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1.
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   

2.
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   

3.
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   

4.
The effect of extenders was studied on the cryopreservation of sperm from African catfish, Clarias gariepinus (Burchell). The following six basic extenders were tested: fructose, glucose, sucrose, NaCl, KCl solutions and the artificial seminal plasma of the African catfish. Each of these extenders was tested both with and without buffer systems (i.e. NaHCO3-CO2 and Tris-HCl) by using 10% dimethyl-sulphoxide (DMSO) as a cryoprotectant. The two-step freezing was carried out in a programmable freezer by using the following freezing rates: (1) 4 °C min–1 between 3 and –4 °C; (2) and 11 °C min–1 between –4 and –80 °C. The best post-thaw motility (25%) was achieved with 333 mmol L–1 fructose solution and NaHCO3 buffer. The fertilization experiments were carried out with unbuffered fructose and glucose extenders using various amounts of sperm and two fertilization methods: (1) dry and (2) wet. The best fertilization rates were achieved with 75 μL of sperm and wet fertilization with glucose extender, or 100 μL of sperm and dry fertilization in case of fructose – both methods fertilized 96% of all eggs.  相似文献   

5.
The effects of cryopreservation on the viability, morphology and capability of spermatozoa in Atlantic cod, Gadus morhua L., were studied. The sperm was cryopreserved in straws using Hanks' balanced salt solution, hens' egg yolks and glycerol in the vapor of liquid nitrogen. Straws of cryopreserved sperm were stored in liquid nitrogen and thawed in seawater (35 C) for 8 sec before use. The motility of cryopreserved sperm was low (range 8–19%) compared to motility before freezing (range 69–76%). The fertilization rate (range 94–95%) in control groups using fresh sperm was significantly higher (P < 0.05) than in test groups (range 48–72%). In cryopreserved sperm, a relatively high percentage (range 82–93%) of the spermatozoa had changes in morphology. Many spermatozoa had no mitochondria; when mitochondria were present, the observed number varied from one and five in cryopreserved spermatozoa, and from two and seven in noncryopreserved spermatozoa. In groups where cryopreserved sperm was used, the hatching rate was lower (range 18–38%) than in control groups (range 41–63%), indicating higher mortality during embryonic development. Paternal effects on progeny performance were noted in the proportion of abnormalities but no negative effects were identified in newly hatched larvae produced using cryopreserved sperm.  相似文献   

6.
This study tested KUROKURA solution (Kurokura et al., 1984, Aquaculture 37, 267–274) and its modifications (by increasing NaCl content to 160, 180 and 200 mM) on immobilizing properties for sampling and short-term preservation of potential motility of tench spermatozoa. The immobilizing solution is used because, when collected, the sperm of most samples is contaminated by urine, causing spermatozoa to be of poor quality, with low motilities and velocities (almost 0), thus resulting in a worsened fertilization and hatching rate. Sperm was sampled with a syringe containing an immobilizing solution (IS), allowing an IS:sperm ratio of 2:1, under aerobic conditions at 0–4°C. This sperm solution was stored for 10 h and untreated sperm was collected prior to fertilization as a control. Spermatozoa quality was evaluated for the cell motility and velocity parameters and also for fertilization ability and hatching rate. Results obtained for tench sperm motility, velocity, fertilization and hatching rate showed that only sperm collected in the various immobilizing solutions can be successfully used for artificial insemination and preservation after 10 h at 0–4°C. The best immobilizing solution was found to be KUROKURA 180 (180 mM NaCl, 2.68 mM KCl, 1.36 mM CaCl2· 2H2O and 2.38 mM NaHCO3), giving a fertility and hatching rate of 41%, with no change in rates after 10 h storage of sperm. Control sperm without immobilizing solution showed a fertilization and hatching rate of only 6–7%.  相似文献   

