Motility parameters of perch spermatozoa (Perca fluviatilis L.) with cryoprotectors addition

The addition of cryoprotectants during the freezing of semen in liquid nitrogen protects spermatozoa from the negative influence of freezing. Every species needs an appropriate cryoprotectant that has to be experimentally selected. Semen obtained from five perches was diluted with the Kobayashi buffer solution at 1:9 ratio. To determine the influence of cryoprotectants on spermatozoa motility parameters, the same type of buffer solution was applied with the addition of methanol, dimethyl sulfoxide (DMSO) and dimethylacetamide (DMA) using the concentration of 10, 5, 2.5 %, respectively, glycerol (15; 7.5 %), sucrose and trehalose (0.45; 0.225; 0.113 M). After the preparation of such tests, parameters of spermatozoa motility were measured, using the CASA system (Image House CRISMAS Company Ltd.). Among used cryoprotectants, methanol did not cause any effect on the sperm motility parameters. The lowest percentage concentrations of DMA, DMSO, glycerol, sucrose and trehalose did not significantly influence the percentage of motile spermatozoa. Higher concentrations of these compounds considerably lowered all motility parameters. As for glycerol and saccharides, their addition resulted in the lowering of the spermatozoa motility possibly due to a higher viscosity of the solution. However, DMA and DMSO were most probably toxic to perch sperm cells. The obtained results indicate that the best cryoprotectant to be used with perch spermatozoa is methanol.     

The effect of K+, Ca2+, and Mg2+ on sperm motility in the perch,Perca fluviatilis

This study investigated the effect of 0.25–5 mM K⁺, Ca²⁺, and Mg²⁺ on sperm motility in the perch, Perca fluviatilis. In 75 mM NaCl, the used motility-activating solution, motility rate, and swimming velocity decreased within the first 4 min after activation, and the rate of locally motile sperm increased. Thereafter, the motility parameters remained constant for periods >20 min. Based on the decrease in sperm motility, two types of semen samples could be distinguished. Semen samples of type I retained a high motility rate of >65 % after 20 min, and the rate of locally motile sperm was <20 %. In semen samples of type II, the motility rate decreased to values <30 % after 20 min, and the rate of locally motile sperm exceeded >50 %. Ca²⁺ and Mg²⁺ concentrations of 0.25–0.5 mM had no effect on the sperm motility parameters 10 s after activation, while 0.25 mM K⁺ increased the swimming velocity. K⁺, Ca²⁺, and Mg²⁺ concentrations ≥1.5 mM had suppressive effects on the sperm motility 10 s after activation. No differences were found between the two semen types. Twenty minutes after activation, type I semen was not affected by the tested cations. On the contrary, 0.25–2.5 mM K⁺, 0.25 mM Mg²⁺, and 0.25–2.5 mM Ca²⁺ significantly increased the sperm motility rate and/or sperm velocity of type II semen. Therefore, supplementation of saline solution with cations might stabilize the motility of perch sperm, which can be a benefit for experimental purposes and for specific handling procedures in aquaculture.     

Protection of common carp (Cyprinus carpio L.) spermatozoa motility under oxidative stress by antioxidants and seminal plasma

The protective influence of seminal plasma and the antioxidants catalase (CAT), superoxide dismutase (SOD), and glutathione (GTH) on quality parameters, oxidative stress indices, and antioxidant activity was studied in common carp (Cyprinus carpio) spermatozoa exposed to the xanthine–xanthine oxidase (X–XO) system. Fish spermatozoa were incubated for 5 and 20 min at 4 °C with X–XO concentrations of 1 mM X–0.1 U/mL, 0.6 mM X–0.05 U/mL, 0.3 mM X–0.025 U/mL, and 0.1 mM X–0.0125 U/mL. A dose-dependent reduction in spermatozoa motility and velocity was observed at concentrations of 0.1 mM X–0.0125 U/mL to 1 mM X–0.1 U/mL XO. Increase in spermatozoa motility parameters was recorded following treatment with antioxidants and seminal plasma. The level of the oxidative stress indices lipid peroxidation (LPO) and carbonyl derivatives of proteins (CP) was significantly reduced after addition of CAT, SOD, or GTH along with seminal plasma. Significant differences in SOD, glutathione reductase, and glutathione peroxidase activity were seen in spermatozoa incubated with, compared to that without, seminal plasma at all studied X–XO concentrations. The data demonstrated that CAT, SOD, or GTH in combination with SP can reduce reactive oxygen species stress in fish spermatozoa and improve spermatozoa quality.     

Sperm quality and selected biochemical markers of seminal plasma at the beginning of the reproductive period of common carp, Cyprinus carpio L.

