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1.
Abstract. The sperm of tench, Tinca tinca L., is characterized by a milky colour and consistency, and is of very low density. After collecting the sperm, motion of spermatozoa was recorded even without water activation. A better motility value (value 4·36 on average) was observed in spermatozoa collected in immobilizing solution (collecting medium) and stored for 3h, when compared with spermatozoa without collecting medium. Average total and relative numbers of spermatozoa were 12·16 × 109 per male and 18·50 × 109 per kg of body weight, respectively. When testing the effect of activating solution in artificial propagation of tench, the highest fertilization rates (81·3 and 85% in two cases) were found for NaCl solution with an osmotic concentration of 34 or 69 mOsmol and for fresh water, respectively. The fertility rate was reduced significantly ( P < 0·01) by any increase above 105 mOsmol in NaCl concentration in the activating solution. In the tests of optimal method of artificial fertilization, the highest hatching rate of sac fry (71·35%) was found in sperm collected into immobilizing solution. The application of immobilizing solution significantly increased the number of sac fry at the levels P < 0·1 and P < 0·01, if compared with intact sperm stored for 3 h and fresh sperm, respectively.  相似文献   

2.
Various procedures for artificial insemination in tench, Tinca tinca (L.) were re-examined with evaluation of fecundity of males and females among different tench strains. The objectives of this study were to enhance fertilization and hatching rates through optimization of the activation solution, the insemination process, the activation of gametes, and the elimination of eggs stickiness. Sperm for all experiments was collected directly into immobilization solution of modified Kurokura solution containing 180 mM of NaCl and stored at 2 °C for 2.5–5 h prior to the experiment. When dechlorinated tap water was used for activation a gamete ratio of 1150 spermatozoa per egg showed the best significant fertilisation and hatching rates. Optimal ratio between eggs (weight in g) and activation solution (in cm3) was 1:1. Different concentrations of activation solutions such as NaCl from 0 to 68 mM (0–136 mOsmol kg−1) without buffer statistically decreased fertilization and hatching rates. The activation solution containing 17 mM of NaCl, 10 mM Tris–HCl, pH 8 and 9 significantly increased fertilization and hatching rates compared to dechlorinated tap water of pH 7 or activation solution containing 17 mM of NaCl, 10 mM Tris–HCl, pH 6 and 7. Adhesiveness of the eggs was successfully removed by incubation in Alcalase and activity: 3.16 Anson units per cm3.  相似文献   

3.
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   

4.
The tench Tinca tinca is an interesting fish from the viewpoint of polyploidy and related atypical reproduction aspects. Triploid tench were produced artificially. Studies of spermiation as well as of sperm motility and structure were performed on several triploid and diploid males simultaneously with individual experimental crosses with diploid females to define their reproductive capacities. The testes of triploids visually looked less developed in the most of cases with lower sperm production (0.05 cm3 sperm per male), GSI and weight of testes compared to diploids (0.58 cm3 sperm per male). Analysis of variance showed significant influence of ploidy level on the percentage of motile spermatozoa. Triploidy did not change percentage of live spermatozoa and velocity of spermatozoa at the first time of sperm movement. The study of sperm structure by scanning electron microscopy revealed that most sperm cells were of normal structure with some anomalies. Sperm heads of triploid and diploid males were mostly round-shaped, 1.86±0.2 and 1.6±0.18 μm in diameter. The midpiece of triploid spermatozoa was slightly narrower than that of diploid ones with typical cylindrical shape. Flow cytometry revealed sperm cells of triploids to be largely aneuploid (1.47 n) with high mosaic DNA, oscillating from haploid DNA content (1.0 n) to diploid DNA content (1.9 n). Experimental crosses between triploid males and diploid females revealed that these males were capable to stimulate effective development with relatively high level of fertilization and hatching rates from 0 to 70%. In conclusion, triploidization does not seem to guarantee sterility of tench.  相似文献   

5.
The spermiation of tench males was stimulated with Supergestran containing mammalian LHRHa lecireline at the following doses: 5, 10, 20 and 40 g kg−1 b.w.; then with carp pituitary suspension (CPS) at a dose of 2 mg kg−1 b.w. and with a control of saline physiological solution. The following days, meaning 24, 48 and 72 h after injection, sperm was collected to evaluate volume and the number of sperm per male per kg body weight (B.W.) The percentage of motile sperm and velocity of spermatozoa were measured 48 h after hormonal injection, and 72 h after hormonal injection the sperm was evaluated for fertilization and hatching ability. All 42 males in experimental groups were diploid. Live weight did not differ significantly among experimental groups. The strongest stimulation of spermiation was achieved with LHRHa in dosage of 20 and 40 g kg−1 b.w. and CPS compared to males of the control group and lower dosage of LHRHa. Analysis of variance showed no significant influence of the treatment on the velocity and percentage of motile spermatozoa. The effect of different treatment on the fertilization capacity (the number of spermatozoa per egg was equilibrated) was significant. Significantly the highest quality of sperm collected 72 h after injection expressed by percentage of fertilization and hatching (62–65% fertilization and 61–64% hatching rates, respectively) was found for LHRHa in dosage of 20 and 40 g kg−1 b.w. Significantly the lowest parameters of fertilization and hatching were found for the control group, on the 12% level.  相似文献   

