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

2.
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   

3.
Cryopreservation of sea cucumber Apostichopus japonicus (Selenka) sperm   总被引：1，自引：0，他引：1  
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.  相似文献   

4.
The commercial‐scale production of fish by use of artificial (induced) spawning would require reliable, large‐volume sources of sperm. Cryopreservation can be used to preserve and store sperm within commercial and research germplasm repositories, but is limited in its application to aquaculture. Straw volume and cooling chamber size restrict the quantity of sperm that can be frozen, and straws must be filled by hand. In contrast, the dairy industry has refined methods for freezing of bull sperm, including automation of straw filling and the use of large cooling chambers. These methods could be used for commercial‐scale cryopreservation of fish sperm, although application would require testing. To supply sperm in large volumes, bags originally developed for swine semen could be cooled using dairy protocols and used as a container for fish sperm. The current study documented the use of commercial‐scale dairy cryopreservation techniques for the production of hybrids of channel catfish Ictalurus punctatus (female) by blue catfish Ictalurus furcarus. Four cryoprotectants (methanol, dimethyl sulfoxide, dimethyl acetamide, and glycerol) were initially evaluated for use with blue catfish sperm. During May 2000 and March to April 2001, suspensions of blue catfish sperm were cryopreserved with 10% methanol in 0.5‐mL French straws and in commercial swine semen bags (Cochette* bags, IMV International. Minneapolis, Minnesota, USA). Cryopreservation took place at a dairy breeding cooperative, using technology employed for bull semen. Sperm motility before freezing was 26 ± 18% during Year 1 (2000) and 62 ± 30% during 2001. Sperm were thawed at 40 C and used to fertilize the eggs of channel catfish (yielding hybrids). Motility after thawing for sperm frozen in 0.5‐mL straws was 11 ± 10% during 2000 and 50 ± 24% during 2001. Motility after thawing was 41 ± 17% for sperm frozen in swine semen bags in 5‐mL aliquots and 43 ± 10% for sperm frozen in 10‐mL aliquots. Neurulation of eggs fertilized with thawed sperm from straws was 83 ± 13% during 2000 and 54 ± 27% during 2001. Neurulation was 57 ± 24% using sperm frozen in swine semen bags in 5‐mL aliquots and 55 ± 10% using sperm frozen in 10‐mL aliquots. There was no correlation between sperm motility before freezing (in 0.5‐mL straws) and after thawing during 2000 (r= 0.52) or during 2001 (r= 0.49). In addition, there was no correlation between initial motility and neurulation of channel catfish eggs fertilized using thawed sperm during 2000 (r= 0.14) or during 2001 (r= 0.29). Sperm of blue catfish can thus be cryopreserved at a commercial scale using dairy protocols and can be made available for the production of hybrid catfish when viable eggs are available.  相似文献   

5.
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.  相似文献   

6.
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   

7.
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   

8.
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   

9.
The effects of extender composition and freezing rate on motility and fertility of frozen‐thawed Arctic char, Salvelinus alpinus, spermatozoa were investigated. Three freezing rates, two semen diluents and three cryoprotectants were tested. Semen frozen in 0.3 mol L?1 glucose diluent with 10% methanol as a cryoprotectant or in a diluent described by Lahnsteiner with 10%N,N‐dimethylacetamide (DMA) resulted in the highest sperm motility. Fertility was the highest for semen frozen in a glucose–methanol extender but was not significantly different than that for semen frozen in Lahnsteiner's diluent with 10% DMA. Dimethyl sulphoxide (DMSO) at 10% was a relatively ineffective cryoprotectant with either semen diluent. Semen frozen at 6 cm above the surface of liquid nitrogen resulted in a higher post‐thaw sperm motility and fertility than semen frozen at 5 cm. The addition of 7% fresh egg yolk to glucose diluent containing methanol or DMSO did not improve the fertility of frozen‐thawed spermatozoa. However, the addition of 7% fresh egg yolk to glucose–DMA extender significantly improved the fertilization percentages of frozen‐thawed spermatozoa. In conclusion, dilution of semen 1:3 in 0.3 mol L?1 glucose with 10% methanol and freezing 6 cm above the surface of liquid nitrogen (freezing rate of 40±8°C min?1, mean±SD from ?5 to ?55°C) is a promising protocol for cryopreservation of Arctic char semen.  相似文献   

