Cryopreservation of filefish (Thamnaconus septentrionalis Gunther, 1877) sperm

The present study examined the possibility of long‐term storage, by cryopreservation in liquid nitrogen, of the sperm of filefish (Thamnaconus septentrionalis). Changes in motility, survival rate, ultrastructure and fertilization rate of the sperm after freezing and thawing were tested. For selection of the immobilizing solution, artificial seawater (ASW) of 250, 350 and 450 mOsmol kg^?1 were tested. Sperm motility was significantly inhibited in 350 mOsmol kg^?1 ASW, and restored entirely after 100% ASW (1200 mOsmol kg^?1) was added. Two cryoprotectants, dimethyl sulphoxide and glycerol, were employed. The sperm was diluted at the ratio of 1:6 with the extenders, and frozen at a freezing rate of ?40°C min^?1 to ?100°C after equilibration for 10 min at room temperature, followed by plunging into liquid nitrogen. The highest post‐thawed sperm motility and survival rate were obtained with 5% glycerol. Afterwards, the effect of different freezing rates was examined using 5% glycerol as a cryoprotectant, and the rate of ?30°C min^?1 to ?100°C showed the best result.     

Use of ACP‐104 at Different Dilutions for Sperm Cryopreservation of Dourado,Salminus brasiliensis

The dourado, Salminus brasiliensis, is an important fish in South American river basins. The fish‐farming potential and concerns for its conservation in watersheds has stimulated studies with this species. This study compared the effect of different dilutions of two cryoprotectant solutions on the quality of cryopreserved dourado sperm (duration of motility/motility rate) post‐thawing using three different activating solutions. The cryoprotectant solution ACP‐104 was compared with the cryoprotectant solution most commonly used for dourado, which consists of a mixture of dimethyl sulfoxide, glucose, egg yolk, and distilled water. The semen was mixed following dilutions of semen : cryoprotectant solution: 1:3, 1:9, 1:15, 1:21, and 1:27. The cryopreserved samples were activated using distilled water, 0.45% NaCl or 1% NaHCO₃. The results showed differences in thawed semen using the different activating solutions and cryoprotectant solutions. The glucose‐based cryoprotectant not only resulted in sperm motility equal to or better than that produced by ACP‐104, but the quality was not affected by the dilution. However, the sperm quality improved with increasing dilutions for ACP‐104. Under the conditions tested, the standard cryoprotectant solution is recommended for the cryopreservation of dourado semen because it requires a lower solution volume and, therefore, reduced storage space for the same volume of semen.     

Spermatozoon ultrastructure and sperm cryopreservation of the Brazilian dry season spawner fish pirapitinga,Brycon nattereri

The aims of this study were to describe the fresh spermatozoon ultrastructure using scanning and transmission electron microscopy and to improve the sperm cryopreservation methodology for the freshwater fish pirapitinga Brycon nattereri. Extenders (BTS^? and NaCl), straw volumes (0.5 and 4.0 mL), thawing temperatures (30 and 60 °C) and activating agents (0.29% NaCl and 1% NaHCO₃) were tested. Methylglycol was used as a cryoprotectant agent and sperm was frozen in nitrogen vapour (dry‐shipper). Post‐thawed sperm motility rate, motility quality (score 0=no movement; 5=rapidly swimming spermatozoa), duration of motility and spermatozoon morphology were evaluated. Fresh spermatozoon was 35.06 μm long, the head was ovoid (2.00 × 1.22 μm) with no acrosome, the midpiece was 2.15 μm long and the flagellum was 30.90 μm long with the typical 9+2 axoneme arrangement. Post‐thawed sperm motility rate (70–79% motile sperm), motility quality (score 3.1–3.7) and morphology (9.3–11.6% abnormal spermatozoa) were not affected by any of the parameters tested. The duration of sperm motility was longer when triggered in 1% NaHCO₃ (392–1031 s) compared with 0.29% NaCl (144–338 s). Brycon nattereri sperm cryopreserved under the conditions described above yields over 70% motility and should last long enough to fertilize oocytes, even after 2 years of freezing.     

