Studies on cryopreservation of eggs from rainbow trout (Salmo gairdneri) and coho salmon (Oncorhynchus kisutch)

The effect of pre-freezing treatments as well as freezing of inseminated, not water-activated eggs from rainbow trout Salmo gairdneri, and coho salmon, Oncorhynchus kisutch, was investigated in relation to survival and further development.Effects above freezing temperatures included: the temperature at insemination, viability of inseminated and unactivated eggs after storage, suitability of an incubation medium and the tolerance of eggs to various levels of the cryoprotectant dimethylsulfoxide (DMSO). Freezing experiments included: investigating the action of DMSO (0, 1, 2 mole) and the tolerance of coho eggs to temperatures between ?4.6 to ?30°C. Insemination temperatures between 0.5°C and 9.8°C (coho eggs) as well as incubation in an artifical medium (1-0°C) for 80 min (rainbow trout eggs) and 170 min (coho eggs) did not influence subsequent fertility. Storage of inseminated and unactivated rainbow trout eggs for 135 min and beyond reduced egg fertility. DMSO at 2 and 4 mole was detrimental to coho eggs (1-0°C). One mole DMSO had no (coho) or reduced (rainbow trout) influence on egg fertility when it was added gradually.In the presence of 1 mole DMSO most eggs remained unfrozen (67–89%) when kept for 10 min in frozen artificial medium (?4.6%) and 27–32% subsequently reached the eyed stage (control = 100). Further cooling (0.3°C/min) to ?10°C was still tolerated (62% unfrozen, 22% eyed eggs) but not to ?20°C (6% unfrozen, no development) and ?30°C (no survival). Use of 2 mole DMSO did not improve the results.     

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.     

Sperm cryopreservation of the noble Scallop Chlamys nobilis by a programmable freezing method: Effect of cryoprotectant

A study on Chlamys nobilis sperm cryopreservation by a programmable freezing method was conducted under laboratory condition. Four cryoprotectant agents (dimethyl sulfoxide [DMSO], methanol [MET], propanediol[PG] and ethylene glycol [EG]) and four concentrations (5%, 10%, 20% and 30%) were evaluated for their ability to retain sperm motility, movement characteristics and fertility. Results showed that cryopreserved sperm total motility produced by DMSO and MET at 5%, 10% and 20% were higher than other cryoprotectant treatment groups (CPA groups), as well as rapid sperm percentage. The curvilinear (VCL) and straight line (VSL) velocity produced by DMSO at 5% significantly higher than other CPA groups (p < 0.05), while no significant differences were found for average path (VAP) velocity. The lateral head displacement (ALH) in all CPA groups was similar and without significant difference (p > 0.05), as well as the beat‐cross frequency (BCF). A significant higher fertilization rate was produced in DMSO than that in MET at same concentration (p < 0.05), and no significant differences were found for differing concentrations of the same cryoprotectant (p > 0.05). Overall, 5%‐20% DMSO was more suitable for Chlamys nobilis sperm programmable cryopreservation when the calcium‐free Hanks’ balanced salt solution was used as the extender, and 10°C/min from 0°C to ?80°C was used as freezing rate. The findings presented in this study will benefit conservation programs for Chlamys nobilis.     

Effect of different permeable and non-permeable cryoprotectants on the hatching rate of rainbow trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>) embryos

The purpose of the present study was to investigate the toxicity of cryoprotectants on the hatching rate of rainbow trout (Oncorhynchus mykiss) embryos. Epiboly and first eye pigmentation stage embryos were immersed in six permeable cryoprotectants, dimethyl sulfoxide (DMSO), glycerol (Gly), methanol (MeOH), propylene glycol (PG), ethylene glycol (EG), and acetamide (Ac), in concentrations of 1–5 M for 5 or 10 min and two non-permeable cryoprotectants, sucrose (Suc) (10%, 15%, 20%) and polyvinyl pyrrolidone (PVP) (5%, 10%, 15%) for 5 min. The embryos were then washed and incubated until hatching occurred. The toxicity of the cryoprotectant was assessed by the hatching rate. The results illustrated that permeable cryoprotectant toxicity for rainbow trout embryos increased in the order of PG < DMSO < MeOH < Gly < EG < Ac. The hatching rate of the embryos treated with permeable cryoprotectants decreased (P < 0.05) with increased concentration and duration of exposure. There were no significant decreases in hatching rate of embryos treated with sucrose and PVP with the increase of concentration; sucrose had higher hatching rates than PVP. Rainbow trout embryos at first eye pigmentation stage exhibited greater tolerance to cryoprotectants than embryos at epiboly stage.     

