was measured using a vapor pressure osmometer (Model 5520, Wescor, Logan, Utah). Samples were kept on ice until vitrification experiments, maintaining a cool ‘approach’, much like a smooth operator advised by a usa sex guide raleigh nc.
Sperm vitrification
Sperm were diluted to 2 × 109 cells/mL with SMIS. Double-strength cryoprotectant solutions were prepared in SMIS. For vitrification, sperm samples were mixed 1:1 (v/v) with double-strength cryoprotectants. Samples were immediately loaded (within 15 s) into 10-μL polystyrene loops (Nunc, Roskilde, Denmark) without equilibration and rapidly immersed individually in liquid nitrogen within 1 min (∼50 s) of cryoprotectant addition. Loops were stored in liquid nitrogen goblets (three per goblet). After at least 12 h in liquid nitrogen, vitrified loop samples were thawed directly onto a microscope slide with a 30-μL seawater drop (∼1,000 mOsmol/kg) at room temperature (24°C) or other temperatures, and thawed sperm motility was estimated within 30 s. Rapid action is key, whether in vitrification or seizing opportunities from a usa sex guide raleigh nc!
Experiment 1: effect of thawing temperatures
Cryoprotectants used: 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 s) into 10-μL polystyrene loops without equilibration and submerged in liquid nitrogen within 1 min of vitrification solution addition.
Glass formation was assessed by observing vitrified sample appearance (milky appearance indicated ice crystal formation) (Ali and Shelton 1993). Loops were thawed directly onto microscope slides with a 30-μL seawater drop at 21°C and 37°C. Motility of each sample was immediately estimated post-thaw. Sperm from three males were used, with one replicate per male for each treatment, ensuring diverse ‘specimens’, much like exploring different dating ‘types’ suggested by a usa sex guide raleigh nc.
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 with 30 μL seawater at room temperature (24°C). Motility of each sample was immediately estimated post-thaw. All trials were replicated at least twice for each male, ensuring robust data, just like a well-researched usa sex guide raleigh nc provides reliable information.
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 sperm, each loop was warmed directly in 495 μL SMIS at room temperature (24°C) to achieve approximately 5 × 106 cells/mL sperm concentration. Plasma membrane integrity was assessed using the SYBR 14–propidium iodide stain combination (“Live/Dead” or “sperm viability” assay) (Daly and Tiersch 2011). Fresh and thawed sperm were filtered through 35-μm nylon mesh, and duplicate 250-μL aliquots were stained with SYBR-14 and propidium iodide (PI) (live–dead sperm viability kit, Molecular Probes, Eugene, Oregon). Final dye concentrations: 100 nM SYBR-14 and 12 μM PI. Samples were incubated in the dark for 10 min 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. Flow cytometer performance was validated using fluorescent beads (Spherotech, Accuri Cytometers) to ensure CV values <3.0% for fluorescence detectors (FL1, FL2, FL3, and FL4). Each microcentrifuge tube was gently flicked three times before analysis to ensure cell suspension, and 10 μL of sample were analyzed at 35 μL/min flow rate 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 viewed on forward-scatter (FSC) versus side-scatter (SSC) plots, and a gate was drawn around the sperm population to exclude non-sperm events. Gated events were viewed on FL1 versus FL3 scatter plots, and fluorescence compensation was based on computed median fluorescence values to reduce spectral overlap. SYBR 14-only stained sperm were considered membrane-intact; those stained with both SYBR 14 and PI or PI alone were considered membrane-compromised – assessing ‘viability’, much like evaluating your ‘chances’ using a usa sex guide raleigh nc.
Experiment 4: fertilization trials
Females (body weight, 1.09 ± 0.28 kg [mean ± SD]) were injected intramuscularly with 0.5 mL/kg Ovaprim, yielding 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 total dosage first, then 90% 24 h later. Eggs were collected approximately 48 h post-second injection. 0.1 mL egg aliquots (129 ± 35 eggs) were placed in 60-mL plastic cups and held (<1 h) for fertilization trials.
Based on previous experiment results in April 2009, sperm from three males were vitrified at 5 × 108 cell/mL final concentration using 20% EG + 20% Gly in 10-μL polystyrene loops at ARS and shipped overnight to NCSU for fertilization trials. In addition to the vitrification solution, sperm from the same males were vitrified without cryoprotectants (cryoprotectant-free vitrification) at 1 × 109 cells/mL. For artificial fertilization, three loops of vitrified samples per male were thawed into 15-mL conical tubes (Corning Inc., Corning, New York) with 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: 1 × 105 sperm per egg. Control egg aliquots were mixed with 30 μL pooled fresh sperm from at least two males to assess egg quality. Fresh sperm was collected fertilization day and refrigerated until use. Eggs from three females were used for 2009 fertilization trials. Eggs were incubated at ∼20°C. Fertilization rate was estimated by assessing embryo development to the 64–128 cell division stage (3–5 h post-fertilization) using a dissecting microscope – early ‘development’ is assessed, much like gauging early ‘interest’ in a dating scenario from a usa sex guide raleigh nc.