Cryopreservation of Persian sturgeon (Acipenser persicus) sperm: effects of cryoprotectants,antioxidant, membrane stabilizer,equilibration time and dilution ratio on sperm motility and fertility

Three experiments were performed to develop protocols for cryopreservation of Persian sturgeon Acipenser persicus, sperm. In the first experiment, sperm from six males was individually split in three subsamples and cryopreserved using Modified Tsvetkova's extender (mT) supplemented with dimethyl sulfoxide (DMSO), methanol (MeOH), glycerol (Gly) and ethylene glycol (EG) at concentration of 5%, 10%, 15% and 20%. In the second set of experiments, the effects of six equilibration times (0, 5, 10, 20, 40 and 60 min) and dilution ratios (volume sperm: volume extender 1:0.5, 1:1, 1:2, 1:3, 1:5 and 1:10) and the additive advantage of bovine serum albumin (BSA; 0, 2.5, 5 and 10 mg mL^?1) and ascorbic acid (0, 2.5, 5 and 10 U mL^?1), on the post‐thaw survival of sperm (triplicate set of six fish) were evaluated. Then, sperm was diluted in 1:1 mT extender with 10 mg mL^?1 BSA with selected cryoprotectants (15% MeOH and 10% DMSO) for 5 min. After a month of storage in liquid nitrogen, post‐thawed sperm motility; fertilization and hatching rate and viability of derived larvae were measured (Exp.3). Evaluation of cryoprotectants efficiency showed that MeOH 15% and DMSO 10% were suitable for cryopreservation of Persian sturgeon sperm. Gly and EG resulted in very low post‐thaw motility rates even at lowest concentration. No significant difference was observed among the four different equilibration times (0, 5, 10, 20 min) (P > 0.05) although higher equilibration times than 20 min resulted low post‐thaw motility (P < 0.05). The motility of frozen–thawed sperm did not significantly change when dilution ratio was increased from 1:0.5 to 1:3 (P > 0.05). However, higher dilution ratios (1:5 and 1:10) reduced the percentage of motile sperm. Supplementation of the cryoprotectant solution with 10 mg mL^?1 BSA significantly improved post‐thaw motility (P < 0.05), but ascorbic acid did not improve post‐thaw motility (P > 0.05). The results of experiment 3 showed that the highest fertilization (30.2 ± 5.75) and hatching rates (28.2 ± 5.25) were observed when samples were frozen with 15% MeOH (P > 0.05). Our study indicates that the use of mT extender consisting of 10 mg mL^?1 BSA in 15% MeOH diluted with sperm at 1:1 ratio for 5 min can be recommended cryopreservation method for Persian sturgeon sperm.     

Cryopreservation of sea cucumber Apostichopus japonicus (Selenka) sperm

A simple and convenient method for the cryopreservation of sea cucumber Apostichopus japonicus (Selenka) sperm was tested in the present study. The highest motility (76.7±2.9%) of post‐thawing sperm was obtained in 15% dimethyl sulphoxide (DMSO) with a 1:9 dilution (semen volume to DMSO volume) when 0.5 mL semen–DMSO mixture was frozen at 6 cm above liquid N₂ in a closed styrofoam box. After thawing, sperm cryopreserved in glycerol almost lost motility entirely. Although there was no significant difference in percentage of motile sperm between 15% and 20% DMSO, the duration of sperm motility of 15% DMSO group was longer than that of 20% DMSO group. The motility of post‐thawing sperm enhanced when the dilution ratio of semen increased from 1:1 to 1:9. Morphological changes such as the loss of mitochondria, swollen plasma membrane and broken or rolled‐up tails were observed in post‐thawing sperm using an eosin–nigrosin staining. The fertility of cryopreserved sperm was significantly lower than that of unfrozen sperm. The 10‐fold increase in sperm to egg ratio resulted in double fertility for cryopreserved sperm, and about 70% fertility relative to the control.     

