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1.
In this study, a non‐programmable freezing technique has been developed with the strip spawned blue mussel (Mytilus galloprovincialis) sperm. The key parameters optimized including (1) cryoprotectant agents (CPAs); (2) cooling temperature; (3) thawing temperature; (4) sugar and amino acid supplementation and (5) sperm to egg ratio. The fertilization rate and/or integrity of plasma membrane and acrosome were used as sperm quality assessment indicators. The highest post‐thaw sperm fertilization rate of 95% was achieved, when sperm were cryopreservated in 8% dimethyl sulfoxide at 7.8 cm above the liquid nitrogen surface and thawed in a 60°C seawater bath. The addition of glucose, sucrose or trehalose in dimethyl sulfoxide did not, whereas 0.8% glycine did significantly improve the post‐thaw sperm fertilization rates. The fluorescent evaluation has demonstrated that the addition of glycine significantly improved the post‐thaw sperm acrosome integrity, revealing a positive role of glycine in the improvement of post‐thaw sperm quality in blue mussels.  相似文献   

2.
This study investigated the effects of broodstock age (2‐ and 3‐yr‐old), and sperm collection time at the beginning, middle, and end of a natural spawning period, on the ability of sperm to tolerate cryopreservation in farmed greenlip abalone, Haliotis laevigata. The quality of sperm was assessed by motility, fertilization rate, plasma membrane integrity, mitochondrial membrane potential (MMP), and acrosome integrity (AI). The sperm collected at the middle of a natural spawning period had significantly higher quality than those collected at the beginning and the end of the spawning period in terms of plasma membrane integrity, MMP, and AI of fresh and post‐thaw sperm, post‐thaw sperm motility, and fertilization rate. No significant difference was found between 2‐ and 3‐yr‐old animals in most quality parameters evaluated. The results suggested that sperm collected during the middle of the spawning season should be used for cryopreservation. The efficiency of genetic improvement programs can be further enhanced by using sperm collected from 2‐yr‐old abalone which has a similar ability to tolerate cryopreservation as 3‐yr‐old counterparts.  相似文献   

3.
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.  相似文献   

4.
Experiments were carried out to develop an optimal cryopreservation protocol for tench sperm by testing the fertilizing capacity and motility parameters including progressive motility, curvilinear velocity (VCL) and linearity (LIN) of cryopreserved sperm. Three experiments were designed to this aim: first experiment where we tested the effects of two extenders (sugar‐based Grayling and ion‐based Kurokura 180) and two cryoprotectants (DMSO and methanol) on fertilization and hatching success; second where we tested the effect of cryoprotectant type (methanol or DMSO) in different concentrations (5%, 10% and 15%) on fertilization and hatching success; and third where we tested the effect of two cryoprotectants (methanol and DMSO) on sperm motility parameters (progressive motility, VCL and LIN) after 4 h post‐thaw storage (4°C). Sperm prepared with the sugar‐based Grayling extender displayed better fertilization and hatching rates independently of the applied cryoprotectant most likely due to glucose present which acted as an external cryoprotectant. Concerning cryoprotectant concentrations, the use of 10% methanol yielded the highest fertilization (85 ± 15%) and hatching (80 ± 13%) rates, which were significantly higher than in all other groups. During the post‐thaw storage time, 5% methanol, 10% methanol and 5% DMSO groups had significantly higher motility parameters than other groups and we observed no significant decline in any of the parameters during the storage time. Overall, we found that a sugar‐based extender in combination with methanol as cryoprotectant is suitable for the cryopreservation of tench sperm and allows storage of cryopreserved sperm for up to 4 h post thaw.  相似文献   

5.
We developed both a cryopreservation method for Japanese sea cucumber spermatozoa and an artificial fertilization method using post‐thaw spermatozoa. Twenty per cent dimethyl sulfoxide (DMSO), 16% foetal bovine serum, and 64% artificial seawater were suitable cryodiluent, and the diluent was pre‐cooled to 0°C. Semen was diluted with the solution and enclosed in a 250 μl straw, cooled to ?50°C at 10.4 ± 0.4°C/min, and immediately immersed in liquid nitrogen. Although this method showed the highest post‐thaw motility in all the conditions we examined, its post‐thaw motility was still less than approximately 15%. Artificial fertilization was carried out by adding post‐thaw semen with a cryodiluent to the oocytes. The fertilization rate of 200 oocytes/ml seawater increased with the amount of post‐thaw semen from 1 to 5 μl but showed a significant decrease at 25 μl. This decrease was considered to be due to DMSO in the cryodiluent, because the fertilization rate of the fresh semen decreased sharply when the DMSO concentration around the oocytes was 1.0% or more. Further improvement in increasing post‐thaw motility and lowering the cryoprotectant concentration is necessary for commercial‐scale artificial fertilization.  相似文献   