10.
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   

11.
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.  相似文献   

12.
《水生生物资源》1998,11(6):387-394
A sperm cryopreservation protocol adapted from turbot, was tested on sea bass using either 250-μL straws or 1.5-mL cryovials. A dilution to 1/3 in Mounib s extender and a cooling rate of −65 °C·min−1 allowed frozen sperm to recover an initial motility similar to that of fresh sperm at thawing; however, significant differences in motility (P < 0.001, n = 10 fish semen) were observed at further post-activation times, the motility decrease being faster in thawed sperm. At the experimental scale, triplicate inseminations of 2-mL aliquots (approximately 2 000 eggs) showed a significant fertility decay of thawed sperm compared to that of fresh sperm (P < 0.01, n = 12 fish semen) when a discriminating 35·103 spermatozoa to egg ratio was applied. When 70·103 and 200·103 spermatozoa per egg were provided in the same experimental conditions, no significant difference appeared between the fertilisation rates of fresh and thawed sperm. In order to validate the procedure for production or cryobank purpose, a scaled-up protocol was established. Two and 50 mL batches of eggs (approximately 2·103 and 50·103 eggs, respectively) were inseminated in triplicate using either fresh or thawed individual sperms of 5 males with 200·103 spermatozoa per egg. The mean fertility decreased by 23.5 % due to cryopreservation. This decline was explained by the loss of fertility of only one sperm, and only in large-volume conditions, probably due to the delay of use after thawing.  相似文献   

13.
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   

14.
The aim of this study was to test the effects of cryoprotectants [dimethyl sulphoxide (DMSO) and methylglycol], extenders (0.9% NaCl, 5% glucose, Beltsville Thawing Solution? and Merck III?), thawing temperatures (30 and 60 °C) and activating agents (0.29% NaCl and 1% NaHCO3) on the cryopreservation process of tiete tetra Brycon insignis sperm. Sperm was loaded in 0.5 mL straws, frozen in nitrogen vapour at ?170 °C and stored in liquid nitrogen. Post‐thaw sperm quality was evaluated in terms of subjective motility rate, quality motility score (0=no movement; 5=rapidly swimming spermatozoa), duration of motility and vitality (eosin–nigrosin staining). Post‐thaw sperm motility rate was greater in methylglycol (76–88%), compared with DMSO (23–59%). In general, the highest quality motility scores were observed when sperm was thawed at 30 °C and triggered in 1% NaHCO3 (3.5–4.3). Duration of motility was longer when triggered in 1% NaHCO3 (95–120 s) compared with 0.29% NaCl (69–107 s). Sperm vitality was not affected by any of the parameters tested and varied from 51% to 69% intact sperm. Brycon insignis sperm frozen in methylglycol combined with any of the extenders tested and using the methods described above yields motility above 57% and that should last long enough to fertilize oocytes.  相似文献   

15.
Cryopreservation of Arctic charr Salvelinus alpinus (L.) semen was investigated using three diluents, three cryoprotectants [10% dimethyl sulphoxide (DMSO), 10% dimethylacetamide (DMA) or 20% glycerol] and three sizes of straw. The three diluents and three cryoprotectants were combined, resulting in nine extenders. One part semen was added to three parts extender, and motility was evaluated to assess the toxicity of six of the extenders. Semen in nine extenders was frozen in 0.5‐mL straws using liquid nitrogen vapour. Semen extended in 0.3 m glucose and each of the cryoprotectants was also frozen in 0.5‐mL, 1.7‐mL (flat) or 2.5‐mL straws. The freezing rate in each size of straw was measured. Fertility trials were conducted to determine the post‐thaw viability of the frozen semen. The motility of activated spermatozoa was higher in the DMA and DMSO extenders than in the glycerol extender. For the trial using 0.5‐mL straws, post‐thaw fertility results were higher for all extenders containing DMSO, or 0.3 m glucose and DMA, than for all other combinations of diluent and cryoprotectant. For the straw size comparison, the highest fertility was obtained for the 1.7‐mL straw using either DMSO or DMA and for the 2.5‐mL straw using DMSO. For all cryopreservation trials, fertility was low for extenders containing glycerol.  相似文献   