The Effects of Osmolality, Cryoprotectant and Equilibration Time on Striped Bass Morone saxatilis Sperm Motility

Four experiments were designed to evaluate the effects of osmolality, cryoprotectant, and equilibration time on striped bass sperm motility. In the first experiment, solutions of NaCI or KCI with osmolalities ranging from 0 to 700 mmol/kg were tested on sperm activation. Over 60% of the sperm were activated by isotonic NaCI and KCI solutions with a treatment osmolality of 350 mmol/kg. Sperm remained motile until osmolality increased to 600 mmol/ kg. In the second and third experiments, Extenders 1, 2 and 3 with osmolalities of 350, 500, and 600 mmol/kg, respectively, were tested. Sperm samples stored in Extender 2 showed significantly higher ( P 0.01) sperm motility after 10 min of exposure as well as greater ( P < 0.01) post-thaw motility when compared to samples stored in Extenders 1 and 3. In the fourth experiment, two trials were carried out to evaluate the effects of cryoprotectant and equilibration time. In the first trial, methanol with a concentration of 5% and 10% yielded the highest ( P < 0.05) sperm motility prior to freezing at all equilibration times examined. However, 5% DMSO yielded the highest ( P < 0.01) post-thaw motility (38 ± 3.6%). DMSO with concentrations of 10% and 15% resulted in 17 ± 2.3% and 6 ± 1.0% post-thaw motility, respectively. Both methanol and DMA, at all concentrations tested, resulted in less than 10% post-thaw motility. In the second trial, four DMSO concentrations with three different equilibration times were examined. We observed a significant ( P < 0.001) interaction effect between DMSO concentration and equilibration time. Post-thaw motility was significantly greater ( P < 0.01) with a concentration of 5% DMSO at all equilibration times examined, compared to 1.25, 2.5, and 10% DMSO. An average post-thaw motility of 40 ± 2.9% was achieved after 10 min equilibration using 5% DMSO.     

Greenlip abalone (Haliotis laevigata Donovan, 1808) sperm cryopreservation using a programmable freezing technique and testing the addition of amino acid and vitamin

This study investigated factors key to the development of sperm cryopreservation in the greenlip abalone Haliotis laevigata using a programmable freezing technique, including (1) permeable cryoprotectant agent (CPA) selection; (2) cooling rate; (3) endpoint temperature; (4) thawing temperature; (5) sperm to egg ratio and (6) sugar, vitamin and amino acid supplementation, using sperm motility, fertilization rate, plasma membrane integrity, mitochondrial membrane potential or acrosome integrity as quality assessment indicators. Results showed that among the permeable CPAs evaluated, 10% dimethyl sulfoxide was the most suitable for greenlip abalone sperm cryopreservation. The highest post‐thaw sperm motility was achieved with the sperm being frozen at a cooling rate of ?5°C min^?1 to ?30°C from 0°C and thawed and recovered in 40°C and 18°C seawater baths respectively. The addition of sugars in 10% dimethyl sulfoxide did not significantly improve the post‐thaw sperm motility and fertilization rate. The addition of 0.6% glycine, 0.2% taurine or 0.02% L‐ascorbic acid, on the other hand, significantly improved the post‐thaw sperm motility. However, only the addition of 0.6% glycine improved the post‐thaw sperm fertilization rate, which was further confirmed by the improvement of the post‐thaw sperm mitochondrial membrane potential and acrosome integrity through flow cytometry analysis.     

Effect of extender composition and freezing rate on post-thaw motility and fertility of Arctic char, Salvelinus alpinus (L.), spermatozoa

The effects of extender composition and freezing rate on motility and fertility of frozen‐thawed Arctic char, Salvelinus alpinus, spermatozoa were investigated. Three freezing rates, two semen diluents and three cryoprotectants were tested. Semen frozen in 0.3 mol L^?1 glucose diluent with 10% methanol as a cryoprotectant or in a diluent described by Lahnsteiner with 10%N,N‐dimethylacetamide (DMA) resulted in the highest sperm motility. Fertility was the highest for semen frozen in a glucose–methanol extender but was not significantly different than that for semen frozen in Lahnsteiner's diluent with 10% DMA. Dimethyl sulphoxide (DMSO) at 10% was a relatively ineffective cryoprotectant with either semen diluent. Semen frozen at 6 cm above the surface of liquid nitrogen resulted in a higher post‐thaw sperm motility and fertility than semen frozen at 5 cm. The addition of 7% fresh egg yolk to glucose diluent containing methanol or DMSO did not improve the fertility of frozen‐thawed spermatozoa. However, the addition of 7% fresh egg yolk to glucose–DMA extender significantly improved the fertilization percentages of frozen‐thawed spermatozoa. In conclusion, dilution of semen 1:3 in 0.3 mol L^?1 glucose with 10% methanol and freezing 6 cm above the surface of liquid nitrogen (freezing rate of 40±8°C min^?1, mean±SD from ?5 to ?55°C) is a promising protocol for cryopreservation of Arctic char semen.     