6.
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.  相似文献   

7.
In two trials, Arctic char (Salvelinus alpinus) semen was frozen in 0.5 mL straws using extenders consisting of 0.3 M glucose and 10%, 12.5% or 15% methanol. Cryopreserved semen was thawed by immersing straws in 25 °C water for 17 s (11.6 °C s?1) or in 5 °C water for 60 s (3.3 °C s?1). The viability of the frozen–thawed semen was measured by determining post‐thaw motility and sperm membrane integrity. Two fertility trials were also conducted. There was no effect of trial or thaw rate on post‐thaw sperm viability or fertility. Use of 15% methanol in the extender resulted in the highest overall percentage of sperm motility and fertility. Use of 12.5% methanol as a cryoprotectant resulted in a higher per cent post‐thaw motility and a lower percentage of dead cells than did 10% methanol. Thus, levels of methanol higher than the commonly used 10% are beneficial for cryopreserving Arctic char sperm.  相似文献   

8.
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.  相似文献   

9.
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.  相似文献   

10.
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.  相似文献   

11.
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.  相似文献   

12.
Sperm were collected in Florida from wild common snook, Centropomus undecimalis (Bloch), and were shipped to Louisiana State University for analysis and cryopreservation. Threshold activation of sperm (10% motility) occurred at 370 mOsmol kg?1, and complete activation occurred at 680 mOsmol kg?1. These values were significantly different. Sperm samples stored at 1°C in Hanks' balanced salt solution (HBSS) or in 0.6% NaCl solution at 200 mOsmol kg?1 retained motility for as long as 22 days. Mean motility remained above 50% for 9 days for sperm stored in HBSS and for 7 days for sperm stored in NaCl solution. Sperm exposed to 5% dimethyl acetamide (62±10%; mean±SD), 10% dimethyl sulphoxide (DMSO) (39±16%), 5% glycerol (26±5%) or 10% glycerol (6±2%) for 30 min had significantly lower motility than did unexposed sperm (89±9%). When used as a cryoprotectant, samples frozen with 5% or 10% DMSO or 5% methanol had significantly higher post‐thaw motility than did samples frozen with other cryoprotectants. Sperm cryopreserved with 10% DMSO (38±12%) had significantly higher post‐thaw motility than did sperm cryopreserved with 15% DMSO (19±10%) or 20% DMSO (4±4%). There were no significant differences in hatch rates of eggs fertilized with fresh sperm (54±29%) or cryopreserved sperm (41±35%). Survival to first feeding was not different between fish produced with fresh sperm (37±30%; range, 0–86%) or with thawed sperm (24±29%; 0–77%). Transport of sperm to a cryopreservation laboratory and back to a hatchery for thawing and use enabled collaboration between groups with specific expertise and provides a model for the application of cryopreservation by transport of fresh and frozen samples.  相似文献   

13.
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.  相似文献   

14.
This study examined the usage of a dry shipper for cryopreservation of Epinephelus septemfasciatus (Thunberg) spermatozoa. Milt was diluted 1:49 with 5% dimethyl sulfoxide plus 95% foetal bovine serum for cryopreservation. Computer‐assisted sperm analysis was used to analyse sperm motility, while fertilization and hatching trials were conducted to gauge the applicability of the cryopreservation method for aquaculture. We showed that cooling rates of the dry shipper were stable for 14 days and could be manipulated by the use of different sized freezing straws and use of a simple polystyrene foam container (5 × 5 × 12 cm and 1 cm thickness on all sides with the upper layer exposed). Dry shipper cryopreserved spermatozoa had significantly lower post‐thaw per cent motility and velocity than fresh sperm, but linearity of movement was unchanged. Fertilization and hatching rates were not significantly different at all tested sperm to egg ratios (3000:1–243000:1). The results indicated that 0.33 mL of milt when cryopreserved was sufficient to fertilize up to 450 g of oocytes. Application of this technology will help improve seed production in aquaculture and further develop breeding and genetics studies.  相似文献   