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.     

Factors Affecting Cryopreservation Efficiency and Enzyme Activity in Northern Pike,Esox lucius,Sperm

Abstract

Two freezing techniques (straws and pellets), three cryo-protectants (DMSO, glycerol, and DMA) in four concentrations, and several extenders were tested to determine their suitability for cryopreservation of northern pike, Esox iucius, sperm. Activity of aspar-tate aminotransferase (AspAT) and acid phosphatase (AcP) in cryo-preserved milt was determined. Fertilization ability of cryopreserved milt was affected by the freezing technique, by type and concentration of cryoprotectant, as well as by the kind of extender used. These factors also influenced AspAT and AcP activity assayed in cryopreserved sperm. Extender containing 0.6 M sucrose + 15% DMSO + 10% egg yolk was most suitable for cryopreservation of pike sperm in pellet form (90.5% of eyed eggs, as compared to control group, which was 89.1%).     

The effect of cryoprotectants on the motility and fertilizing capacity of cryopreserved African catfish Clarias gariepinus (Burchell 1822) sperm

The effect of six cryoprotectants was investigated on the cryopreservation of African catfish Clarias gariepinus (Burchell) sperm. Fructose (6%) solution buffered with NaHCO₃‐CO₂ was used as the diluent in the experiments. Glycerol (5–11%), ethylene glycol, methanol and propylene glycol (5–15%) and, finally, dimethyl sulphoxide (DMSO) and dimethylacetamide (DMA) (10%) were tested using various equilibration times (2–30 min). Sperm was frozen in 250‐μL straws in a programmable freezer (Cryocell‐15, BLS, Hungary) from 3 °C to ?4 °C at 4 °C/min and from ?4 °C to ?80°C at 11 °C/min. Thawing was carried out in a 40 °C water bath for 5 s. Fertilization and hatching trials were performed only with DMSO and DMA using 200 and 100 μL of diluted sperm (100 and 50 μL of pure sperm) and the dry and the wet fertilization methods. Ethylene glycol, glycerol, methanol and propylene glycol yielded poor results. An average post‐thaw motility rate of 44.0 ± 9.7% and 22.6 ± 18.1% was achieved after 10 min equilibration using DMSO and DMA respectively. Highest average fertilization (86.8 ± 3.1%) and hatching (67.1 ± 11.9%) rates were achieved with DMA and DMSO, respectively, 200 μL of diluted sperm and the wet fertilization technique. The use of cryoprotectants increased the percentage of malformed larvae compared with the control groups. We found that DMA at a 10% concentration was equally as suitable for the cryopreservation of African catfish sperm as DMSO.     

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.     

Cryopreservation of summer flounder,Paralichthys dentatus L., sperm

The summer flounder, Paralichthys dentatus L., is a high‐value species and considerable research has been conducted to determine practices conducive for its culture. As milt can be limited in this species, experiments were conducted to develop a practical sperm cryopreservation protocol for hatchery use. Two dilution ratios (1:2 and 1:4; sperm:extender), 2 diluents (saline and sucrose‐based), 2 cryoprotectants (10% DMSO and 12% glycerol) and 3 freezing rates (?5, ?10 and ?15°C min^?1) were evaluated using differential staining to assess post‐thaw sperm survival. Seven combinations of the factors examined reduced post‐thaw viability by less than 30%. The average viability of sperm from fresh, pooled flounder milt (67.2 ± 2.9%) was not different from that of thawed milt diluted 1:4 with sucrose diluent (10% DMSO) frozen at ?5°C min^?1 (38.4 ± 7.7%) and fertilization and hatch success were not different in trials using fresh or thawed, cryopreserved sperm. From these experiments a practical sperm cryopreservation method was developed, but further refinement of the freezing protocol is necessary to optimize results.     