7.
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   

8.
Understanding the effects of environmental factors in sperm qualities will be helpful in the development of optimal artificial reproduction methods and contributes towards the knowledge base of better short‐ and long‐term fish semen preservation conditions The objectives of this study were to determine properties and activities of wild‐caught striped jewfish Stereolepis doederleini sperm contaminated with blood or seawater and compare them with data reported in the literature on other freshwater and marine fish species, for effective short‐ and long‐term storage of fish semen. Overall, we observed that the sodium, chloride, glucose, total protein concentrations of normal sperm were not significantly different from blood‐ or seawater‐contaminated sperm. The salinity and osmolality concentration of sperm contaminated with blood were lower than sperm contaminated with seawater and were not significantly different from normal sperm. In addition, the spermatozoa motility (SM) and duration of spermatozoa motility (DSM) in blood‐contaminated sperm were higher than seawater‐contaminated sperm and also not significantly different from normal sperm. The best condition for SM and DSM in normal sperm was dilution rate of 1:50. Sperm was immotile in distilled water, and cationic factors were shown to stimulate the initiation of spermatozoa activation. The maximum SM and DSM were observed in solution containing 0.4 M NaCl, 0.6 M KCl, 0.6 M CaCl2 and 0.4 M MgCl2. This study provides some basic and important knowledge about striped jewfish sperm sensitivity to a cationic condition. In this regard, Na+ is the major inhibitory factor of spermatozoa motility in this fish species.  相似文献   

9.
The dourado, Salminus brasiliensis, is an important fish in South American river basins. The fish‐farming potential and concerns for its conservation in watersheds has stimulated studies with this species. This study compared the effect of different dilutions of two cryoprotectant solutions on the quality of cryopreserved dourado sperm (duration of motility/motility rate) post‐thawing using three different activating solutions. The cryoprotectant solution ACP‐104 was compared with the cryoprotectant solution most commonly used for dourado, which consists of a mixture of dimethyl sulfoxide, glucose, egg yolk, and distilled water. The semen was mixed following dilutions of semen : cryoprotectant solution: 1:3, 1:9, 1:15, 1:21, and 1:27. The cryopreserved samples were activated using distilled water, 0.45% NaCl or 1% NaHCO3. The results showed differences in thawed semen using the different activating solutions and cryoprotectant solutions. The glucose‐based cryoprotectant not only resulted in sperm motility equal to or better than that produced by ACP‐104, but the quality was not affected by the dilution. However, the sperm quality improved with increasing dilutions for ACP‐104. Under the conditions tested, the standard cryoprotectant solution is recommended for the cryopreservation of dourado semen because it requires a lower solution volume and, therefore, reduced storage space for the same volume of semen.  相似文献   

10.
In order to develop artificial reproduction in freshwater fish for potential species to be developed in South American aquaculture, milt quality and sperm morphology were studied in yamú (Brycon siebenthalae) under captive conditions during the natural middle spermiation period. The volume of milt collected for each male was 1.8±1.2 mL and the sperm concentration was 13.9±4.0 × 109 spermatozoa mL?1. Spermatocrit (41.5±10.8%) was positively associated (r2=0.30) with sperm density calculated using a corpuscle counting chamber. Sperm motility was 88±9% and the average duration of forward motility was 41±7 s. Fertilization rate was 84±8% and there was no association between this trait and sperm motility (r2=0.009) or with sperm density (r2=0.073). These results suggest that captive B. siebenthalae broodstock can be reproduced successfully.  相似文献   

11.
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   

12.
The aims of this study were to evaluate the efficiency of simple and complex extenders in prolonging the cold storage of sperm (Experiments 1 and 2) and to test the diluted‐cooled sperm in the best extender with regard to sperm quality parameters (Experiment 3) in the streaked prochilod, Prochilodus lineatus. In all the experiments, aliquots of 0.3 mL of sperm were diluted 1:10 in extenders and stored at 4–6 °C. Sperm diluted in simple extenders (NaCl and glucose solutions) yielded 0–26% sperm motility, whereas sperm diluted in complex extenders (BTS?, M III? and Androstar?) yielded 62–81% sperm motility on day 4 after cold storage. When Androstar? was further investigated, the following was observed on day 4: 53% motility with 94 s of duration; 47% live spermatozoa; 26–61% fertility rate; and 22–60% hatching rate. The use of Androstar? improves the sperm fertility of the streaked prochilod during a 4‐day storage period and can therefore be used to facilitate artificial reproduction.  相似文献   