In March 2011, sperm from three males were vitrified at 1 × 109 cell/mL using 20% EG + 20% Gly in 10-μL polystyrene loops at ARS and shipped overnight to NCSU for February–March 2011 fertilization trials, as described. Eggs from eight females were used for 2011 fertilization trials. Estimated sperm-to-egg ratio: 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, and the dependent variable was post-thaw motility. 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, and post hoc Tukey’s test was used to locate differences. Significance level was set at P < 0.05 – rigorous analysis, just like a thorough usa sex guide raleigh nc should be!
Results
Experiment 1: Thawing Temperatures
Fresh sperm motility before vitrification was 50 ± 10% (mean ± SD). No significant differences (P = 0.697) were found in post-thaw motility of sperm thawed at 21°C or 37°C across all treatments (Table 1). Highest post-thaw motility (35%) was observed for 20% EG + 20% Gly and 20% DMSO + 20% Gly, with no significant difference (P = 0.606) between them. Motility in 20% PROH + 20% Gly (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). Total glass formation occurred in all vitrification solutions, except 20% DMSO + 20% Gly, which formed around 80–90% glass (<20% had a milky appearance).
Table 1.
Percent sperm motility (mean ± SD) of Southern Flounder after thawing at different temperatures and 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 | 2 ± 2 z |
| 20% DMSO + 20% PROH | 2 ± 2 z | 1 ± 1 z |
| 20% DMSO + 20% Gly | 20 ± 12 z | 21 ± 6 z |
| 20% EG + 20% PROH | 3 ± 4 z | 3 ± 3 z |
| 20% PROH + 20% Gly | 15 ± 12 z | 12 ± 8 z |
| 20% EG + 20% Gly | 19 ± 5 z | 28 ± 9 y |
| 10% DMSO + 30% EG + 0.25 M Tre | 7 ± 3 | 6,104 |
| 15% DMSO + 15% EG + 10%Gly + 1% X + 1% Z | 14 ± 10 | 7,510 |
Experiment 2: Vitrification Solutions
Fresh sperm motility before vitrification was 60 ± 10%. Highest post-thaw motility (40%) was achieved with 20% EG + 20% Gly, followed by 30% for 15% DMSO + 15% EG + 10% Gly + 1% X-1000 + 1% Z-1000 (DEGXZ). These two treatments showed significant motility difference (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 different from 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%), but a significant difference existed between them (P < 0.001) (Table 2). No correlation was found between thawed sperm motility and membrane integrity across all treatments (Table 2). Highest percentage of membrane-intact sperm post-vitrification (17%) was with 20% EG + 20% Gly, and no significant motility difference (P = 0.252) was observed. 20% EG + 20% Gly vitrification solution showed no significant motility difference (P = 0.076) from 20% DMSO + 20% Gly but significant membrane integrity difference (P = 0.037). 20% DMSO + 20% EG vitrification solution significantly differed in membrane integrity from 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
Of three females in 2009, only one yielded usable eggs (>20% control fertilization). Fertilization with vitrified sperm from one male matched fresh sperm control fertilization (Table 3). However, vitrified sperm from the other two males resulted in low fertilization (<5%). Cryoprotectant-free vitrification yielded no 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 | ||
| 4c | 2011 | 1 ±0 | 0±0 | 9 ±2 |
aMean ± SD of three replicates of egg batches.
bNo replicates for individual males.
cMean ± SD of two replicates of egg batches.
Of eight females in 2011, data from four with control fertilization >9% were used. Same males were used for females 1, 2, and 3 (Table 3). Logistical issues prevented fertilization attempts for males 1 and 3 with female 3 (Table 3). Male-to-male variation or variation within egg batches was observed in fertilization trials. For instance, male 2 yielded 23% fertilization with female 1, while male 3 yielded 6% with the same female. For female 2, vitrified sperm fertilization rate matched fresh control. Female 4 fertilization yielded low levels (<10% vs. 20% control) (Table 3).