Cryopreservation of Paddlefish Polyodon spathula Milt

Abstract.— A practical procedure for cryopreserving milt of paddlefish Polyodon sparhula was developed to obtain thawed spermatozoa that would fertilize eggs and permit hatching of normal larvae. Milt was mixed with a cryoprotectant medium containing DMSO (2.4 M) in a ratio of 3:1(milt: medium; final concentration of DMSO 0.6 M), stored in 5.0-mL freezing straws, and frozen in dry ice (15 min) and then in liquid nitrogen. A total of three replicates were made; the milt of a different male was used in each replicate. Motility of the thawed spermatozoa decreased to 50%-25% as compared to 100% motility of the fresh (control) spermatozoa. Hatching of paddlefish (16.3 ± 2.2%) from eggs fertilized with thawed spermatozoa was significantly lower ( P ≤ 0.01) than the hatch rate (90.8 ± 2.5%) for the control. It was suggested that an increase in viable motile spermatozoa to egg would result in better fertilization and hatching of paddlefish.     

Cryopreservation of silver barb Puntius gonionotus (Bleeker) spermatozoa: effect of extender composition,cryoprotective agents and freezing rate on their postthawing fertilization ability

The effects of extender composition, cryoprotectant concentration and freezing and thawing on the fertilization efficiency of cryopreserved spermatozoa of Puntius gonionotus were evaluated. Computer‐aided motility analysis of semen was conducted to check the suitability of spermatozoa for cryopreservation after mixing with different extenders and cryoprotective agents (CPAs). Extender‐4 with an osmolality 260 mOsmol kg⁻¹and pH 7.6 was used for the cryopreservation study. Among the CPAs, dimethyl sulphoxide (DMSO) was least toxic and more than 60% fertilization was achieved when used at 1.4 M at 0 °C for 10 and 30 min, whereas the toxicity of all CPAs to spermatozoa was evident when tested at 30 °C. Semen frozen at −16 °C min⁻¹ with 1.4 M DMSO showed 70% fertilization, which was significantly higher (P<0.05) than other freezing rates. Samples thawed at 35 °C water showed a fertilization rate comparable with that of fresh semen. Computer‐assisted semen analysis of fresh and frozen semen after thawing showed variations in different types of motility in spermatozoa and in their class. There was no significant difference in motility before or after cryopreservation; however, significant differences could be observed in the average path velocity (VAP), straight line velocity (VSL) and curve linear velocity (VCL). Semen of silver barb could be cryopreserved with extender‐4 by addition of 1.4 M DMSO to a final cryopreservation medium (MED 2) cooled at a rate of −16 °C min⁻¹, stored in liquid nitrogen (−196 °C) and utilized after thawing at 35±2 °C.     

Cryopreservation of trochophore larvae from the hard clam Mercenaria mercenaria: Evaluation of the cryoprotectant toxicity,cooling rate and thawing temperature

Aquaculture of hard clams Mercenaria mercenaria is a $65 million industry along the east coast and Gulf of Mexico coast in the United States. The goal of this study was to develop a preliminary protocol to cryopreserve trochophore larvae of hard clams. The objectives were to evaluate the: 1) toxicity of cryoprotectants, including dimethyl sulfoxide (DMSO), propylene glycol, ethylene glycol and glycerol, at 5, 10, 15 and 20% for exposure time of 1, 15, 30, 45, 60 and 75 min; 2) effects of cooling rates (5, 10, 20 and 30°C/min for the first trial; and 1, 3 and 5°C/min for second trial from 4 to ?80°C), thawing temperature (30, 40 and 50°C) and their interactions on post‐thaw viability. A basic protocol was concluded as: 15‐hr trochophore larvae mixed with DMSO or propylene glycol (5, 10%), equilibrated for 15 min, cooled in a programmable freezer from 4 to ?80°C at a cooling rate of 5°C/min and thawed at 50°C for 6 s. With this protocol, the immediate post‐thaw trochophore survival was 23 ± 14%, and survival to D‐stage was 27 ± 14%. This is the first report on larval cryopreservation in the hard clam and would have application for genetic breeding and seed production.     