A Strategy for Sperm Cryopreservation of Atlantic Salmon,Salmo salar,for Remote Commercial‐scale High‐throughput Processing

Sperm cryopreservation is an essential tool for long‐term storage of genetic resources for aquaculture fishes. The goal of this study was to develop an efficient and streamlined protocol for high‐throughput processing for sperm cryopreservation in Atlantic salmon, Salmo salar. The objectives were to evaluate: (1) osmolality of blood serum for determining extender osmolality, (2) effects of extenders for fresh sperm dilution and refrigerated storage, (3) effects of methanol and dimethyl sulfoxide (DMSO) on fresh sperm motility, and (4) motility and fertility after thawing. In this study, sperm samples were collected at a hatchery site in Canada and shipped to a freezing site located 2200 miles (3550 km) away in the USA. Evaluation of three extenders indicated that Mounib solution was suitable for diluting dry sperm for sample processing. Ten percent of methanol or DMSO was less toxic to sperm cells than was 15% within 30 min. Further testing with methanol at 5, 10, and 15%, and sperm solution : extender dilutions (v:v) of 1:1, 1:3, and 1:19 (at concentrations of 5 × 10⁷, 3 × 10⁸, and 1 × 10⁹ cells/mL) indicated that methanol at 5 and 10% showed less toxicity to fresh sperm within 1 h at sperm:extender dilutions of 1:1 and 1:3. Post‐thaw motility of sperm cryopreserved with 10% methanol was significantly higher than that with 10% DMSO, and fertility reflected those results (0–1% in DMSO vs. 38–55% in methanol). Further evaluation of sperm cryopreservation with 10 and 15% methanol at sperm dilution ratios of 1:1, 1:3, and 1:19 indicated that post‐thaw motility in 10% methanol was significantly higher than that in 15% methanol, and post‐thaw fertility in 10% methanol at 1:1 and 1:3 dilution ratios had fertilization rates similar to that of fresh sperm controls. Sperm samples from 12 males cryopreserved with 10% methanol showed male‐to‐male variation in post‐thaw motility (0–36%). Overall, a simplified standard protocol was established for cryopreservation of shipped sperm of Atlantic salmon using extender without egg yolk and yielded satisfactory post‐thaw motility and fertilization rates. This procedure can be readily adopted by aquaculture facilities to take advantage of high‐throughput cryopreservation capabilities at remote service centers. Most importantly, this approach lays the groundwork for an alternative commercial model for commercial‐scale production, quality control, and development of industrial standards. Control of male variability and sperm quality remain important considerations for future work.     

Cryoprotectant microinjection toxicity and chilling sensitivity in gilthead seabream (Sparus aurata) embryos

Cryopreservation of fish embryos requires an optimal distribution of cryoprotectants inside all embryo compartments. Traditional techniques for the incorporation of cryoprotectants (CPAs) have failed to protect all fish compartments, especially the yolk sac which has been considered the principal point of embryo chilling sensitivity. In the present study, microinjection was used to incorporate cryoprotectants into the yolk sac of gilthead seabream (Sparus aurata) embryos at tail bud stage. The effect of microinjection viability, cryoprotectant toxicity and chilling resistance was evaluated through the hatching rate. Larval survival at first feeding was also determined in microinjection viability and cryoprotectant toxicity studies. Permeabilized seabream embryos were microinjected with 2.35 nl dimethyl sulfoxide (Me₂SO), methanol (MeOH), ethylene glycol (EG) (5 M, 10 M and pure) or sucrose (10% and 15%). In a second experiment, 29.5 nl and 154.0 nl of the highest concentration of each cryoprotectant were used in the same embryo stage. To test the effect of microinjected cryoprotectants on embryo chilling resistance, 29.5 nl of pure Me₂SO or 15% sucrose was microinjected into the yolk sac of tail bud stage embryos and then at a later stage, (tail-bud-free), were exposed to 3 M Me₂SO solution at − 10 °C for 30 min. Our results showed that microinjection technique did not affect the viability of tail bud stage embryos as is shown by the high hatching and survival rates. Hatching and larval survival rate at first feeding were not affected with any of the CPAs tested, showing percentages higher than 75% and 90%, respectively, when embryos were microinjected with a smaller quantity of cryoprotectant. Sucrose was the cryoprotectant better tolerated at higher concentration and volume. Cryoprotectant concentration inside the yolk higher than 1.18 M for Me₂SO, 1.5 M for EG and 2 M for methanol decreased the hatching rate. Microinjection allowed the delivery of high concentrations of CPAs into the yolk sac without deleterious effects on the embryo, but did not provide a significant level of protection for the whole embryo against chilling injury.     