Changes in semen quality and selected biochemical markers were analyzed during a week of spawning season of common carp Cyprinus carpio L. Semen was obtained twice, on May 30 and on June 7, and each time it was collected 24 h after hormonal stimulation using Ovopel [(d-Ala⁶, Pro⁹ NEt)-mGnRH + metoclopramide] in 1 pellet kg^?1. The total volume of semen (ml), volume of semen per kg of body weight (ml kg^?1 b.w.), sperm concentration (×10⁹ ml^?1), total number of sperm per kg of body weight (×10⁹ kg^?1 b.w.), pH of semen, pH of seminal plasma, seminal plasma osmotic pressure (mOsm kg^?1) and the total protein content in seminal plasma (mg ml^?1) were determined. A 10 mM Tris buffer containing 100 mM NaCl with 0.5 % BSA (pH 9.0, osmolality 200 mOsm kg^?1) was used to activate sperm. The following computer-assisted sperm analysis (CASA) parameters were determined: percentage of motile sperm (MOT, %), progressively motile sperm (PRG, %), curvilinear velocity (VCL, μm s^?1), straight-line velocity (VSL, μm s^?1), movement linearity (LIN, %), wobbling index (WOB, %), amplitude of lateral head displacement (ALH, μm) and beat cross-frequency (BCF, Hz). The volume of semen per kg of BW, total number of sperm per kg of BW and semen pH were significantly lower at the second semen sampling compared to the first semen sampling. Volume of semen at the second sampling correlated positively with CASA parameters. A lack of differences among CASA parameters between both collection periods indicates good quality of carp sperm hormonally stimulated with Ovopel twice at a 1-week interval.     

Relationship between biochemical constituents of fish semen and fertility: the effect of short-term storage

Spermatozoa and seminal plasma obtained from rainbow trout and whitefish were analyzed in respect to their aspartate aminotransferase (AspAT) and alkaline phosphatase activities. In particular, the experiments characterized AspAT optimum pH, optimization of assay conditions and action of coenzyme, pyridoxal 5-phosphate (vitamin B₆). The effect of short-term semen storage at 0°C on biochemical indicators and fertilization rate was examined in both species. The concentrations of reduced and oxidized ascorbic acid in seminal plasma of both species were several folds higher than in spermatozoa and blood plasma of fish. Highly significant correlations were found for both species between AspAT activity (sperm or seminal plasma) and fertilization rate (% of eyed-stage or hatched embryos). For rainbow trout, highly significant correlations were found between sperm concentration, motility and fertilization rate. These results suggest that several biochemical indicators of seminal plasma can be used as measures of sperm quality of fish. Some common biochemical parameters for fish and mammal's semen provide evidence for using fish sperm as a model in biomedical research.     

Effect of the addition of six antioxidants on sperm motility,membrane integrity and mitochondrial function in red seabream (<Emphasis Type="Italic">Pagrus major</Emphasis>) sperm cryopreservation

The present study was to evaluate the effects of six antioxidants on frozen-thawed sperm motility, viability, membrane integrity and mitochondrial function in red seabream (Pagrus major) by computer-assisted sperm analysis system and flow cytometry, respectively. All the parameters tested in this study were determined using one-way ANOVA and identified using the SNK test (P < 0.05). The results demonstrated that on the first day, the highest motility and longevity occurred in 100 mM trehalose (78.34 ± 3.41 %, 29 ± 4.00 days) and 50 mM taurine (77.46 ± 1.54 %, 29.33 ± 4.04 days), followed by 25 mM vitamin C (79.03 ± 5.37 %, 17 ± 1.00 days), 25 mM vitamin E (69.64 ± 1.64 %, 27.67 ± 1.53 days) and 25 mM vitamin A (78.89 ± 2.81 %, 9.33 ± 1.53 days), which were all higher than frozen-thawed sperm without antioxidant (control) (66.80 ± 5.55, 5.67 ± 1.15 days). Especially, the percentages of class A sperm with the addition of 100 mM trehalose (40.39 ± 5.20 %) and 50 mM taurine (37.78 ± 3.22 %) were significantly improved compared to the control (19.63 ± 5.44 %). The viability of all groups on the third and sixth day showed a similar trend. Moreover, during the 4 °C storage process, the decrease of frozen-thawed sperm motility was closely associated with the decrease in membrane integrity and mitochondrial function. In conclusion, the present study indicated that antioxidant (100 mM trehalose and 50 mM taurine) provided the most pronounced protective effect in improving frozen-thawed quality of red seabream sperm. The addition of antioxidant may be capable of scavenging the ROS generated during the cryopreservation process and 4 °C storage.     

Characterization of rainbow trout milt collected with a catheter: semen parameters and cryopreservation success

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.     

Application of Computer‐assisted Sperm Analysis in Selecting the Suitable Solution for Common Carp,Cyprinus carpio L., Sperm Motility

The common carp, Cyprinus carpio L., sperm motility parameters were analyzed by using computer‐assisted sperm analysis system. The percentage of motile sperm (MOT, %), progressively motile sperm (PRG, %), curvilinear velocity (VCL, µm/sec), average path velocity (VAP, µm/sec), the wobbling index (WOB, %), movement linearity (LIN, %), beat cross frequency (BCF, Hz), and amplitude of lateral head displacement (ALH, µm) were determined. Five activation solutions (As) were used to activate sperm movement. As 1 solution: 68 mM NaCl, 50 mM urea, 0.5% bovine serum albumin (BSA), pH: 7.7, 181 mOsm/kg; As 2 buffer: 100 mM NaCl, 10 mM Tris, 0.5% BSA, pH: 9.0, 199 mOsm/kg; As 3 solution: 86 mM NaCl, 0.5% BSA, pH: 7.4, 167 mOsm/kg; As 4 buffer: 5 mM KCl, 45 mM NaCl, 30 mM Tris, 0.5%, pH: 8.0, 160 mOsm/kg; and As 5 solution: distilled water with the addition of 0.5% BSA, pH: 7.3, <3 mOsm/kg. Among five tested solutions, a buffer with a pH of 9.0 and osmolality of approximately 200 mOsm/kg (As 2) was the most suitable. After its activation, a significant increase in MOT and ALH values was observed, which can be of importance to the effectiveness of egg fertilization .     