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.     

Cryopreservation of scaly carp (Cyprinus carpio) sperm: effect of different cryoprotectant concentrations on post-thaw motility, fertilization and hatching success of embryos

The aim of the present study was to determine the effect of various cryoprotectants on post-thaw sperm quality and fertilizing capacity of cryopreserved scaly carp (Cyprinus carpio) semen. The present study focused on freezing of scaly carp sperm utilizing a practical and inexpensive protocol for aquaculture. Semen was diluted with Kurokura’s extender composing 3.6 g/l NaCl, 10 g/l KCl, 0.22 g/l CaCl₂, 0.08 g/l MgCl₂ and 0.2 g/l NaHCO₃. The extender contained three different cryoprotectants (DMSO, DMA and egg yolk) at ratios of 5, 10 and 15 %. Semen was placed into 0.25-ml straws and exposed to liquid nitrogen vapor (?120 °C) using an insulated box with an adjustable tray for 10 min and then plunged into liquid nitrogen (?196 °C) tank. The thawing process was performed in a water bath at 40 °C for 10 s. The results indicated that type of cryoprotectants and their concentrations are rather effective in scaly carp sperm cryopreservation on post-thaw sperm quality, while they are very important in order to obtain high fertilization rates. The highest fertilization rate was determined as 96.4 ± 0.15 % with 15 % egg yolk, while the highest hatching rate was determined as 99.3 ± 0.80 with 15 % DMA. In conclusion, the applied cryopreservation method for scaly carp sperm is suitable to fertilize high amounts of eggs.     

不同冻存液对长江鲟鳍条细胞冷冻效果的影响

为了探究不同浓度配比冻存液对长江鲟(Acipenser dabryanus)鳍条细胞(Dabry′s sturgeon fin-derived cells,DSFCs)冷冻效果的影响,选择传代培养处于对数生长期的DSFCs,将第8代DSFCs置于液氮(-196℃)中冻存。根据冻存液中培养基(MEM)、胎牛血清(Fetal bovine serum,FBS)及二甲基亚砜(Dimethyl sulfoxide,DMSO)的配比不同,分为8个实验组:①含不同浓度DMSO配比实验组:5%DMSO+45%MEM+50%FBS,10%DMSO+40%MEM+50%FBS,20%DMSO+30%MEM+50%FBS;②含不同浓度FBS配比实验组:10%DMSO+90%FBS+0%MEM,10%DMSO+70%FBS+20%MEM,10%DMSO+50%FBS+40%MEM,10%DMSO+30%FBS+60%MEM,10%DMSO+10%FBS+80%MEM,对各组分采用形态学观察、CCK-8法、流式细胞仪检测细胞凋亡和细胞周期,综合分析不同冻存液组分对DSFCs冻存后活力的影响。结果显示,不同浓度DMSO配比实验组中,DMSO的浓度为20%时,细胞复苏后难以贴壁,存活率下降非常明显,仅为38.3%;而5%和10%DMSO细胞生长状态良好,存活率分别为80.2%和78.7%。不同浓度FBS配比实验组中,90%、30%和10%实验组细胞活力显著高于70%和50%实验组。FBS浓度为30%和10%的冻存组细胞凋亡率显著高于90%的冻存组(8.24%,15.35%vs 3.54%),3组之间相互比较有明显差异(P<0.05);细胞周期结果显示,与FBS浓度为90%的冻存组比较,FBS浓度为30%和10%冻存组G 0/G 1期细胞比例均明显增加(P<0.05),S期和G 2+M期细胞比例均显著降低(P<0.05),FBS浓度30%与10%冻存组比较差异不明显(P>0.05)。研究表明,长江鲟细胞的长期冷冻保存宜采用稍低浓度的DMSO(5%~10%)和高浓度的FBS。     