Cryopreservation of sperm from spotted wolffish

The effects of different concentrations of cryoprotectant (dimethyl sulfoxide; DMSO), cooling rate and straw size on the post-thaw motility of frozen sperm from spotted wolffish, Anarhichas minor, were studied. There was no significant difference in the post-thaw motility of sperm treated with three different concentrations of DMSO (10, 20 and 30%). Similarly, there was no significant difference in the post-thaw motility of spermatozoa when using different freezing rates (i.e. distance of straws from the surface of liquid N₂, 4.7, 5.5 and 7.1°C min⁻¹) and the straw size (0.5 and 1.0 ml) did not affect survival. The cryopreservation of sperm can be used to make up for the frequent lack of sperm and/or the unsynchronised timing of sperm production in spotted wolffish males and the ovulation time in females. The results show that sperm from spotted wolffish can be frozen to secure access to viable sperm, but further experiments are needed in order to reveal the effect of different parameters on the post-thawing mortality and define the optimum conditions for cryopreservation.     

Cryogenic and Refrigerated Storage of Rainbow Smelt Osmerus mordax Spermatozoa

The effects of extender composition, cryoprotectant, and freezing rate on post-thaw rainbow smelt Osmerus mordax sperm motility were examined, and the fertilization capacity of fresh and post-thaw sperm were compared. The highest post-thaw motility (75%) was obtained when milt was diluted 1:3 with an extender containing 600 mM sucrose supplemented with 10% dimethyl sulfoxide and 1.5% bovine serum albumin and frozen at a rate of –20 C/min. Post-thaw motility for sperm stored in this extender was similar to fresh sperm and did not change after 90 d of storage. Furthermore, there were no differences in fertilization rate or embryo survival to the eyed stage between fresh and post-thaw sperm frozen in this extender. The lowest post-thaw motility was observed when sperm were frozen with methanol at a rate of -30 C/min. Refrigerated sperm diluted 1:3 with the 600 mM sucrose extender remained motile for 30 d. These data demonstrate that rainbow smelt spermatozoa can be effectively used following short and long-term storage using a simple, sucrose-based extender.     

The Cryopreservation of Striped Bass Morone saxatilis Semen

Abstract.— Two experiments were designed to improve upon existing methods for cryopreserving striped bass Morone saxatilis , semen. In the first experiment, two extenders, two cryoprotectant concentrations, and two freezing rates were evaluated on the basis of post-thaw semen motility after 1, 7, and 30 d of storage at −196 C. Semen samples cryopreserved at a freezing rate of −40 C/m resulted in a significantly higher percentage of motile sperm ( P < 0.001) and longer duration of spermatozoa motility ( P < 0.001) than samples cryopreserved at a freezing rate of -30 Chin. Also, the cryoprotectant dimethyl-sulfoxide yielded a significantly higher percentage of motile sperm ( P < 0.001) and longer duration of spermatozoa motility ( P < 0.001) when a 5% concentration was used instead of 7.5%. In the second experiment, the two extenders from Experiment I were re-evaluated and a new extender, which was a modified version of Extender 1, was tested. The samples were cryopreserved at -40 C/min with 5% DMSO and thawed in a 25 C water bath. Spermatozoa motility and fertilization ability were evaluated, and semen cryopreserved in Extender 2 yielded the longest duration of spermatozoa motility ( P < 0.001). the highest percentage of motile sperm ( P < 0.001). and the highest percentage of fertilized eggs ( P < 0.002) in comparison to Extenders I and 3.     

