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1.
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   

2.
The cryopreservation of semen from the Northern pike, Esox lucius L., was investigated with a method that was originally developed for the Salmonidae. Because the amounts of semen obtained by stripping were insufficient, the suitability of testicular sperm was tested for cryopreservation. Frozen-thawed testicular sperm had fertilization rates similar to frozen-thawed semen obtained by stripping (74.2-84.7%), and at sperm to egg ratios of S= 4.5 × 105 spermatozoa per egg, the post-thaw fertilization rates were also similar to fresh, untreated semen controls. Out of all the fertilization solutions investigated, a 100-mm NaCl, 10-mm Tris (pH 9) solution resulted in the highest post-thaw fertilization rates. To facilitate the fertilization of large egg batches, 1.2-mL straws were used for cryopreservation with a similar efficiency to 0.5-mL straws.  相似文献   

3.
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   

4.
人工养殖西伯利亚鲟精子超低温冷冻保存研究   总被引：7，自引：6，他引：7  
刘鹏  庄平  章龙珍  王斌  闫文罡 《海洋渔业》2007,29(2):120-127
研究了人工养殖西伯利亚鲟精子的生物学特征及超低温冷冻保存方法。西伯利亚鲟的产精量为113.67±39.86 ml,精子密度为(6.49±3.10)×108/ml,精子活力为(85.4±9.5)%,精子寿命为353±23 s。精子密度与精子快速运动时间、精子寿命之间均存在线性相关,用方程分别表示为:y=1.0384x+1.5089(R2=0.7325);y=2.9069x+74.289(R2=0.6967)。结果表明精子密度可作为一项精子质量评价的标准。通过比较西伯利亚鲟精子在不同稀释液、不同抗冻剂和抗冻剂浓度、降温速率、解冻温度下的保存效果,结果表明:配方2作为稀释液,18%甲醇作为抗冻剂,二步法超低温(-196℃)冷冻保存精子,40℃水浴解冻取得最好的冻后活力,解冻后活力为(51.8±5.8)%。西伯利亚鲟授精的最佳精卵比为106∶1。在此精卵比下用冻精授精分别得到了(72.3±3)%的受精率和(52.9±4.1)%的孵化率,其中受精率与鲜精没有显著性差异,孵化率与鲜精有显著差异(P<0.05)。  相似文献   

5.
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   

6.
Milt from individual males of northern pike, Esox lucius L., was separately cryopreserved. Concentration of spermatozoa in fresh milt and spermatozoa motility before freezing and after thawing was evaluated. Activity of aspartate aminotransferase (AspAT, E.C. 2.6.1.1.) and acid phosphatase (AcP, E.C. 3.1.2.2.) in fresh and thawed sperm were determined. In comparison with the control group, egg fertilization with cryopreserved milt varied from 6.6% to 96.0%, depending on the donor male. Fertilization success with cryopreserved pooled milt was 71.8%. Freezing and thawing procedure caused loss of proteins from injured spermatozoa, resulting in significantly lower enzymatic activity in spermatozoa. Intensity of enzyme leakage in thawed milt correlated negatively with fertilization success. Concentration of spermatozoa could be a possible accessory quality indication, useful when selecting sperm samples appropriate for cryopreservation.  相似文献   

7.
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   

8.
This paper reports an initial trial to cryopreserve semen from two freshwater South American fishes, the curimbatá (Prochilodus scrofa) and the dourado (Salminus maxillosus). Motility and duration of motility were observed in curimbatá and dourado fresh sperm. Semen mixed with extender (0.8% NaCl) was frozen using vials (1 ml) with subsequent storage in liquid nitrogen. Samples were thawed in 1% NaHCO3 or in 0.8% NaCl solutions. Post-thawing motility and duration of motility were verified. A simple extender consisting of 0.8% NaCl plus 10% DMSO was able to initiate motility in fresh spermatozoa. The percentage of motile cells and duration of motility were similar in both thawing solutions, but lower than in fresh sperm.  相似文献   