Sperm cryopreservation of silver barb (Barbodes gonionotus): cryoprotectants,cooling rate and storage time on sperm quality

Effectiveness and efficiency of frozen sperm on fertilization and hatching success of eggs from silver barb was examined in relation to cryoprotectants, freezing rate and storage period. Sperm was diluted in calcium‐free Hank's balanced salt solution, equilibrated with dimethylsulphoxide (DMSO), propylene glycol, sucrose or methanol at 5%, 10%, 15% or 20% final concentrations, and frozen in 250‐μL straws using a one‐step freezing procedure (1, 5 and 8°C min^?1 from 25 to ?40°C). Highest post‐thaw sperm motility was found from a treatment using 10% DMSO and 5°C min^?1 (82.2 ± 2.1%), similar to that of 10% DMSO and 8°C min^?1 (87.8 ± 3.2%). Post‐thaw motility of sperm frozen at 5 or 8°C min^?1 was significantly higher than 1°C min^?1. Relative sperm motility declined significantly after 10 months of cryostorage while viability did not change during a 12‐month cryostorage. Average fertilization rates of sperm after 1 and 4 months of storage were 64.5 ± 4.6% and 61.3 ± 3.4%, respectively, similar to those of fresh sperm (69.6–72.3%). Hatching rates of cryopreserved sperm (45.4–51.2%) were similar to those of fresh sperm (51.8–57.8%). This study developed suitable methods for cryopreservation of silver barb sperm that can be used to facilitate hatchery operation.     

Cryopreservation of male and female gonial cells by vitrification in the critically endangered cyprinid honmoroko <Emphasis Type="Italic">Gnathopogon caerulescens</Emphasis>

We investigated the feasibility of cryopreservation of spermatogonia and oogonia in the critically endangered cyprinid honmoroko Gnathopogon caerulescens using slow-cooling (freezing) and rapid-cooling (vitrification) methods. Initially, we examined the testicular cell toxicities and glass-forming properties of the five cryoprotectants: ethylene glycol (EG), glycerol (GC), dimethyl sulfoxide (DMSO), propylene glycol (PG), and 1,3-butylene glycol (BG), and we determined cryoprotectant concentrations that are suitable for freezing and vitrification solutions, respectively. Subsequently, we prepared the freezing solutions of EG, GC, DMSO, PG, and BG at 3, 2, 3, 2, and 2 M and vitrification solutions at 7, 6, 5, 5, and 4 M, respectively. Following the cryopreservation of the testicular cells mainly containing early-stage spermatogenic cells (e.g., spermatogonia and primary spermatocytes), cells were cultured for 7 days and immunochemically stained against germ cell marker protein Vasa. Areas occupied by Vasa-positive cells indicated that vitrification led to better survival of germ cells than the freezing method, and the best result was obtained with 5 M PG, about 50% recovery of germ cells following vitrification. In the case of ovarian cells containing oogonia and stage I, II, and IIIa oocytes, vitrification with 5 M DMSO resulted the best survival of oogonia, with equivalent cell numbers to those cultured without vitrification. The present data suggest that male and female gonial cells of the endangered species G. caerulescens can be efficiently cryopreserved using suitable cryoprotectants for spermatogonia and oogonia, respectively.     

Evaluation of Commercial‐scale Approaches for Cryopreservation of White Crappie,Pomoxis annularis,Sperm

