Systematic factor optimization for cryopreservation of shipped sperm samples of diploid Pacific Oysters, Crassostrea gigas

Despite some 26 published reports addressing oyster sperm cryopreservation, systematic factor optimization is lacking, and sperm cryopreservation has not yet found application in aquaculture on a commercial scale. In this study, the effects of cooling rate, single or combined cryoprotectants at various concentrations, equilibration time (exposure to cryoprotectant), straw size, and cooling method were evaluated for protocol optimization of shipped sperm samples from diploid oysters. Evaluation of cooling rates revealed an optimal rate of 5 degrees C/min to -30 degrees C followed by cooling at 45 degrees C/min to -80 degrees C before plunging into liquid nitrogen. Screening of single or combined cryoprotectants at various concentrations suggested that a low concentration (2%) of polyethylene glycol (FW 200) was effective in retaining post-thaw motility and fertilizing capability when combined with permeating cryoprotetcants such as dimethyl sulfoxide (DMSO), methanol (MeOH), and propylene glycol (P-glycol). However, polyethylene glycol alone was not as effective as MeOH, DMSO, and P-glycol when using the same methods. The highest post-thaw motility (70%) and percent fertilization (98%) were obtained for samples cryopreserved with 6% MeOH. However, this does not exclude other cryoprotectants such as DMSO or P-glycol identified as effective agents in other studies. There was no significant difference in post-thaw motility between straw sizes of 0.25- and 0.5-ml. Equilibration time (exposure to cryoprotectant) of 60 min could be beneficial when the cryoprotectant concentration is low and solution is added in a step-wise fashion at low temperature. Differences in post-thaw sperm quality (e.g., motility or percent fertilization) among individual males were evident in this research. As a consequence, a generalized classification describing males with different tolerances (broad, intermediate, and narrow) to cryopreservation was developed. This classification could be applied to strain or species differences in tolerances to the cryopreservation process. The present study demonstrated that oyster sperm could be collected and shipped chilled to another facility for cryopreservation, and that it could be shipped back to the hatchery for fertilization performed at a production scale yielding live larvae with >90% fertilization. Given the existence of facilities for commercial-scale cryopreservation of dairy bull sperm, the methods developed in the present study for oysters provide a template for the potential commercialization of cryopreserved sperm in aquatic species.     

Post-thawed motility and fertility from Atlantic salmon (Salmo salar L.) sperm frozen with four cryodiluents in straws or pellets

The cryopreservation of salmonid sperm is a complex process involving the interplay of many factors. Although cryopreservation protocols can be evaluated through a range of responses at various stages in the process, the number of progeny is the ultimate indicator of success. We compared reproductive success from freezing Atlantic salmon (Salmo salar L.) sperm using the eight combinations of (1) the penetrating cryoprotectants, 10% dimethyl sulfoxide (DMSO) or methanol (MeOH); (2) the nonpenetrating cryoprotectants glucose (0.3 M) or sucrose (0.6 M), and freezing in 0.1 mL pellets or 0.25 mL straws. All cryodiluents were supplemented with 10% (v/v) of hen's egg yolk. Response variables were the percentage and degree of motility of thawed and activated sperm using computer assisted sperm analysis (CASA), and rates of eyed embryos, hatch and egg sac larvae. Growth rates of alevins were assessed to two months post hatch. Atlantic salmon milt cryopreserved in straws had higher spermatozoa motility and fertilization success than milt cryopreserved in pellets (P < 0.05). Type of sugar tested did not significantly affect the response variables. In the MeOH treatment, thawed spermatozoa achieved higher speed and a higher fertilization rate evaluated at the eyed embryo stage than spermatozoa subjected to the DMSO treatment. Higher mortality rate (especially before hatching) of MeOH offspring than DMSO offspring led to equal numbers of progeny for the two treatments from the swimming stage to the end of the study. Moreover, during feeding fish from the MeOH group produced significantly lower weight larvae than the DMSO and control groups. Even so, the weight of the MeOH group was satisfactory. Length and the condition factors did not differ significantly among the larvae groups. Significant positive correlations were found between fertilization success (measured in number of eyed eggs) and both motility (rs = 0.81), and velocity (rs = 0.49). Freezing in straws gave betters results than freezing in pellets for cryopreservation of salmon milt; whereas type of sugar tested (glucose vs sucrose) did not have significant effects. Penetrating cryoprotectants DMSO and MeOH differed in their effect on post-thawed sperm velocity, fertilization rate and mortality rate of progeny, suggesting the need for further research on the influence of these cryoprotectants on frozen sperm and and post-fertilization devopmental processes.     