6.
The objective of this study was to investigate the effects of extenders and storage time on motility, viability and fertilization of preserved black sharkminnow, Labeo chrysophekadion spermatozoa. Sperm were diluted 1:3 in one of five extenders: modified Cortland solution (MC); Hanks' balanced salt solution (HBSS); 0.9% sodium chloride (NaCl); Kurokura solution (KU); and modified extender, and undiluted sperm samples were used as control and stored at 4°C for 5 days. Motility, viability and fertilization rates were evaluated every day. After a storage time of three days, the highest motility, viability and fertilization rates (61.27 ± 2.26%, 58.60 ± 2.29% and 40.58 ± 0.57, respectively) were achieved with sperm diluted with modified extender. Motility, viability and fertilization rates decreased significantly (P < 0.05) with increasing storage time in all treatments. In addition, this study found that motility, viability and fertilization had a positive significant correlation (P < 0.01). The results indicate that isotonic extender is suitable for the short‐term preservation of black sharkminnow spermatozoa.  相似文献   

7.
《水生生物资源》2003,16(5):450-456
This study summarizes optimization of techniques for common carp artificial propagation including improvements of activation solution (AS), the process of insemination, and elimination of egg stickiness. The optimum gamete ratio for good fertilization and hatching rate ranged from 8490 to 23 672 spermatozoa per egg, when dechlorinated tap water was used. Optimal ratio between eggs (weight in g) and AS (in ml) was defined as 1:1 to 1:2. Different concentrations of AS such as NaCl from 0 to 34 mM (0–68 mOsmol kg–1) did not change fertilization and hatching rates. An AS adopted for carp spermatozoa (45 mM NaCl, 5 mM KCl, 30 mM Tris–HCl, pH 8) was compared with other saline AS; only the 51 mM (102 mOsmol kg–1) NaCl solution decreased fertilization and hatching rate. The AS containing 20 mM Tris–HCl at pH 9 increased fertilization and hatching rates compared to dechlorinated tap water of pH 7 or to AS of pH 6 and 7. Adhesiveness from the eggs was successfully removed by incubation in Alcalase DX (PLN 04715) using two successive steps with different enzyme concentrations. The first step with an enzyme concentration of 2 ml l–1 was applied from 8 to 20 min after fertilization. Later in a second step, the best time for application of alcalase enzyme at a concentration of 20 ml l–1 was for 45 and 60 s at 20 min after fertilization leading to fertilization and hatching rates of 80–87%. The α-Chymotrypsin (EC 3.4.21.1. Merck) was also found effective for elimination of stickiness. Results with α-Chymotrypsin enzyme indicate that the response to success in elimination of stickiness is highly variable mainly due to differences in the environment, quality of water and carp strains.  相似文献   

8.
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   

9.
为提高黄颡鱼(Pelteobagrus fulvidraco)人工授精的稳定性,分别用卵子和精子同步激活法以及预激活精子法,在6个梯度浓度(0%～0.5%)范围的低渗溶液中进行人工授精试验,比较精子活力、受精率和孵化率。结果显示:低渗溶液浓度与精子活力、精子激活率和受精率密切相关,在相同浓度的低渗溶液组中采用预激活精子法的受精率显著高于卵子和精子同步激活法(P<0.05),受精率相对应提高了7.7%～14.5%;低渗溶液的浓度和人工授精方式对孵化率无显著影响。结果表明在浓度0.3%的低渗溶液中采用预激活精子法的效果最好。  相似文献   

10.
Yellow catfish (Pelteobagrus fulvidraco) is a candidate freshwater fish for aquaculture in China with its high consumer demand. The aim of this study was to examine the possibility of storage of the sperm of yellow catfish by cryopreservation in liquid nitrogen. Experiments were designed to investigate the effects of the different combinations of three extenders (Ringer extender, Kurokura-1 extender and D-15 extender) and three cryoprotectants (DMSO, Glycerol and Methanol) on the cryopreservation of yellow catfish sperm. Post-thaw sperm motility, fertilization and hatching rate were detected to evaluate the reliability of sperm cryopreservation. The results demonstrated that Ringer extender and 10% methanol was the best combination for protecting the sperm during freezing in liquid nitrogen by a three-step method and thawing in a water bath at 37 °C for 60 s. In this combination for cryopreservation, sperm maintained the highest post-thaw motility (65 ± 5%), fertilization (90.47 ± 3.67%) and hatching rate (88 ± 4%). And more interestingly, the fertilization and hatching rate were similar to those of fresh sperm (97.55 ± 2.74% and 92 ± 5%). Successful sperm cryopreservation techniques for yellow catfish have been developed for hatchery purpose.  相似文献   