Preservation of trout sperm in liquid or frozen stateFlüssig- und gefrierkonservierung von regenbogenforellensperma

A series of experiments to study the preservation of rainbow trout semen in the liquid and frozen state was carried out.For the preservation of liquid semen both undiluted and diluted semen was stored in total darkness at 20° or 4°C under a number of different gasses (air, O₂, O₂ + N₂ (1:1), N₂, CO₂, N₂ + H₂ + CO₂ (8:1:1)). The best results were obtained when storing semen in an undiluted state at 4°C under O₂ (hatching rate after 15 days was 80.6%, controls were 98.2%). Good results were also obtained under air (after 9 days 94.7% vs 94.9% for the controls). Anaerobic conditions, dilution or an increase in temperature all rendered less favourable results.For the purpose of cryopreservation a diluent was developed on the basis of the composition of rainbow trout seminal plasma. The semen was mixed with the diluent immediately before freezing at a ratio of 1:1 or 1:3. The pellet-technique (Nagase, 1964) was found to be more convenient and successful than freezing in straws. The pellets were thawed in a 1% NaHCO₃-solution. The hatching results after storage periods of 1–4 months were very variable (2.6–80.3%).     

Physio-Chemical Characteristics of Seminal Plasma and Development of Media and Methods for the Cryopreservation of European eel Sperm

The high sperm density, together with the short spermatozoa swimming time, makes European eel sperm manipulation and assessment for quality difficult. Two diluting media (K15 and K30) previously designed for Japanese eel sperm were tested. After 24 h, European eel sperm showed significant reduction in the percentage of motile spermatozoa after activation and different motility parameters (VAP, angular velocity; VCL, curvilinear velocity; VSL, straight line velocity; BCF, beating cross frequency), concluding that these media are not suitable to preserve the sperm of this species. After a hormonal treatment to induce spermiation, sperm volume, density and motility were recorded at weekly samplings. The variation of the osmolality (325–330 mOsm kg⁻¹), pH (8.4–8.6) and the ionic composition (concentration of Na⁺, K⁺, Mg²⁺ and Ca²⁺) of the seminal plasma were registered. Physio-chemical results were related with sperm quality throughout the treatment, to determine which must be the suitable characteristics of one extender for the sperm of this species, and to find the best conditions to obtain suitable cryopreservation media for European eel sperm. K⁺ concentration increased, while Ca²⁺ and Mg²⁺ concentrations showed a progressive reduction in correlation with the sperm quality improvement. Na⁺ showed a decreasing, but not significant tendency. P1 and P2 freezing media were designed considering the physio-chemical parameters as well as the ionic composition shown by the best quality sperm samples, and then compared with the previously described solutions, TNK and K30. Sperm quality was determined, checking the percentage of motile spermatozoa and motility parameters using computer-assisted sperm analysis (CASA) software. Samples were frozen after dilution (1:5, 1:20, 1:100) in different freezing media supplemented with 10% dimethyl sulfoxide (DMSO). After thawing, samples frozen with low dilution ratio (1:5) in TNK and P1 media showed higher, although not significant, spermatozoa survival (35.5 ± 14.5 and 36.6 ± 6.7%). The addition of l-α-phosphatidylcholine to the media seems to have a positive effect, as reported in the Japanese eel.     

Cryopreservation of Atlantic cod (Gadus morhua) sperm in large‐volume straws: applications for commercial production and gene banking

In our study, we used a full factorial analysis of variance design to examine the effects of diluent [Mounib's sucrose‐based diluent+hen's egg yolk (EY) and Hanks' balanced salt solution (HBSS)+EY], freezing rate (?2.5, ?5.0 and ?7.5 °C min^?1) and thawing rate (2.5, 5.0 and 7.5 °C min^?1) on motility and velocity of Atlantic cod sperm cryopreserved in 2.5 mL cryogenic straws. We found that post‐thaw sperm performance was strongly influenced by the presence of higher‐order interactions of the factors we tested. For all models broken down by diluent, the 2.5 °C min^?1 thawing rate had the lowest sperm motility recovery index. Mounib's sucrose‐based diluent+EY had the highest motility recovery index at all thawing rates. Mean per cent motility for fresh sperm (87.7±2.9%) was not significantly different than of sperm cryopreserved using Mounib's sucrose‐based diluent+EY, frozen at ?2.5 °C min^?1 and thawed at 5.0 °C min^?1 (77.1±2.9%). For Mounib's sucrose‐based diluent+EY, velocity was significantly higher with sperm thawed at 7.5 °C min^?1, than sperm thawed at 2.5 °C min^?1, while thawing rate had no effect for HBSS+EY. Our findings have implications for cod mariculture and aiding in conservation efforts for a dominant marine fish species.     