Viability and fertilizing capacity of cryopreserved sperm from three North American acipenseriform species: a retrospective study

Populations of sturgeon across the globe are threatened due to unregulated harvest and habitat loss, and the status varies among species across North America. Ready access to viable and functional sperm would contribute to recovery programmes for these species. In this study, we examined the motility, viability (cell membrane integrity) of cryopreserved sperm from three North American acipenseriform species and fertilizing capacity. Milt samples were collected from captive shortnose sturgeon (Acipenser brevirostrum), wild paddlefish (Polyodon spathula) and pallid sturgeon (Scaphirhynchus albus) and cryopreserved using combinations of Modified Tsvetkova’s (MT) extender, Original Tsvetkova’s extender, and modified Hanks’ balanced salt solution, along with the cryoprotectants methanol (MeOH) or dimethyl sulfoxide (DMSO). A dual‐staining technique using the fluorescent stains SYBR‐14 and propidium iodide was employed with flow cytometry to determine the percentages of spermatozoa that were viable by virtue of having intact membranes. The percentage of viable spermatozoa ranged from 5% to 12% in shortnose sturgeon, 30–59% in paddlefish, and 44–58% in pallid sturgeon. In the first experiment with shortnose sturgeon sperm, methanol allowed for higher values for dependent variables than did DMSO, and sperm viability generally correlated with post‐thaw motility. However, fertilization rate, neurulation, or hatching rates were independent from these factors. In the second experiment with shortnose sturgeon, 5% MeOH combined with MT yielded higher values for all parameters tested than the other combinations: viability was correlated with motility, fertilization rate, and hatching rate. Overall, viability and post‐thaw motility was not affected by the use of hyperosmotic extenders (OT) or cryoprotectants (DMSO), but their use decreased fertilization percentages. For paddlefish sperm (experiment 3), MT combined with 10% MeOH was clearly a good choice for cryopreservation; viability and motility results were correlated, but independent of fertilization. For pallid sturgeon sperm (experiment 4), MT with 5–10% MeOH showed significantly higher sperm quality and fertilization parameters. Membrane integrity can be used as a predictor of fertilization by cryopreserved sperm, however additional sperm quality parameters, supplementary to motility and membrane integrity, would be useful in the refining and optimizing cryopreservation protocols with acipenseriform sperm.     

Cryopreservation of flounder (Paralichthys olivaceus) sperm with a practical methodology

A simple and convenient protocol for the cryopreservation of the flounder (Paralichthys olivaceus) sperm was established for "on the spot" cryopreservation of large quantities of semen. The use of three cryoprotectants, dimethyl sulphoxide (DMSO), glycerol (Gly) and methanol was tested in the method. The percentage of motile sperm present in semen after it had been frozen and thawed in the presence of DMSO, Gly or methanol was 60.5+/-3.6, 79.17+/-4.5 and 13.25+/-4.7%, respectively. The fertilization rates of this sperm were 67.06+/-15.1, 76.20+/-10.0 and 44.93+/-22.6%, while the hatching rates of eggs fertilized with this sperm were 37.40+/-8.3, 48.18+/-25.7 and 23.35+/-10.8%, respectively. It was found that Gly and DMSO were better cryoprotectants than methanol, with Gly giving the best overall results. Under scanning electron microscopy, it could be seen that while the majority of the frozen-thawed sperm remained morphologically normal, some exhibited lost or dilated mitochondria, swollen mid-pieces, broken tails, or damaged cell membrane, which probably caused the decrease in motility and fertility of the frozen-thawed sperm.     