11.
The spermatozoa of oviparous fish, such as feral carp (Cyprinus carpio), are immotile in the presence of semen plasma or isotonic solutions, and to obtain good motility, they must be diluted with suitable medium. The objective of this study was to identify the best activating solution for feral carp sperm. Sperm motilities were compared in the new activating solution (a): (50 mM NaCl, 30 mM KCl, 30 mM Tris, pH = 8.5) and activating solution (b): (50 mM NaCl, 40 mM KCl, 30 mM Tris, pH = 8.5) based on effect of pH with everyone of Na+ and K+ ions versus four other activating solutions Billard’s saline solution, Poupard’s saline solution, distilled water and hatchery water that is routinely used for extending carp semen. Our results showed that maximum total motility period and percentage of motile sperm were seen in selected saline solution (a). The present study describes an activating solution that prolongs feral carp sperm motility.  相似文献   

12.
Effects of NH3 concentration in sea water and pH of sea water on the motility of spermatozoa obtained from testes were examined in the Japanese pearl oyster Percent motility at 30 s after dilution increased with increasing NH3 concentration in sea water from 0.75–2.0 mM. When spermatozoa were diluted with sea water containing 0.75 mM NH3, which is widely used as the insemination fluid in the hatchery of this species, the percent motility increased with time elapsed after dilution, and peaked at 5 min. For spermatozoa diluted with sea water containing 2.0 mM NH3, the percent motility increased rapidly and peaked at 30 s. The pH of sea water increased with increasing NH3 concentration from 8.2 (0 mM NH3) to 9.9 (5.0 mM NH3). When spermatozoa were diluted with artificial sea water at various pH (buffered without NH3 at 6.0–10.0), only spermatozoa diluted with artificial sea water of pH 10.0 were motile, and the percent was considerably lower than those in ammonical sea water. These results indicate that sea water containing 2.0 mM NH3 is a suitable solution for evaluating sperm motility, and that NH3 and/or ammonium ions may activate sperm motility in this species.  相似文献   

13.
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   

14.
The motility and fertilizing ability of the Persian sturgeon, Acipenser persicus, spermatozoa were investigated. Optimum ionic content (Na+, K+, Ca2+ and Mg2+) and pH of activation solution as well as the optimum dilution rate were determined. The results show optimum motility characteristics of spermatozoa in buffered solutions containing 25, 0.2, 3 and 10 mM L?1 Na+, K+, Ca2+ and Mg2+, respectively, at dilution rate 1:50 and pH 8.0. To test the fertilizing ability of sperm, two buffered saline solutions were used as activation solution of sperm motility. The present study indicated (1) spermatozoa motility is one of key factors that influence on fertilizing ability of sperm, (2) a high fertilizing ability of sperm is obtained after dilution in saline solutions rather than in freshwater and (3) a maximum fertilization rate occurs in buffered saline solution containing 0.2 mM L?1 K+. There is also a good correlation between biochemical characteristics of seminal plasma and fertilizing ability of sperm.  相似文献   

15.
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   

16.
With the aim of improving artificial androgenesis in teleost fishes, we tested two methods for producing androgenetic diploids of amago salmon (Oncorhynchus masou ishikawae), namely, fertilization of gamma-ray irradiated eggs with fused spermatozoa (sperm-fusion method) and the fertilization of irradiated eggs with untreated sperm followed by the blocking of cell division (mitosis-inhibition method). Our results showed that the optimal condition for sperm fusion was to treat the sperm with 50% polyethylene glycol (molecular weight 7500) for 100 s. The efficiency of the two methods of androgenesis was compared in terms of fertilization rate, hatching rate, and larval survival after hatching. The rate of fertilization was lower with the sperm-fusion method than with the mitosis-inhibition method, but the reverse was true for the hatching rate. The survival rate of hatched larvae was the same with the two methods. Androgenesis was confirmed with a recessive albino color marker, and all viable offspring were found to be heterozygous based on analysis of the microsatellite markers. Our results suggest that androgenesis with the sperm-fusion method is a promising approach with potential applications in both aquaculture breeding programs and the preservation of endangered freshwater fishes.  相似文献   