16.
Long‐term cryopreservation of the giant freshwater prawn, Macrobrachium rosenbergii, spermatophores using glycerol (Gly) and ethylene glycol (EG) as cryoprotective agents (CPAs) was studied. The tolerance of sperm to cryopreservation was evaluated on the basis of sperm survival and fertilizing ability. The survival of the sperm was determined by trypan blue staining, while the fertilizing ability was assessed from artificial insemination of the cryopreserved spermatophores. The rates of embryo survival on day 5 after spawning and of spermatophores capable of producing embryos survived to hatching were determined. Storage of spermatophores at ?20°C without CPA for a short period of up of 1–5 days decreased the sperm survival significantly and did not preserve fertilizing ability. Preservation at ?20°C in the presence of 10% or 20% Gly or of 10% or 20% EG offered a simple and efficient short‐term storage up to 10 days. For a long‐term storage, cryopreservation in the presence of 20% EG at ?196°C was more efficient than at ?20°C. High sperm survival rates and high fertilizing ability were recorded from those cryopreserved at ?196°C for up to 150 days. High sperm survival rates with moderate levels of fertilizing ability were obtained from those cryopreserved at ?20°C for not more than 30 days. The results indicate that preservation at ?196°C with 20% EG is a suitable procedure for long‐term storage of the giant freshwater prawn spermatophores.  相似文献   

17.
To clarify factors reducing the motility and fertility of cryopreserved spermatozoa of the Japanese pearl oyster Pinctada fucata martensii, the structure of spermatozoa before and after cryopreservation was observed by scanning electron microscopy. Testicular spermatozoa were diluted with cryopreservation diluent (10% methanol+18% fetal bovine serum+72% sea water), and dispensed into 0.25-mL straws. The straws were cooled at a rate of approximately −20 °C/min to −50°C, and subsequently immersed in liquid nitrogen. Percentage motility of spermatozoa before cryopreservation was 69.9±4.2%, and that of cryopreserved spermatozoa was 24.0±1.8%, respectively. In cryopreserved spermatozoa, the percentage that lacked or had a deformed flagellum was 56.6±3.9%, while in fresh spermatozoa this was 8.7±2.0%. In cryopreserved spermatozoa, the percentage of deformed acrosomes was 76.6±5.2%, while in fresh spermatozoa this was only 0.9±0.3%. Cryopreserved spermatozoa with a normal acrosome and flagellum were only 15.4±3.5% of those in fresh spermatozoa. These results indicate that lesion of the flagellum and deformation of the acrosome occurred through the cryopreservation procedure, and both types of damage lead to loss of the motility and fertility in thawed spermatozoa.  相似文献   