Effect of glycerol and dimethyl sulfoxide on cryopreservation of rhesus monkey (Macaca mulatta) sperm

Glycerol and dimethyl sulfoxide (DMSO) are widely used as penetrating cryoprotectants in the freezing of sperm, and various concentrations are applied in different species and laboratories. The present study aimed to examine the effect of these two cryoprotectants at different concentrations (2%, 5%, 10%, and 15% glycerol or DMSO) on rhesus monkey sperm cryopreservation. The results showed that the highest recovery of post-thaw sperm motility, and plasma membrane and acrosome integrity was achieved when the sperm was frozen with 5% glycerol. Spermatozoa cryopreserved with 15% DMSO showed the lowest post-thaw sperm motility, and spermatozoa cryopreserved with 15% glycerol and 15% DMSO showed the lowest plasma membrane integrity among the eight groups. The results achieved with 5% glycerol were significantly better for all parameters than those obtained with 5% DMSO. The functional cryosurvival of sperm frozen with 5% glycerol was further assessed by in vitro fertilization (IVF). Overall, 85.7% of the oocytes were successfully fertilized, and 51.4% and 5.7% of the resulting zygotes developed into morulae and blastocysts, respectively. The results indicate that the type and concentration of the penetrating cryoprotectant used can greatly affect the survival of rhesus monkey sperm after it is frozen and thawed. The suitable glycerol level for rhesus monkey sperm freezing is 5%, and DMSO is not suitable for rhesus monkey sperm cryopreservation.     

Gene banking of the neotropical fish Leporinus obtusidens (Valenciennes, 1836): a protocol to freeze its sperm in the field

A practical sperm cryopreservation protocol using a dry-shipper and a diluent of simple composition is described for the neotropical fish Leporinus obtusidens (Valenciennes, 1836). The cooling rate of the dry-shipper and its period of useful time, established under laboratory conditions, were respectively 25.7-30.8 degrees C/min (between 0 and -60 degrees C) and 9 days after charging. Sperm donors were selected on the basis of their hyperemic genital papilla and the ability to ooze milt under gentle manual pressure, during the reproductive months of November to January. Milt volume (1.3+/-0.3 mL; n=9 fish), fresh sperm motility rate (93.3+/-2.5%; n=6 fish), and sperm concentration (10.9+/-3.0 x 10(9)spermatozoa/mL of milt) were obtained. The sperm cryopreservation experiments were conducted with the following cryoprotectants (all at 10%, before mixing with milt): dimethyl sulphoxide (DMSO; n=10 fish), methanol (n=6 fish), propanediol (n=6 fish) and ethylene glycol (n=5 fish). Glucose (5%) and hen's egg yolk (10%) made up the diluents containing DMSO, ethylene glycol or propanediol. Milk powder (10%) replaced hen's egg yolk in the diluent containing methanol. Distilled water (up to 100%) completed the diluent solutions. Milt freezing (in 0.5-mL straws) was performed in the dry-shipper after 1:5 (milt:diluent) dilution. Thawed sperm cryopreserved in DMSO-containing diluent and activated by 119 mM NaHCO(3) gave the highest motility rate (62+/-14%). The fertilizing capacity of L. obtusidens sperm was tested using the combination of DMSO-containing diluent as the cryoprotectant and 119 mM NaHCO(3) as the activating solution. Oocytes were obtained from artificial spawning and fertilized with different proportions of spermatozoa. The greatest rate of fertilization (74%) occurred when the ratio of about 112,000 motile spermatozoa:oocyte was used. Thus, a protocol to freeze L. obtusidens sperm can be elaborated as follows. Milt (<1.5 mL fish(-1)) was readily available only in November to January; a simple solution, composed of 10% DMSO (concentration before adding milt), 5% glucose, and 10% hen yolk egg, in distilled water, was used as sperm diluent; cooling rate of 25-30 degrees C/min, yielded in a portable dry-shipper, was adequate to freeze diluted milt (1:5; milt:diluent), in 5-mL straws; about 112,000 thawed motile spermatozoa:oocyte activated by 119 mM NaHCO(3) assured a fertilization rate of 74%.     