Cryopreservation of sperm in farmed blacklip abalone (Haliotis rubra Leach, 1814)

This study developed a technique of sperm cryopreservation using liquid nitrogen (LN) vapour in farmed blacklip abalone Haliotis rubra through evaluating the following five key factors: (1) cryoprotectant agent (CPA) toxicity; (2) cooling temperature; (3) thawing temperature; (4) sperm to egg ratio and (5) sugar addition, using sperm motility or fertilization rate as quality assessment indicators. The results demonstrated that 6% dimethyl sulfoxide (DMSO) was the best single CPA for sperm cryopreservation in this species. The highest post‐thaw sperm motility was achieved when sperm were exposed to LN vapour for 10 min at 5.2 cm above the LN surface and thawed at 60°C and recovered at 16°C in seawater baths. Post‐thaw sperm motility was found to be significantly higher when 6% DMSO was used in combination with 1% or 2% glucose than 6% DMSO alone. Further evaluation of fertilization rate between these CPAs showed that 6% DMSO+2% glucose achieved the highest fertilization rate of 70% at a sperm to egg ratio of 10 000:1.     

Cryopreservation of Mekong catfish,Pangasius bocourti Sauvage, 1880 spermatozoa

The effects of three extenders (Ginzburg fish ringer, Calcium‐free Hank's balanced salt solution, C‐F HBSS and sodium chloride, 0.9% NaCl) and four cryoprotectants (dimethyl sulphoxide, DMSO; dimethyl acetamide, DMA; methanol, MeOH and glycerol) in different concentrations (5%, 10% and 15%) on the motility, viability and fertilization rates of Mekong catfish (Pagasius bocourti) sperm were investigated. Sperm samples were transferred into 250‐μL French straws and sealed with a heated haemostat. The straws were then placed in a cryochamber. A computer‐controlled rate freezer (CL 3300) and programmable Cryogenesis, version 4 were used to regulate the freezing rate. The sperm samples were frozen at a rate of 10°C min^?1 from 4 to ?80°C and then evaluated after 72 h. Of the three extenders used with each cryoprotectant, C‐F HBSS had the highest fertilization rate of 75% (93% of control). This was not significantly different from the control treatment (fresh sperm) when tested with DMSO as the cryoprotectant. The lowest fertilization rate of 27% (38% of control) was resulting from the combination of 15% glycerol and C‐F HBSS. This study found that fertilization, motility and viability rates in all of the experiments had a positive significant correlation (P < 0.001).     

Effect of cryoprotectants, extenders and freezing rates on the fertilization rate of frozen striped catfish, Pangasius hypophthalmus (Sauvage), sperm

The effects of four cryoprotectants (methanol, MeOH; dimethyl sulphoxide, DMSO; dimethyl acetamide, DMA; and ethylene glycol, EG), three extenders (calcium‐free Hanks' balanced salt solution, C‐F HBSS, Hanks' balanced salt solution, HBSS and sodium chloride, NaCl) and two different freezing procedures (one‐ and two‐step) on the cryopreservation of striped catfish (Pangasius hypophthalmus (Sauvage)) sperm were investigated. Sperm were frozen using a controlled‐rate freezer in 250 μL straws and stored for 2 weeks in a liquid nitrogen (LN₂) container. They were then airthawed at room temperature, and fertilization, motility and viability were assessed. The highest fertilization rate of 41% (81% of control) was achieved with the combination of 12% DMSO and 0.9% NaCl using a one‐step freezing procedure (10°C min^?1). Also, DMA resulted in a higher fertilization rate (30% or 51% of the control) than MeOH (18% or 38% of the control) or EG (8% or 12% of the control). In addition, the three extenders used did not affect fertilization rates after cryopreservation with each cryoprotectant. There were no significant differences among the three cryoprotectant concentrations and between the one‐ and two‐step freezing procedures. However, fertilization rates of cryopreserved sperm were significantly lower than the controls (P<0.05). The results of this study indicate that high fertilization rates of striped catfish eggs can be achieved using cryopreserved sperm when frozen at 10°C min^?1 in DMSO or DMA with either 0.9% NaCl or C‐F HBSS.     

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

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.