18.
Preliminary techniques of mass propagation of grey mullet were established in Taiwan some years ago. Among the many problems concerned, the cryogenic preservation of grey mullet sperm has been studied since 1971. The aim of this study is not only to ensure the availability of mullet semen anywhere and anytime it is needed, but also to contribute to the international cooperation of cross breeding of grey mullet in the future. Results of the study on cryogenic preservation of grey mullet sperm made during the experimental mass propagation of mullet in the Tungkang Marine Laboratory for the past 3 years are summarized in this paper.The gonadosomatic index (GSI) of male grey mullet migrating near the coast of Tungkang ranged from 4 to 20 during the spawning season. The pH value of the semen was 7.4. Each spermatozoon was composed of a head part measuring 2.3 μ × 1.4 μ and a tail part four to five times as long as the head. There were about 5.3 × 1010 sperms in each ml of semen. Eosin-nigrosin staining was used for clearer identification. Sperm motility was preserved for up to 23 days in the case of raw semen at 5° C. Cryoprotective agents were needed at the ultra-low temperature (?196° C) of preservation in liquid nitrogen. Feasible procedures of freezing the grey mullet sperm were determined. Fresh semen diluted with cryoprotective agents was dispensed into 0.5 ml straws which were then sealed. These pretreatments prior to cryopreservation had to be done within the correct equilibration time of 1 h or less. Semen in straws was precooled in liquid nitrogen vapor until a temperature of ?80° C was reached. Straws in the canister were then put into liquid nitrogen for long-term preservation. The optimum effect of cryoprotective agents was found with 5–10% glycerine or dimethylsulfoxide (DMSO) at 1:1, 1:5, and 1:10 dilutions. In this condition, both good motility and fertility before freezing and cryoprotection were obtained. So far the best result of frozen thawed mullet sperm was moderate motility and 2.7% fertility of the semen cryopreserved for 1 year and 4 days.  相似文献   

19.
The aim was to evaluate the effect of different concentrations of caffeine added in activating solution over sperm motility in fresh and thawed semen of pacu, Piaractus mesopotamicus, and curimba, Prochilodus lineatus. The activating solutions were prepared with sodium bicarbonate solution of 0.76% (NaHCO3) and caffeine was added at concentrations of 2.5, 5.0, 10.0, and 20.0 mM. As control, a solution of NaHCO3 0.76 without caffeine was used. Eight males of pacu and 20 males of curimba were used. Aliquots of 200 μL of semen were diluted in 800 μL extender solution (DMSO 10% and BTS 5%), placed in 0.5 mL straws and cryopreserved for 7 d in a liquid nitrogen tank. There was a linear increase in sperm motility for fresh semen of pacu, and for curimba fresh and thawed semen (P < 0.05), due to the increase in the concentration of caffeine. There was a quadratic response for duration of motility for thawed semen of pacu and for fresh semen of curimba (P < 0.05), respectively. These results indicate that addition of caffeine in the activator solution can improve sperm motility parameters, however, is dependent on the species and concentration used.  相似文献   

20.
The effects of four cryoprotectants (methanol, MeOH; dimethyl sulphoxide, DMSO; dimethyl acetamide, DMA; and ethylene glycol, EG), three extenders (calcium‐free Hanks' balanced salt solution, C‐F HBSS, Hanks' balanced salt solution, HBSS and sodium chloride, NaCl) and two different freezing procedures (one‐ and two‐step) on the cryopreservation of striped catfish (Pangasius hypophthalmus (Sauvage)) sperm were investigated. Sperm were frozen using a controlled‐rate freezer in 250 μL straws and stored for 2 weeks in a liquid nitrogen (LN2) container. They were then airthawed at room temperature, and fertilization, motility and viability were assessed. The highest fertilization rate of 41% (81% of control) was achieved with the combination of 12% DMSO and 0.9% NaCl using a one‐step freezing procedure (10°C min?1). Also, DMA resulted in a higher fertilization rate (30% or 51% of the control) than MeOH (18% or 38% of the control) or EG (8% or 12% of the control). In addition, the three extenders used did not affect fertilization rates after cryopreservation with each cryoprotectant. There were no significant differences among the three cryoprotectant concentrations and between the one‐ and two‐step freezing procedures. However, fertilization rates of cryopreserved sperm were significantly lower than the controls (P<0.05). The results of this study indicate that high fertilization rates of striped catfish eggs can be achieved using cryopreserved sperm when frozen at 10°C min?1 in DMSO or DMA with either 0.9% NaCl or C‐F HBSS.  相似文献   

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