Cryopreservation of European eel (Anguilla anguilla) sperm using different extenders and cryoprotectants. Short communication

Experiments were carried out on the sperm cryopreservation of artificially induced eels. The effects of several extenders and two cryoprotectants on the motility of spermatozoa were investigated. The highest post-thaw motility was observed with the combination of Tanaka's extender and DMSO as cryoprotectant. Further dilution after thawing resulted in complete loss of motility in samples frozen in presence of DMSO while sperm frozen with methanol as cryoprotectant retained its motility after further dilution.     

Quality control of refrigerated and cryopreserved semen using computer-assisted sperm analysis (CASA), viable staining and standardized fertilization in African catfish (Clarias gariepinus)

A new integrated approach including computer-assisted sperm analysis (CASA), viability staining and fertilization was used to study the quality of cryodiluents used in fish sperm cryopreservation. As an example the sperm quality of an African catfish, Clarias gariepinus (Burchell, 1822), was assessed by its fertilizing ability, motility and viability at day 0 (fresh), after 2 days' storage at 4 degreesC and after 2 days, 5 months and 10 months frozen at -196 degreesC using solutions containing dimethyl sulphoxide (DMSO) or glycerol as permeating cryoprotectants. Four of the best freezing solutions were used, namely, Steyn's extender (S1, S4) and Mounib's extender (M3, M4) associating 10% hen's egg yolk. Progressive sperm movement measured by CASA and expressed by the straight line velocity (VSL), the average path velocity (VAP) and the curvilinear velocity (VCL) was highly correlated with hatching rates obtained from fertilization using minimal sperm:egg ratios. After 2 days, the motility of spermatozoa frozen with DMSO and 10% egg yolk had deteriorated less than that of spermatozoa stored at 4 degreesC. Post-thaw hatching rates reflected the post-thaw sperm viability, which was cryodiluent dependent: 14.9+/-2.0% (S4), 17.0+/-4.2% (S1), 25.9+/-3.7% (M4) and 52.1+/-3.4% (M3) after 5 months of cryopreservation. The percent motility of 10-months-frozen spermatozoa was high in M3 (70.7+/-11.4%) and M4 (64.0+/-2.0%) cryoprotected sperm when measured between 5 and 20 sec after activation, but decreased rapidly to 24.3+/-8.3% (M3) and 23.0+/-9.0% (M4) between 21 and 35 sec after activation. Mounib's extender (M3, M4) provided the best cryoprotection to the spermatozoa for all post-thaw sperm quality measurements and at all freezing durations. Sperm motility was positively related to fertility. Our method will make it possible to develop even better extenders and cryoprotectants.     

Osmotic tolerance and membrane permeability characteristics of rhesus monkey (Macaca mulatta) spermatozoa

Biophysical characteristics of the plasma membrane, such as osmotic sensitivity and water and cryoprotectant permeability are important determinants of the function of spermatozoa after cryopreservation. A series of experiments was conducted with rhesus macaque spermatozoa at 23 degrees C to determine their: (1) cell volume and osmotically inactive fraction of the cell volume; (2) permeability coefficients for water and the cryoprotectants dimethyl sulfoxide, glycerol, propylene glycol, and ethylene glycol; (3) tolerance to anisosmotic conditions; and (4) motility after a one step addition and removal of the four cryoprotectants. An electronic particle counter and computer aided semen analysis were used to determine the cell volume and permeability coefficients, and motility, respectively. Rhesus spermatozoa isosmotic cell volume was 27.7+/-3.0 microm3 (mean+/-SEM) with an osmotically inactive cell fraction of 51%. Hydraulic conductivity in the presence of dimethyl sulfoxide, glycerol, propylene glycol, and ethylene glycol was 1.09+/-0.30, 0.912+/-0.27, 1.53+/-0.53, and 1.94+/-0.47 microm/min/atm, respectively. Cryoprotectant permeability was 1.39+/-0.31, 2.21+/-0.32, 3.38+/-0.63, and 6.07+/-1.1 (x10(-3)cm/min), respectively. Rhesus sperm tolerated all hyposmotic exposures. However, greater than 70% motility loss was observed after exposure to solutions of 600 mOsm and higher. A one step addition and removal of all four cryoprotectants did not cause significant motility loss. These data suggest that rhesus sperm are tolerant to hyposmotic conditions, and ethylene glycol may be the most appropriate cryoprotectant for rhesus sperm cryopreservation, as it has the highest permeability coefficient of the tested cryoprotectants.     