9.
Collection of fish milt by stripping risks the danger of milt contamination by urine. This may seriously influence milt characteristics and quality, including usefulness for cryopreservation. Urine contamination of milt may be avoided by using a catheter for sperm collection. The objectives of this study were to provide basic characteristics of milt collected with a catheter, to test the usefulness of this milt for cryopreservation, and to correlate characteristics of fresh and cryopreserved semen with sperm fertility rates. Milt from 25 rainbow trout Oncorhynchus mykiss (Walbaum) males were used. All samples were cryopreserved using the pellet method within 1 h of collection, using 0.6 m sucrose and 10% dimethyl sulphoxide (DMSO) as an extender. Catheterization resulted in semen of very good motility (> 90% motile spermatozoa) and high fertilization rates after cryopreservation (mean fertilization rate 81.8 ± 13.3% of control, at a sperm/egg ratio of 2.4 ± 0.3 × 106). Osmolality of seminal plasma and concentrations of sodium, potassium and magnesium ions had low variability, which suggests that they are important for creating a stable environment for sperm storage in the sperm duct. Higher variability of certain seminal plasma characteristics, such as protein concentration and antiproteinase activity, suggests that these characteristics are related to individual semen features of particular males. A strong correlation of seminal plasma zinc concentration with protein concentration may reflect an importance of zinc in semen biology. Cryopreservation caused a significant release of protein and acid phosphatase from spermatozoa. Our results did not reveal any single characteristic of semen collected by catheter that could be used as a powerful predictor of cryopreservation success, presumably because all samples were of high quality.  相似文献   

10.
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   

11.
Cryopreservation of sperm in marine fish   总被引：10，自引：0，他引：10  
Since the first work of Blaxter in 1953, fish sperm cryopreservation has been attempted on about 30 marine species. The present paper reviews the techniques used and the results published in these species. Particular attention is paid to the handling procedure of sperm before freezing, the problems of semen ageing and semen contamination with urine. The quality of frozen–thawed semen was evaluated using previously standardized biotests, such as a two‐step motility activation technique adapted for the different species and fertilization assays using a discriminating insemination technique. Most extenders used in marine fish are saline or sugar solutions. From the investigated cryoprotectants, dimethyl sulphoxide (DMSO) generally leads to the best results. Cooling rates range from 8 °C to 99 °C min?1; the thawing rate is generally high. Compared with freshwater species, a high percentage of spermatozoa survives cryopreservation. Therefore, and because of the simplicity of the techniques, the cryopreservation of marine fish sperm is suited for application in aquaculture.  相似文献   

12.
The present study investigated motility, acrosome reaction, fertility and cryobiological parameters of the semen of the sterlet, Acipenser ruthenus L. Sperm motility persisted for about 4 min in water, and the main swimming type was the linear motion. Motility was prolonged at osmolalities of 12.5 mosmol kg?1 and in the presence of magnesium ions, while calcium had no effect. Also a pH in the range of 7.0–9.0 had no effect on ` motility. At osmolalities of 25–50 mosmol kg?1 the sperm motility was partly inhibited, at osmolalities of 100 mosmol kg?1, completely and irreversibly. In 50 mosmol kg?1 solutions with 2.5–5 mM L?1 KCl the motility inhibition was total, but reversible. The acrosome reaction was not induced by one of the described solutions, but the percentage of spermatozoa with reacted acrosomes was low (<20%) and highly variable in all experiments. The optimal extender base for cryopreservation was a solution consisting of 50 mM L?1 NaCl, 5 mM L?1 KCl, 10 mM L?1 Tris (pH 8.5). From the tested cryoprotectants only dimethylsulphoxide (DMSO) and methanol provided sufficient cryoprotection. After freezing and thawing, the motility rates and swimming velocities were higher with DMSO than with methanol. However, the fertility was very significantly reduced with DMSO (10.3±0.5%) while with methanol fertilization rates in a similar range (32.7±4.4%) as with fresh semen (33.90±0.8%) could be obtained. Optimal freezing conditions for sterlet semen were in the vapour of liquid nitrogen 3–5 cm (?95°C to ?85°C) above its surface, the optimal thawing conditions at 25°C for 30 s. The acrosome reaction was not induced by these cryopreservation protocols.  相似文献   

13.
The aims of this study were to describe the fresh spermatozoon ultrastructure using scanning and transmission electron microscopy and to improve the sperm cryopreservation methodology for the freshwater fish pirapitinga Brycon nattereri. Extenders (BTS? and NaCl), straw volumes (0.5 and 4.0 mL), thawing temperatures (30 and 60 °C) and activating agents (0.29% NaCl and 1% NaHCO3) were tested. Methylglycol was used as a cryoprotectant agent and sperm was frozen in nitrogen vapour (dry‐shipper). Post‐thawed sperm motility rate, motility quality (score 0=no movement; 5=rapidly swimming spermatozoa), duration of motility and spermatozoon morphology were evaluated. Fresh spermatozoon was 35.06 μm long, the head was ovoid (2.00 × 1.22 μm) with no acrosome, the midpiece was 2.15 μm long and the flagellum was 30.90 μm long with the typical 9+2 axoneme arrangement. Post‐thawed sperm motility rate (70–79% motile sperm), motility quality (score 3.1–3.7) and morphology (9.3–11.6% abnormal spermatozoa) were not affected by any of the parameters tested. The duration of sperm motility was longer when triggered in 1% NaHCO3 (392–1031 s) compared with 0.29% NaCl (144–338 s). Brycon nattereri sperm cryopreserved under the conditions described above yields over 70% motility and should last long enough to fertilize oocytes, even after 2 years of freezing.  相似文献   