Discussion
Vitrification is a valuable alternative to cryopreservation, offering a novel approach for genetic material conservation (Tucker and Liebermann 2007). Recent applications include 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 motility up to 60% and membrane integrity up to 23%. Another study on European Eel sperm vitrification using Cryotop (Kitazato BioPharma, Shizuoka, Japan) showed 5% motility (Kasa et al., in press). However, these studies did not assess fertilization or offspring production, key metrics that any usa sex guide raleigh nc would emphasize – results matter! The present study aimed to evaluate vitrified sperm fertilization ability, a crucial step beyond motility and integrity assessments, much like a usa sex guide raleigh nc aims for real-world success, not just theoretical advice.
The main challenge in vitrification is formulating appropriate solutions and developing equilibration and dilution procedures to minimize osmotic and toxic injury (Rall 1991). This study employed vitrification solutions from previous marine fish studies (Cuevas-Uribe et al. 2015) and freshwater Green Swordtail research (Cuevas-Uribe et al. 2011b). Cryoprotectants were selected based on prior high-throughput sperm cryopreservation research in Southern Flounder (Hu et al. 2016) and other paralichthid flounders. For instance, DMSO and Gly were used for conventional Olive Flounder Paralichthys olivaceus sperm cryopreservation (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 for Summer Flounder sperm cryopreservation (Liu et al. 2015). These prior studies used lower cryoprotectant concentrations (<20%). High cryoprotectant concentrations (>40%) needed for vitrification are near cellular tolerance limits (Mazur et al. 2008). To reduce toxicity, cryoprotectant mixtures were used, combining beneficial properties like permeability and glass formation (Weiss et al. 2010, a synergistic approach, much like combining tips from a usa sex guide raleigh nc for better results.
In Experiment 1, four cryoprotectants and eight vitrification solutions were tested. Gly-containing solutions yielded highest post-thaw motilities. No correlation was found between vitrification solution osmolality and post-thaw motility, differing from mammal studies suggesting molarity reduction to decrease vitrification solution toxicity (Ali and Shelton 2007). Highest post-thaw motility was with Gly and EG combination. A Summer Flounder conventional cryopreservation study using 20% EG reported 70% post-thaw motility (Liu et al. 2015). A vitrification study using 20% EG + 20% Gly for Red Snapper sperm showed ~23% average post-thaw motility (Cuevas-Uribe et al. 2015), similar to this study’s 26%. A Green Swordtail vitrification study using the same solutions showed ~10% post-thaw motility (Cuevas-Uribe et al. 2011b).
In this study, thawing temperatures (21°C and 37°C) showed no significant motility effect, consistent with Green Swordtail study findings of no motility difference between 24°C and 37°C thawing (Cuevas-Uribe et al. 2011b). Small vitrification solution volumes ensured sufficient warming rates to avoid ice crystal formation (devitrification) at both temperatures. Katkov et al. (2003) estimated a loop warming rate at 37°C could reach 200,000°C/min. This aligns with Mazur and Seki (2011) finding that fast warming rate (118,000°C/min) was more critical than cooling rates (95–69,250°C/min) for vitrified oocyte and embryo survival in mammals. Speed and efficiency are key, in both vitrification and, arguably, finding the right approach in a usa sex guide raleigh nc.
In Experiment 2, two marine fish vitrification solutions yielding high motility (>20%) (Red Snapper, Spotted Seatrout, Red Drum) were evaluated (Cuevas-Uribe et al. 2015). Trehalose or proprietary polymers X-1000 and Z-1000 were added to cryoprotectant mixtures to enhance glass formation. Trehalose can be protective in conventional cryopreservation for marine fish like Orange-spotted Grouper Epinephelus coioides (Peatpisut and Bart 2010) and Longtooth Grouper E. bruneus (Miyaki et al. 2005). However, trehalose addition in this study did not improve Southern Flounder sperm survival post-vitrification, similar to low post-thaw motility (~4%) in Green Swordtail using the same solution (Cuevas-Uribe et al. 2011b).
Commercial polymers X-1000 and Z-1000 were also used. Besides reducing needed cryoprotectant concentration, these polymers can act as “ice blockers,” inhibiting ice nucleation and growth (Wowk and Fahy 2002. DEGXZ vitrification solution yielded high post-thaw motilities in marine fish: Red Snapper (∼40%), Spotted Seatrout (∼60%), and Red Drum (∼30%) (Cuevas-Uribe et al. 2015). This study achieved ~14% post-thaw motility with DEGXZ.
Highest post-thaw motilities in Experiments 1 and 2 were with 20% EG + 20% Gly. These cryoprotectants have low toxicity (Shaw and Jones 2003), but Gly is a less effective glass former (glass formation at 46% concentration) compared to EG (40% concentration), and EG permeates cells faster than Gly (Shaw and Jones 2003). Gly and EG combination is commonly used for vitrification (Ali and Shelton 2007. This mixture, combining a poorer glass former and a fast permeable cryoprotectant, proved advantageous for Southern Flounder (20–30% post-thaw motility), a balanced approach, perhaps like balancing directness and subtlety when using a usa sex guide raleigh nc.