Flow cytometry and ultrastructure of cryopreserved red seabream (Pagrus major) sperm

The objectives were to assess motility, fertilizing capacity, structural integrity, and mitochondrial function in fresh versus frozen-thawed (15% DMSO was used as a cryoprotectant) sperm from red seabream (Pagrus major). Mean (+/-S.D.) rates of motility, fertilization and hatching of frozen-thawed sperm were 81.0+/-5.4, 92.8+/-1.9, and 91.8+/-5.2%, respectively; for fresh sperm, they were 87.5+/-7.7, 95.8+/-2.4, and 93.8+/-4.2%. Although motility was lower in frozen-thawed versus fresh sperm (P<0.05), there was no effect (P>0.05) of cryopreservation on fertilization or hatching. Based on scanning and transmission electron microscopy, 77.8+/-5.6% of fresh sperm had normal morphology, whereas for frozen-thawed sperm, 63.0+/-7.2% had normal morphology, 20.6+/-3.1% were slightly damaged (e.g. swelling or rupture of head, mid-piece and tail region as well as mitochondria), and 16.4+/-4.2% were severely damaged. Sperm were stained with propidium iodide and Rhodamine 123 to assess plasma membrane integrity and mitochondrial function, respectively, and examined with flow cytometry. For fresh sperm, 83.9% had an intact membrane and functional mitochondria, whereas for frozen-thawed sperm, 74.8% had an intact membrane and functional mitochondria, 12.7% had a damaged membrane, 9.9% had nonfunctional mitochondria, and 2.6% had both a damaged membrane and nonfunctional mitochondria. In conclusion, ultrastructure and flow cytometry were valuable for assessment of frozen-thawed sperm quality; cryopreservation damaged the sperm but fertilizing ability was not significantly decreased.     

Post-coital sperm recovery and cryopreservation in the Sumatran rhinoceros (Dicerorhinus sumatrensis) and application to gamete rescue in the African black rhinoceros (Diceros bicornis)

Sumatran rhinoceros (Dicerorhinus sumatrensis) sperm samples were collected from a post-copulatory female and characterized to determine their potential for sperm preservation and future use in artificial insemination. Five samples of acceptable quality from one male were used to compare the effect of two cryoprotectants (glycerol and dimethyl sulfoxide (DMSO)) and two post-thaw protocols (untreated and glass wool column) on sperm quality. The percentage of motile spermatozoa, sperm motility index (0-100) and sperm morphology were evaluated subjectively, and viability and acrosomal status were assessed using fluorescent markers. Evaluations of frozen-thawed spermatozoa were performed over a 6 h incubation interval. Post-coital semen samples (n = 5; 104.0 +/- 9.1 ml; 2.5 +/- 0.8 x 10(9) total spermatozoa; mean +/- SEM) exhibited a sperm motility index of 56.7 +/- 3.3, and contained 40.2 +/- 6.3%, 72.0 +/- 3.2% and 79.8 +/- 6.5% normal, viable and acrosome-intact spermatozoa, respectively. Glycerol and DMSO were equally effective as cryoprotectants and, regardless of post-thaw protocol, samples retained greater than 80% of all pre-freeze characteristic values. Processing semen samples through glass wool yielded higher quality samples, but only half the total number of motile spermatozoa compared with untreated samples. High values for pre-freeze sperm characteristics were also maintained after cryopreservation of epididymal spermatozoa from one black rhinoceros (Diceros bicornis) using the same protocol. In summary, Sumatran rhinoceros spermatozoa of moderate quality can be collected from post-copulatory females. Rhinoceros sperm samples show only slight reductions in quality after cryopreservation and thawing and have potential for use in artificial insemination.     