14.
Using the cryopreservation method of Lahnsteiner, Berger, Weismann & Patzner (1995, Aquaculture Research 26 , 801-807) the influence of allowable variations of methodical parameters (storage of semen before cryopreservation, dilution ratios in the extender, equilibration in the extender, cooling rates, storage of deep-frozen semen in liquid nitrogen, storage of frozen/thawed semen, minimal sperm/egg ratio) was investigated under the aspect of routine utilization. Under optimized experimental conditions, fertilization rates were 90-100% of controls in Oncorhynchus mykiss (Walbaum), Salmo trutta L. f. lacustrisSalmo truttaL.f. fario and Salvelinus fontinalis (Mitchill). The following results were obtained: 1. Storage of untreated semen for more than 1 h before cryopreservation decreased the postthaw fertility. 2. Equilibration of semen up to 20 min in the extender did not affect the postthaw fertility. 3. Optimal dilution ratio of semen in the extender was threefold in Oncorhynchus mykiss and Salvelinus fontinalis. Lower dilution ratios decreased the postthaw fertility, higher dilution ratios did not affect the postthaw fertility. In Salmo trutta f. lacustris and Salmo trutta f. fario, which have a higher sperm density, optimal dilution ratio of semen in the extender was fivefold to sevenfold. 4. In Oncorhynchus mykiss, as in Salmo trutta f lacustris and Salmo trutta f. fario, the optimal freezing height was at 1.5 cm above the level of liquid nitrogen (-110 ± 2oC); in Salvelinus fontinalis it was 2.5 cm above the level of liquid nitrogen (-92 ± 2oC). Changes in the freezing height of 0.5 cm (about 10oC) resulted in a significant decrease of postthaw fertility. 5. Storage of deep-frozen semen for up to 370 days in liquid nitrogen had no influence on its postthaw fertilization rate. 6. Storage of frozen/thawed semen for 30 s before insemination significantly decreased its postthaw fertility. 7. Reliable minimal sperm:egg ratio to obtain fertilization rates of 90-100% of control was 3-5 X 105 spermatozoa egg-1.  相似文献   

15.
The aim of this study was to develop a protocol for semen storage of piracanjuba (Brycon orbignyanus) by both cool storage at 4 °C and cryopreservation at − 196 °C. Semen was diluted in some fish semen extenders (Exp. 1) or in extenders combined with the antibiotic gentamycin sulfate (Exp. 2) and stored at 4 °C. Sperm motility was estimated every 24 h. Then, the effects of egg yolk (0 and 5%), cryoprotectants (dimethyl sulphoxide — DMSO, methanol, and methylglycol) and extenders (NaCl 154 mM, BTS™ Minitub and M III™ Minitub) on semen cryopreservation were evaluated (Exp. 3). Semen was added to each of eighteen cryosolutions (2 yolk concentrations × 3 cryoprotectants × 3 extenders), aspirated into 0.5-mL straws, frozen in nitrogen vapor (Taylor-Wharton, CP 300, “dry shipper”) and stored at − 196 °C. Sperm motility was evaluated after thawing at 60 °C-water bath for 8 s. The three cryosolutions that produced the highest post-thaw sperm motility were used again to freeze semen. Post-thaw semen quality was then evaluated under three tests: sperm motility, the percentage of live spermatozoa and hatching rate (Exp. 4). Piracanjuba semen diluted (1:10 total volume) in NaCl 200 mM or in Saad solution (NaCl 200 mM, Tris 30 mM) maintained motility above 35% for as long as 7 days, at 4 °C. Motility of only 7% was observed on undiluted semen after 3 days at 4 °C. There was neither beneficial nor detrimental effect of gentamycin on sperm motility at 250 μg/mL. Egg yolk addition to the cryosolution was beneficial in samples cryopreserved in NaCl 154 mM and in M III™, but detrimental for samples cryopreserved in BTS™. Methylglycol was the most effective cryoprotector compared to DMSO and methanol. Motility and percentage of live spermatozoa were similar among semen cryopreserved in NaCl–yolk, M III™–yolk and BTS™, all containing 10% methylglycol, but lower than fresh control. Hatching rates of eggs fertilized with sperm cryopreserved in NaCl–yolk or BTS™ were higher than for eggs fertilized with sperm cryopreserved in M III™–yolk, but lower than control fertilizations. The semen cryopreservation protocols developed here will be used to set up a gene bank for endangered piracanjuba populations.  相似文献   