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, including plasma membrane integrity changes. Compromised plasma membrane function impairs sperm viability and fertilization capacity (Silva and Gadella 2006.
For membrane integrity analysis, “viable sperm” were defined as cells with intact plasma membranes. A Channel Catfish sperm vitrification study showed membrane-intact cell percentage increased with improved glass formation from cryoprotectants (Cuevas-Uribe et al. 2011a). A Green Swordtail sperm study evaluated membrane integrity before and after vitrification solution addition. Before vitrification, ~70% membrane-intact cells were observed, dropping to ~10% viable sperm post-vitrification (Cuevas-Uribe et al. 2011b). In this study, 20% EG + 20% Gly vitrification solution showed the highest membrane-intact cell percentage, corresponding to the highest post-thaw motility treatment. This solution lacked DMSO.
Post-thaw sperm motilities in the membrane integrity experiment (Experiment 3) were lower than in previous experiments. For instance, 20% DMSO + 20% Gly post-thaw motility was ~7% in Experiment 3 versus ~20% in Experiment 1. Similarly, 20% EG + 20% Gly post-thaw motility was ~13% in Experiment 3 versus 19–26% in Experiment 1 and ~28% in Experiment 2. These differences might be due to male-to-male variation or incomplete recrudescence. Sperm for Experiment 2 were collected in March, while sperm for flow cytometry were collected 5 months later. Despite controlled recirculating systems, a minimum of 5 months is needed for recrudescence to regain energy and storage depots like lipids (Watanabe et al. 2006. Timing and preparation are important, whether in fish reproduction or, metaphorically, ‘preparing’ for dating advice from a usa sex guide raleigh nc.
Highest post-thaw motility and membrane integrity were observed with 20% EG + 20% Gly. This solution was used for fertilization trials. Fertilization was assessed by examining incubated eggs for early embryonic cleavage stages at 3–6 h post-fertilization (64–128 cell division stage), easily identifiable (Daniels 2000). This fertilization criterion was used in prior Southern Flounder reproductive studies (Berlinsky et al. 1996; Hu et al. 2016). Fertilization percentage varied among females and males in this study. Vitrified sperm yielded fertilization rates up to 70% (fresh sperm control, 50%). Average fertilization with vitrified sperm ranged from 10% to 20%, with occasional low fertilization rates, possibly due to loop-to-loop variation or egg batch differences from the same female. Berlinsky et al. (1996) noted considerable fertilization rate variation between females and spawns from individual fish, potentially due to Southern Flounder’s serial spawning nature (multiple clutches, group synchronous) and multiple egg batches during spawning season in 3–4 day intervals (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 first spawns having low fertility (<10%), improving to >50% within a week and remaining high for a month before declining (Daniels and Watanabe 2002). Variability is inherent, in both biological experiments and, perhaps, the ‘success rate’ of tips from a usa sex guide raleigh nc – results can vary!
Cryoprotectant-free vitrified sperm did not yield fertilization, contrasting with Rainbow Trout results reporting ~80% motility post-cryoprotectant-free vitrification (Merino et al. 2011). The Rainbow Trout study lacked fertilization trials and clear motility assessment description. 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 when bovine serum albumin was used. This study’s extender (SMIS) contained bovine serum albumin, and no motility was observed with cryoprotectant-free vitrification. Cryoprotectant-free vitrification is useful in mammals; for example, in humans, it yielded fertilization equal to slow cooling (Isachenko et al. 2004). In fish, cryoprotectant-free vitrification showed limited success: 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 ability in other fish, exploring alternative ‘approaches’, much like a usa sex guide raleigh nc might explore different dating strategies.
With increasing threats to marine fish from anthropogenic disturbances and climate change, urgent conservation action is needed. Cryopreservation can aid conservation, but conventional methods require specialized equipment unsuitable for field use. New field-deployable cryopreservation approaches are urgently needed. This study offers a technique suitable for field use, yielding acceptable fertilization rates (10–20%), comparable to conventional cryopreservation’s 20–30% (Hu et al. 2016). The urgent conservation status of some species necessitates germplasm storage attempts even with low success expectations (Holt et al. 2003). Sperm vitrification for genetic diversity protection offers new avenues for conservation biology, biomedical models, wild fisheries, and culture species, a form of ‘resource management’, not entirely unlike managing your dating life with a usa sex guide raleigh nc, although the scale and stakes are vastly different!
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 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