Successful fertilization of Varicorhinus macrolepis eggs with sperm subjected to two freeze-thaw cycles

Although sperm from several fish species have been successfully cryopreserved, few studies have been done in small and/or endangered species. The aim of the present work was to develop a method of freezing and refreezing Varicorhinus macrolepis semen in 1.8 mL cryovials. The effect of extenders and cryoprotectants on the motility of post-thaw sperm was examined. The motility of frozen-thawed sperm in extender D-15 was higher than that in MPRS and fish Ringer solution (P<0.05). Dimethyl sulfoxide (DMSO) and glycerol provided greater protection to sperm than methanol during freezing and thawing; the most effective concentration of DMSO and glycerol was 10%. The fertilization rate of frozen-thawed sperm was not significantly different from that of fresh sperm. Furthermore, mean (+/-S.D.) hatching rate did not differ significantly between frozen-thawed (82.7+/-12.4%) and fresh sperm (90.7+/-4.5%). Although frozen-thawed sperm that was immediately refrozen had 0% post-thaw motility, frozen semen that was refrozen after dilution with D-15 (containing DMSO at a ratio of 1:2) had post-thaw motility of 38.3+/-2.9%. Motility was lower for refrozen than for frozen sperm (P<0.05). Furthermore, fertilization and hatching rates of refrozen sperm were 42.9+/-6.7 and 34.1+/-10.5%, respectively, which were lower than that of fresh sperm (P<0.05).     

Development of a simplified and standardized protocol with potential for high-throughput for sperm cryopreservation in zebrafish Danio rerio

Sperm cryopreservation offers potential for long-term storage of genetic resources. However, the current protocols for zebrafish Danio rerio are cumbersome and poorly reproducible. Our objective was to facilitate adoption of cryopreservation by streamlining methods from sperm collection through thawing and use. First, sperm activation was evaluated, and motility was completely inhibited when osmolality of the extender was >/=295-300mOsmol/kg. To evaluate cryoprotectant toxicity, sperm were incubated with dimethyl sulfoxide (DMSO), N,N-dimethyl acetamide (DMA), methanol, or glycerol at 5, 10, and 15% concentrations. Based on motility, DMSO, DMA, and methanol (相似文献   

软鳍新光唇鱼精子的超低温冷冻保存

2011年,对软鳍新光唇鱼(Neolissochilus benasi)进行了精子超低温冷冻保存研究。以解冻后的精子活力为指标,采用稀释液D-15,设计不同的抗冻剂种类和浓度,以及不同的实验条件(包括冷冻体积、4℃平衡时间和解冻温度等)探索软鳍新光唇鱼精子的超低温冷冻保存方法。筛选出了适合软鳍新光唇鱼精子超低温冷冻保存的两种抗冻保护剂及其浓度分别为10%MeOH和15%EG,确定了精液与稀释液的最适稀释比例为1:7、4℃平衡时间区间为10～60min、冷冻体积为60μL,以及复苏方法为37℃水浴快速解冻30s。当鲜精活力为(62.33±2.05)%,综合以上最佳实验条件进行保存,解冻后精子的最高活力为(29.67±0.47)%,但效果不理想,不能达到广泛生产运用水平;产生这一结果,可能与异地保育物种的饲养管理有关。因此,在亲鱼培育管理中要最大限度地降低捕获诱发的压力,尽量提供适合的养殖条件。在珍稀鱼类异地保育时,繁殖用雄鱼的培育与雌鱼同等重要,是获得大量高质量仔鱼的关键。     

Cryopreservation of red snapper (Lutjanus argentimaculatus) sperm: Effect of cryoprotectants and cooling rates on sperm motility, sperm viability, and fertilization capacity