16.
The present study describes a uniform method for cryopreservation of semen of Salmonidae (Oncorhynchus mykiss (Walbaum), Salmo trutta f. fario L., Salmo trutta f. lacustris L., Coregonus sp.). It presents a new type of extender and experiments demonstrating that warming of frozen/thawed semen to 20°C prior to fertilization significantly increases the fertilization rate. Freezing is performed in straws in the vapour of liquid nitrogen and for insemination a diluent technique is used. The consistency of the method was tested by repeating the experiments with different batches of semen and eggs. The following fertilization rates (% of control) were obtained: Oncorhynchus mykiss: 89.6 ± 16.0% (mean ± standard deviation, n= 25, n of control = 20, sperm/egg ratio of 1.6 ± 0.2 × 106 spermatozoa/ egg). Salmo trutta f. fario: 93.8 ± 6.4% (n= 12,9.9 ± 1.2 × 106spermatozoa/egg), Coregonus sp.: 92.8 ± 2.4% (n= 6, 0.5 × 106 spermatozoa/egg), Salmo trutta f. lacustris: 85.0 ± 8.4% (n= 12, 4.8 ± 1.4 × 106 spermatozoa/egg).  相似文献   

17.
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   

18.
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   

19.
The yamú Brycon siebenthalae is an endemic fish of the Orinoco river basin, but wild stocks are decreasing because of the disruption of their habitat. We evaluated a protocol for the cryopreservation of yamú sperm to contribute to the preservation of this endangered genetic resource. Milt was mixed with a cryoprotectant medium (5.5% glucose, 12% egg yolk, and 5%, 10%, or 15% dimethyl sulfoxide - DMSO) in a ratio 1:4 (milt:medium), stored in 0.5-mL French straws, frozen in nitrogen liquid vapor (-76 C), then immersed and stored in liquid nitrogen for 10 d or 12 mo. Motility of thawed spermatozoa was higher ( P < 0.001) in 10% DMSO medium than 5% DMSO or 15% DMSO mediums; but lower than the control ( P < 0.001). With sperm cryopreserved, the highest level of fertilization was achieved with 10% DMSO ( P < 0.001) after 10 d or 12 mo of cryopreservation. Fertilization of eggs inseminated with 6.4 × 109 spermatozoa per g of eggs was higher ( P <0.05) than with 1.6 × 109 spermatozoa per g of eggs. There was no difference (P > 0.05) in fertilization between insemination doses of 3.2 × 109 and 6.4 × 109 spermatozoa per g of eggs. Cryopreservation of yamu milt can be performed successfully with a simple medium combined with 10% of DMSO as cryoprotectant. The highest level of fertility was achieved using between 3 × 109 and 6 × 109 spermatozoa per g of fresh eggs.  相似文献   

20.
In spite of the fact that egg yolk from different avian species has successfully been used as an additive for the cryopreservation of sperm in mammalian species, its efficacy for cryopreserving fish sperm has not previously been tested comparatively. Therefore, the present study was carried out to determine the effect of egg yolks from different avian species, namely domestic chicken (Gallus gallus domesticus), turkey (Meleagris gallopavo) and quail (Coturnix coturnix), on post-thaw motility and fertilization ability of cryopreserved common carp spermatozoa. Egg yolks from chicken, turkey and quail were analysed for moisture, total fat, protein, cholesterol and phospholipid profile. Total fat and cholesterol contents of the turkey egg yolk were higher than chicken and quail egg yolks (p < 0.05). Semen was frozen according to conventional slow freezing procedure. The extender contained 350 mM glucose, 30 mM Tris and 5 % glycerol supplemented with different ratios of avian egg yolk (10, 15 and 20 %). Semen was equilibrated at 4 °C for 15 min and placed into 0.25-ml straws and frozen in liquid nitrogen vapour (for 10 min at ?120 °C) and finally stored in liquid nitrogen (?196 °C) tank. The frozen spermatozoa were thawed in a water bath at 35 °C for 30 s. Fertilization was conducted using a ratio of 1 × 105 spermatozoa/egg. Cryopreservation experiments resulted in higher post-thaw motility and fertilization rates. Mean post-thaw motility of cryopreserved spermatozoa was between 45 and 80 %, and fertilization rates, expressed as the percentage of eyed embryos, ranged from 70 to 95 %. In conclusion, the present study showed that turkey and quail egg yolks are suitable alternatives to the chicken egg yolk for the cryopreservation of common carp spermatozoa.  相似文献   

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