13.
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   

14.
Abstract Four parameters were examined in order to define sperm quality in turbot Scophthalmus maximus L., sperm: (1) sperm motility, measured by direct counts of the number of active spermatozoa, expressed as % of total spermatozoa; (2) retention of motility after activation, measured by direct counts, 0–60min after activation, expressed as a % of the initial level of activity; (3) resistance to thermal stress, measured as change in retention of motility, and (4) adenosine phosphate (ATP) concentration, determined for samples of non-activated sperm. The proportion of motile spermatozoa at activation ranged from 34·8% to 97·6% (mean 76·3%) for the individual males tested. Turbot sperm retained on average 52% (range 27–90%) of its initial activity one hour after activation. Sperm samples which were stressed by cooling to –27°C retained only 8·6% (range 0–25%) of initial activity, compared to control samples which retained 49% (range 38–63%) of initial activity. The retention of motility after activation was not significantly related to the initial motility or the levels of ATP. Concentrations of ATP in turbot sperm (mean 0·46mg ATP/106 spermatozoa, equivalent to 9·2nmol ATP/108 spermatozoa) were comparable to those measured in mammals.  相似文献   

15.
The role of the seminal fluid antioxidant system in protection against damage to spermatozoa during in vitro sperm storage is unclear. This study investigated the effect of in vitro storage of sterlet Acipenser ruthenus spermatozoa together with seminal fluid for 36 h at 4 °C on spermatozoon motility rate and curvilinear velocity, thiobarbituric acid reactive substance level, and components of enzyme and non-enzyme antioxidant system (superoxide dismutase and catalase activity and uric acid concentration) in seminal fluid. Spermatozoon motility parameters after sperm storage were significantly decreased, while the level of thiobarbituric acid reactive substances, activity of superoxide dismutase and catalase, and uric acid concentration did not change. Our findings suggest that the antioxidant system of sterlet seminal fluid is effective in preventing oxidative stress during short-term sperm storage and prompt future investigations of changes in spermatozoon homeostasis and in spermatozoon plasma membrane structure which are other possible reasons of spermatozoon motility deterioration upon sperm storage.  相似文献   

16.
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   

17.
《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   

18.
Morphological investigations on the changes in flagellar beating was carried out on native (taken from the milt) and thawed sperm of the Siberian sturgeon Acipenser baerii (Brandt). Immediately after activation, the pattern of flagellar wave formation and distribution was the same in native and thawed sperm but, after 27–42 s, depending on the samples, the thawed flagella showed asymmetric and poorly developed waves. The swimming trajectories recorded during 1‐s exposure were much shorter in thawed than in native sperm after 26–28 s motility. In native sperm, the flagella remained in the same axis as the head during the entire motility course, while the head of thawed sperm showed a right angle after 47 s. It is concluded that the freezing/thawing procedure induces some alteration in the dynamics of flagellar beating in many sperm, but these sperm still show progressive displacement. Therefore, the change in morphology of the flagellum during motion is a parameter that should be taken into account in the evaluation of the impact of various treatments on sperm motility.  相似文献   

19.
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   

20.
Abstract.— Two experiments were designed to improve upon existing methods for cryopreserving striped bass Morone saxatilis , semen. In the first experiment, two extenders, two cryoprotectant concentrations, and two freezing rates were evaluated on the basis of post-thaw semen motility after 1, 7, and 30 d of storage at −196 C. Semen samples cryopreserved at a freezing rate of −40 C/m resulted in a significantly higher percentage of motile sperm ( P < 0.001) and longer duration of spermatozoa motility ( P < 0.001) than samples cryopreserved at a freezing rate of -30 Chin. Also, the cryoprotectant dimethyl-sulfoxide yielded a significantly higher percentage of motile sperm ( P < 0.001) and longer duration of spermatozoa motility ( P < 0.001) when a 5% concentration was used instead of 7.5%. In the second experiment, the two extenders from Experiment I were re-evaluated and a new extender, which was a modified version of Extender 1, was tested. The samples were cryopreserved at -40 C/min with 5% DMSO and thawed in a 25 C water bath. Spermatozoa motility and fertilization ability were evaluated, and semen cryopreserved in Extender 2 yielded the longest duration of spermatozoa motility ( P < 0.001). the highest percentage of motile sperm ( P < 0.001). and the highest percentage of fertilized eggs ( P < 0.002) in comparison to Extenders I and 3.  相似文献   

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