17.
Large yellow croaker, Pseudosciaena crocea, exhibit sexually dimorphic growth, with females growing faster and reaching larger adult sizes than males. Thus, development of techniques for preferentially producing females is necessary to optimize production of these species. We have established a protocol to produce all-female croaker P. crocea through induction of meiotic gynogenesis with homologous sperm. The first set of experiments investigated the ultra-violet (UV) irradiation on sperm motility and duration of sperm activity to determine the optimal UV dosage for genetic inactivation of sperm, yet retaining adequate motility for activation of eggs. Milt from several males was diluted 1:100 with Ringer’s solution and UV irradiated with doses ranging from 0–150 J cm−2. The results indicated that motility and duration of activity generally decreased with increased UV doses. At UV doses greater than 105 J cm−2, after fertilization, motility was <10% and fertilization rates were significantly lower. Highest hatching rate was obtained at 75 J cm−2. A second set of experiments was carried out to determine appropriate conditions of cold shock for retention of the 2nd polar body in P. crocea eggs after fertilization with UV-inactivated sperm by altering the timing, temperature and duration of shock. At 20°C, shock applied at 3 min after fertilization resulted in higher survival rate of larvae at 6 h after hatching. Results of different combinations of three shock temperatures (2°C, 3°C or 4°C) and five shock durations (4 min, 8 min, 12 min, 16 min or 20 min) at 3 min after fertilization demonstrated that shocks of 12 min gave highest production of diploid gynogens. Statistical analysis revealed that maximum production of diploid gynogens (44.55 ± 2.99%) were obtained at 3°C. The results of this study indicate that the use of UV-irradiated homologous sperm for activation of P. crocea eggs and cold shock for polar body retention is an effective method for producing gynogenetic offspring.  相似文献   

18.
The effects of extender composition, cryoprotectant, and freezing rate on post-thaw rainbow smelt Osmerus mordax sperm motility were examined, and the fertilization capacity of fresh and post-thaw sperm were compared. The highest post-thaw motility (75%) was obtained when milt was diluted 1:3 with an extender containing 600 mM sucrose supplemented with 10% dimethyl sulfoxide and 1.5% bovine serum albumin and frozen at a rate of –20 C/min. Post-thaw motility for sperm stored in this extender was similar to fresh sperm and did not change after 90 d of storage. Furthermore, there were no differences in fertilization rate or embryo survival to the eyed stage between fresh and post-thaw sperm frozen in this extender. The lowest post-thaw motility was observed when sperm were frozen with methanol at a rate of -30 C/min. Refrigerated sperm diluted 1:3 with the 600 mM sucrose extender remained motile for 30 d. These data demonstrate that rainbow smelt spermatozoa can be effectively used following short and long-term storage using a simple, sucrose-based extender.  相似文献   

19.
This study reports on the spermatological properties, and on the development of a protocol for refrigerator storage (4°C) of Labeo calbasu (Hamilton, 1822) sperm for artificial breeding. Volume, motility, concentration and pH of the freshly collected sperm were 2.21 ± 0.53 (μL g?1 of fish weight) (mean ± SD), 95 ± 1 (%), 1.93 ± 0.44 × 109 (cells mL?1) and 7.56 ± 0.17 respectively. Sperm activation was evaluated at different osmolalities of NaCl solution, and motility ceased completely when osmolality of the extender was ≥287 mOsmol kg?1. Sperm retained motility for 24, 72 and 108 h, after refrigerator storage when sperm were undiluted, suspended in Alsever's solution and suspended in Alsever's solution containing 5% methanol respectively. Fertilization rate of fresh eggs with sperm stored at 4°C in Alsever's solution and Alsever's solution containing 5% methanol was 77% and 60% with a hatching rate of 60% and 43% respectively. The fertilization and hatching success of the stored sperm suggests potential to use refrigeration for transporting genetic material to hatcheries for artificial breeding of L. calbasu in Bangladesh.  相似文献   

20.
Basic characteristics of the European smelt (Osmerus eperlanus) sperm are reported here for the first time. Smelt spermatozoa had a bullet-shaped head (1.42 μm length), a short midpiece and a long flagellum (27.72 μm). Two mitochondria were located along the flagella. The volume of smelt sperm was small (30-60 μl) and the duration of sperm motility was short (22 s in distilled water and 41 s in 20 mM sodium bicarbonate solution). Sodium chloride at concentrations ranging from 0-120 mM did not influence the percentage of motile spermatozoa but caused a steady increase in the duration of sperm movement. Potassium ions clearly reduced the percentage of motile sperm at a concentration of 10 mM. Spermatozoa were motile through a broad range of pH with an optimum from 7.5 to 8.5. Testicular spermatozoa had a different motility pattern compared to stripped spermatozoa (the latter exhibiting a reduction of motile spermatozoa by 30%, lower ALH and VCL and higher LIN and VSL). These results indicate that maturation of smelt spermatozoa occurring in sperm ducts is related not only to an increase of the percentage of motile spermatozoa but also to changes in the sperm motility pattern. Maintaining males with females resulted in stimulation of milt production. Our results indicate that European smelt sperm characteristics are similar to those of ayu (Osmeridae).  相似文献   

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