Sperm cryopreservation of red snapper (Lutjanus argentimaculatus) is essentially unexplored, although many species of the Lutjanidae family are considered to be high-value commercial species. The objective of this study was to develop a species-specific cryopreservation protocol for red snapper (L. argentimaculatus) sperm by optimizing cryoprotectants and cooling rates in the cryopreservation procedure. Ten cryoprotectants at four concentrations and two freezing protocols were examined in two separate experiments. In the first experiment, toxicity studies of dimethyl sulfoxide (DMSO), glycerol, propylene glycol (PG), ethylene glycol (EG), formamide, methanol, ethanol, sucrose, trehalose, and dimethylacetamide (DMA) on sperm motility were performed. Semen diluted 1:1 in Ringer solution were exposed to cryoprotectants at four final concentrations of 5%, 10%, 15%, or 20% for periods of 10, 20, 30, 40, 50, 60, 90, and 120 min at room temperature (25 °C). The cryoprotectants and concentrations that showed the least toxic effect on sperm motility were selected for cryopreservation trials. In the second experiment, selected cryoprotectants were then assessed for freezing capacity of sperm as follows: DMSO 5% and 10%, PG 5% and 10%, EG 5% and 10%, ethanol 5%, and methanol 5%. Semen was diluted 1:1 in Ringer solution and equilibrated with selected cryoprotectants for 10 min at room temperature. Sperm were frozen in a controlled-rate programmable freezer at four cooling rates of 3, 5, 10, and 12 °C/min from an initial temperature of 25 °C to final temperatures of −40 or −80 °C before plunging into liquid nitrogen. Sperm equilibrated in 10% DMSO and cooled at a rate of 10 °C/min to a final temperature of −80 °C had the highest motility (91.1 ± 2.2%) and viability (92.7 ± 2.3%) after thawing. The fertilization rate of frozen-thawed sperm (72.4 ± 2.4%) was not different (P > 0.05) from that of fresh sperm (75.5 ± 2.4%). This study apparently represents the first reported attempt for cryopreservation of L. argentimaculatus sperm.     

The effects of cryoprotectants on sperm motility of the Chinese pearl oyster,Pinctada fucata martensii

Cryopreservation has been widely employed to preserve genetic material of aquatic animals. Although of common use in bivalves, resulting effects due to the toxicity of the cryoprotectants dimethyl sulfoxide (DMSO), propanediol (PG), methanol (MET) and ethylene glycol (EG), upon sperm motility in the Chinese pearl oyster, Pinctada fucata martensii, has remained undocumented. This study endeavors to identify the least toxic among the effective cryoprotectant agents by observing and comparing their toxic effects on sperm motility under varying concentrations and duration of exposure. Sperm samples were exposed during controlled experiments, for 1, 3, 6, 9, 12 and 15 min durations, to each of the listed cryoprotectants at 5, 10, 15, and 20% (volume:volume) concentrations. Sperm motility was observed to diminish when exposed to all cryoprotectant solutions, and observations demonstrated that toxicity increased relative to both concentration and equilibration time. After 6 min of exposure to the cryoprotectants, sperm motility was seen to have diminished significantly in DMSO at just 5% concentration, and in MET, PG and EG at 10% concentrations, respectively (the values of the lowest observed effect concentrations). The relationship between the quantity of immotile sperm and the cryoprotectant concentration was described using the logarithmic regression equation. MET exhibited the lowest effective concentration required to inhibit sperm motility by 50% (EC₅₀), followed by EG, PG and DMSO, in order. Therefore, MET proved most toxic under the test conditions for sperm of P. fucata martensii, whereas DMSO, PG and EG were observed as comparatively safer, suggesting that DMSO, PG and EG warrant further study in the application of cryopreservation of Chinese P. fucata martensii sperm.     

Cryopreservation of yellowfin seabream (Acanthopagrus latus) spermatozoa (Teleost, Perciformes, Sparidae)

The effects of both osmolality and cation in the initiation of sperm motility were examined in yellowfin seabream, Acanthopagrus latus. Various factors involved in the cryopreservation of yellowfin seabream spermatozoa on motility are discussed. Extender containing only glucose proved to be a suitable medium for freezing yellowfin seabream spermatozoa to -196 degrees C. Glycerol seems to have a direct osmotic effect on yellowfin seabream sperm cells, and it induced sperm motility before freezing and during thawing. However, this exhausted the energy needed for sperm motility for fertilization. Dimethyl sulfoxide (DMSO) proved superior to ethylene glycerol, propylene glycerol, glycerol and methanol as a cryoprotectant. Prolonged equilibration time had a detrimental effect on both prefreezing and post-thawing sperm motility. The estimated optimum freezing rate was in the range of -20 to -154 degrees C/min. More frozen-thawed than fresh spermatozoa are required to achieve comparable fertilization rates.     