15.
The aim of this study was to optimize the cryopreservation protocols for the sperm of red seabream, Pagrus major. The 2‐mL cryovials and programmable freezer were employed for cryopreservation. Six extenders, six cryoprotectants in various concentrations ranging from 6 to 20% (v/v), four cooling rates, and three thawing temperatures were evaluated by postthaw sperm motility and fertility. The ratio of sperm to egg for postthaw sperm fertilization trials was experimentally standardized and was optimal at 500:1. The best motility of postthaw sperm (79.4 ± 4.7% to 88.6 ± 8.0%), fertilization rates (89.6 ± 2.9 to 95.6 ± 1.9%), and hatching rates (85.3 ± 5.1% to 91.4 ± 4.3%) were achieved when Cortland extender, dimethyl sulfoxide (15, 18, and 20%) or ethylene glycol (9, 12%) as cryoprotectants, 20 C/min as the cooling rate, and 40 C as the thawing temperature were employed. Moreover, the results on embryonic development were not significantly different between cryopreserved sperm and fresh sperm during incubation process. In conclusion, these methods of cryopreservation of red seabream sperm are suitable for routine aquaculture application and preservation of genetic resources.  相似文献   

16.
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.  相似文献   

17.
Zebrafish sperm cryopreservation is a fundamental methodology to manage and back-up valuable genetic resources like transgenic and mutant strains. Cryopreservation usually requires liquid nitrogen for storage, which is expensive and hazardous. Our objective was to evaluate if electric ultrafreezers (??150 °C) are a viable alternative for zebrafish sperm storage. Zebrafish sperm was cryopreserved in the same conditions (??20 °C/min), stored either in liquid nitrogen or in an ultrafreezer, and thawed after 1 week, 1 month, and 3 months. Sperm motility, membrane integrity, and fertilization ability were assessed. There were no significant differences in motility and hatching rate throughout storage time. Additionally, we aimed at understanding if cryopreservation directly in an ultrafreezer (??66 °C/min) could improve post-thaw sperm quality. Freezing at ??20 °C/min was performed as before, and compared to samples cryopreserved with a fast cooling rate by placing directly in an ultrafreezer (??66 °C/min). Sperm quality was assessed according to motility, viability, DNA fragmentation, and apoptosis (annexin V). The ??66 °C/min cooling rate showed significantly higher membrane and DNA integrity, and lower number of cells in late apoptosis in comparison to the other treatments. This study showed that zebrafish sperm cryopreservation and storage in an ultrafreezer system is possible and a fast cooling rate directly in ultrafreezer improves post-thaw sperm quality.  相似文献   

18.
In this study, the effects of three commonly used chemicals, dimethyl sulphoxide (DMSO), ethylene glycol (EG), propylene glycol (PG) and their combinations with trehalose, were evaluated on the cryopreservation of D‐larvae of the blue mussel Mytilus galloprovincialis. The larvae were harvested 30 h post‐fertilization at 21 °C and cryopreserved using a standard protocol in 5%, 10% or 15% of DSMO, EG and PG either as single chemical solutions or in combination with 0.2 M trehalose. Among these cryoprotectants, 5% DMSO resulted in the highest post‐thaw survival rate of 55.3±7.8%, although it did not significantly differ from those with 10% and 15% EG. The addition of 0.2 M trehalose did not improve the post‐thaw larval survival rates in all the combinations assessed. The cryo‐effects on subsequent development were evaluated using the D‐larvae frozen with 5% DMSO. The results showed that cryopreservation affected both larval survival and growth in this species. The relative daily mortality rate was significantly higher in treated than control groups over the period from 3 h post‐thaw to day 11 post‐fertilization. On day 6 post‐fertilization, the average larval length in the treated group was significantly smaller than that in the control. From day 11 post‐fertilization, and onwards, differences in these two traits were not significant between treated and control groups. On day 21 post‐fertilization, about 80% of the larvae in both treated and control groups developed eyes and the normalized survival rate in the treated group was 12.5%.  相似文献   

19.
We used flow cytometry to determine the quality of small abalone, Haliotis diversicolor, sperm before freezing and after thawing. We investigated the effects of cryopreservation on the characteristics of small abalone semen and determined the motility and fertilization capacity of the pre-freezing and post-thawed sperm. The percentages of motility and fertility were 61 ± 2% and 67 ± 1%, respectively, for the post-thawed sperm and 90 ± 4% and 92 ± 0%, respectively, for the pre-freezing sperm. Sperm cells were stained with specific fluorescent dyes to measure plasma and acrosomal membrane integrity, mitochondrial status, oxidation level, DNA compaction, and viability through flow cytometry. The frozen–thawed sperm exhibited significantly higher mitopotential activity (p < 0.05, damaged mitochondria; 25.01 ± 1.18%) and oxidation value (p < 0.01, free radicals; 63.79 ± 3.93%) compared with the pre-freezing sperm. The oxidation level was the most sensitive signal of the cryopreservation-induced small abalone sperm damage. Flow cytometry is valuable for the objective, accurate, and rapid assessment of pre-freezing and post-thaw small abalone sperm quality.  相似文献   

20.
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.  相似文献   

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