Crappie, Pomoxis spp., are popular game fish throughout North America and are produced by public and private hatcheries. However, production is limited by a lack of information on tank culture and induced spawning methods. Development of techniques for storage of sperm and in vitro fertilization would increase flexibility in spawning. Therefore, techniques for sperm cryopreservation were examined in white crappie, Pomoxis annularis. Sperm from adult wild white crappie were used to evaluate sperm extender, cryoprotectant agent and concentration, and cooling technique based on post‐thaw sperm motility. Percent egg fertilization was also compared between sperm stored in the two best cryopreservation protocols and two different osmotic activator solutions. Sperm were cryopreserved using treatment combinations of two extenders (350 mOsmol/kg Hanks' balanced salt solution [HBSS] and 350 mOsmol/kg Ca²⁺free HBSS) and two cryoprotectants (dimethyl sulfoxide [DMSO] and methanol) at concentrations of 5, 10, and 15% that were cooled at four different rates: 5, 10, 20, and 40 C/min. Post‐thaw sperm motility and fertilization rates indicated white crappie sperm can be cryopreserved using either extender, cryoprotectants of either 5% DMSO or 10% methanol, and cooling at 40 C/min. A follow‐up experiment demonstrated sperm in suspensions on ice retained viability after overnight transport.     

Motility parameters of perch spermatozoa (Perca fluviatilis L.) with cryoprotectors addition

The addition of cryoprotectants during the freezing of semen in liquid nitrogen protects spermatozoa from the negative influence of freezing. Every species needs an appropriate cryoprotectant that has to be experimentally selected. Semen obtained from five perches was diluted with the Kobayashi buffer solution at 1:9 ratio. To determine the influence of cryoprotectants on spermatozoa motility parameters, the same type of buffer solution was applied with the addition of methanol, dimethyl sulfoxide (DMSO) and dimethylacetamide (DMA) using the concentration of 10, 5, 2.5 %, respectively, glycerol (15; 7.5 %), sucrose and trehalose (0.45; 0.225; 0.113 M). After the preparation of such tests, parameters of spermatozoa motility were measured, using the CASA system (Image House CRISMAS Company Ltd.). Among used cryoprotectants, methanol did not cause any effect on the sperm motility parameters. The lowest percentage concentrations of DMA, DMSO, glycerol, sucrose and trehalose did not significantly influence the percentage of motile spermatozoa. Higher concentrations of these compounds considerably lowered all motility parameters. As for glycerol and saccharides, their addition resulted in the lowering of the spermatozoa motility possibly due to a higher viscosity of the solution. However, DMA and DMSO were most probably toxic to perch sperm cells. The obtained results indicate that the best cryoprotectant to be used with perch spermatozoa is methanol.     

Studies on the semen biology and sperm cryopreservation in the sterlet, Acipenser ruthenus L.

The present study investigated motility, acrosome reaction, fertility and cryobiological parameters of the semen of the sterlet, Acipenser ruthenus L. Sperm motility persisted for about 4 min in water, and the main swimming type was the linear motion. Motility was prolonged at osmolalities of 12.5 mosmol kg^?1 and in the presence of magnesium ions, while calcium had no effect. Also a pH in the range of 7.0–9.0 had no effect on ` motility. At osmolalities of 25–50 mosmol kg^?1 the sperm motility was partly inhibited, at osmolalities of 100 mosmol kg^?1, completely and irreversibly. In 50 mosmol kg^?1 solutions with 2.5–5 mM L^?1 KCl the motility inhibition was total, but reversible. The acrosome reaction was not induced by one of the described solutions, but the percentage of spermatozoa with reacted acrosomes was low (<20%) and highly variable in all experiments. The optimal extender base for cryopreservation was a solution consisting of 50 mM L^?1 NaCl, 5 mM L^?1 KCl, 10 mM L^?1 Tris (pH 8.5). From the tested cryoprotectants only dimethylsulphoxide (DMSO) and methanol provided sufficient cryoprotection. After freezing and thawing, the motility rates and swimming velocities were higher with DMSO than with methanol. However, the fertility was very significantly reduced with DMSO (10.3±0.5%) while with methanol fertilization rates in a similar range (32.7±4.4%) as with fresh semen (33.90±0.8%) could be obtained. Optimal freezing conditions for sterlet semen were in the vapour of liquid nitrogen 3–5 cm (?95°C to ?85°C) above its surface, the optimal thawing conditions at 25°C for 30 s. The acrosome reaction was not induced by these cryopreservation protocols.