Effect of cryoprotectants on hatching rate of red seabream (Pagrus major) embryos

The objectives were to investigate the effect of cryoprotectants on the hatching rate of red seabream embryos. Heart-beat embryos were immersed in: five permeable cryoprotectants, dimethyl sulfoxide (DMSO), glycerol (Gly), methanol (MeOH), 1,2-propylene glycol (PG), and ethylene glycol (EG), in concentrations of 5-30% for 10, 30, or 60min; and two non-permeable cryoprotectants: polyvinylpyrrolidone (PVP), and sucrose (in concentrations of 5-20% for 10 or 30min). The embryos were then washed and incubated in filtered seawater until hatching occurred. The hatching rate of the embryos treated with permeable cryoprotectants decreased (P<0.05) with increased concentration and duration of exposure. In addition, PG was the least toxic permeable cryoprotectant, followed by DMSO and EG, whereas Gly and MeOH were the most toxic. At a concentration of 15% and 30min exposure, the hatching rate of the embryos immersed in PG was 93.3+/-7.0% (mean+/-S.D.), however, in DMSO, EG, Gly, and MeOH, it was 82.7+/-10.4, 22.0+/-5.7, 0.0+/-0.0, and 0.0+/-0.0%, respectively. Hatching rate of embryos treated with PVP decreased (P<0.05) with the increase of concentration and exposure time, whereas for embryos treated with sucrose, there was no significant decrease in comparison with the control at the concentrations used.     

Cryopreservation of sperm from the endangered formosan landlocked salmon (Oncorhynchus masou formosanus)

The Formosan landlocked salmon (Oncorhynchus masou formosanus) are at a high risk of extinction, and the sustained maintenance of the population will soon depend on aquaculture systems, which use cryopreservation of spermatozoa to increase genetic diversity. We investigated the effectiveness of dimethyl sulfoxide (DMSO), dimethyl-acetamide (DMA), and methanol as cryoprotectants in combination with 300 mM glucose as extender on the freezing of Formosan landlocked salmon spermatozoa. We also evaluated the morphological changes of Formosan landlocked salmon spermatozoa after their immediate dilution in the 300 mM glucose-DMSO extender and after freeze-thawing. The spermatozoa frozen with DMSO as a cryoprotectant showed significantly higher post-thaw motility and fertility than spermatozoa frozen with DMA or methanol. The fertilization capacity of frozen-thawed Formosan landlocked salmon was comparable to that of fresh spermatozoa. Intersubspecies fertilization trials between cryopreserved Formosan landlocked salmon spermatozoa and Amago salmon eggs showed high fertilization rates. Based on the findings, the potential value of using sperm bank to safeguard this endangered species is discussed.     

Effect of cryopreservant combinations on the motility and morphology of curimba (Prochilodus lineatus) sperm

This study investigated the application of intra- and extra-cellular cryoprotectant combinations on the quality of curimba Prochilodus lineatus semen subjected to cryopreservation. Semen treatments were tested with 8% DMSO or methanol as intracellular cryoprotectant, 5% egg yolk or lactose as extracellular cryoprotectant and 5% BTS. These cryoprotectant combinations are suitable for curimba but have not been tested at the lesser concentrations proposed or in combination with BTS. Semen samples collected from 19 curimbas were diluted into one of four cryoprotectant combinations: DMSO+yolk; DMSO+lactose; methanol+yolk; and methanol+lactose. After dilution, semen samples were cryopreserved in 0.5 mL straws for 10 days in a liquid nitrogen tank. Semen was thawed in a water bath at 60°C for 8s. We evaluated the quality of fresh, diluted (pre-freezing) and post-freezing semen according to sperm motility rate (%) and duration (s). Sperm morphology was also analyzed in thawed semen. Sperm motility rate decreased progressively after dilution and thawing. The motility rate in post-freezing semen was higher in the treatments using DMSO+lactose and methanol+yolk. Sperm motility duration in post-freezing sperm was greater in the treatments using methanol rather than DMSO as intracellular cryoprotectant, irrespective of the extracellular cryoprotectant used. Abnormality frequency in thawed sperm was less in semen treated with egg yolk than with lactose. Thus the use of methanol intracellular cryoprotectant is recommended along with yolk extracellular cryoprotectant in the cryopreservation process for curimba semen.