Changes in osmotic pressure that trigger the initiation of spermmotility in the river sculpin Cottus hangiongensis

The effects of changes in osmotic pressure on the initiation of spermmotility in the river sculpin were studied. When the fresh milt was diluteddirectly with solutions of mannitol at various concentrations, 22% ofthe spermatozoa became motile in isotonic (300 mM) solution, 63%became motile in a solution of 200 mM, and more than 90% becamemotile in a solution of 100 mM and in 20 mM HEPES-NaOH. When the spermatozoawere transferred to various osmotic conditions after the termination ofswimming in the isotonic solution, 27.9% of spermatozoa became motilein the solution of 200 mM, while more than 80% of spermatozoa becamemotile in the solution of 100 mM and in the buffer solution. Aftertermination of swimming in the solutions of 300 mM and 200 mM, more than80% of spermatozoa became motile again when they were transferred toa solution of 100 mM and to the buffer solution. After the termination ofswimming in the solution of 100 mM, most of spermatozoa were immotile evenwhen they were transferred to the buffer solution. These results suggest theexistence of multiple osmotic switches in each sperm cell that initiate themotility; one reacts in response to the osmotic conditions above 200 mOsmkg^-1, with the threshold varying for each cll, while anotherswitch, which is common to all the cells, reacts in response to osmoticpressure below 200 mOsm kg^-1. The existence of the latterswitch was confirmed by the motility in the solution of 100 mM and in thebuffer solution in this study.     

The antioxidant system of seminal fluid during in vitro storage of sterlet <Emphasis Type="Italic">Acipenser ruthenus</Emphasis> sperm

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.     

Optimisation of sodium and potassium concentrations and pH in the artificial seminal plasma of common carp <Emphasis Type="Italic">Cyprinus carpio</Emphasis> L.

The effect of sodium and potassium concentrations as well as optimal pH on the motility of common carp Cyprinus carpio L. sperm during short-term storage in artificial seminal plasma (ASP) was investigated. Sperm was collected from individual males (n?=?5) and each sample diluted tenfold (1:9) in ASP (sperm:extender) containing 2 mM CaCl₂, 1 mM Mg₂SO₄ and 20 mM Tris at pH 8.0 and supplemented by the following concentrations of sodium and potassium (mM/mM): 0/150, 20/130, 40/110, 75/75, 110/40, 130/20 and 150/0. The osmolality of all ASP variants was set at 310 mOsm kg^?1. Sperm motility was measured using a CASA system during 72 h of storage. Immediately after dilution, sperm motility was high (90%) both in each variant and in the control group (fresh sperm). After 72-h storage, the highest sperm motility was noted in ASP containing 110 mM NaCl and 40 mM KCl. No differences were found in the motility of samples preserved within the pH range of 7.0–9.0. Our data suggest that for the short-term storage of common carp sperm, whereas the pH of the solution does not play a crucial role, a specific potassium concentration of around 40 mM is required.     

Effects of cysteine supplementation on the quality of cryopreserved sperm of South American silver catfish

The cryopreservation promotes cellular damage that could compromise sperm quality in terms of motility and fertility rates, which may be caused by oxidative stress. Thus, the aim of this study was to assess the effects of cysteine addition on post‐thaw sperm quality, DNA damage and indices of oxidative stress of the South American silver catfish (Rhamdia quelen) sperm, compared with the cryoprotectant solution without cysteine addition. Sperm collected from five males were cryopreserved in cryoprotectant solution (fructose 50 g/L, powdered milk 50 g/L and methanol 100 ml/L) containing different cysteine concentrations (0, 2.5, 5, 10 and 20 mM). After thawing, the following were measured: sperm motility, morphology, sperm viability, DNA damage, lipid peroxidation, concentration of carbonyl and sulfhydryl groups and the activity of SOD, CAT, GST and GPx enzymes. The lowest sperm motility was determined for semen cryopreserved with addition of 20 mM of cysteine. The control group had the lowest DNA damage and lipid peroxidation. The findings of this study show that cysteine addition had no positive effect on evaluated parameters. Therefore, the concentrations tested are not recommended for the supplementation of cryoprotectant solution for semen of R. quelen.     

The Cryopreservation of Striped Bass Morone saxatilis Semen

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.     

Semen biology and stimulation of milt production in the European smelt (Osmerus eperlanus L.)

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).     

Sperm of feral carp <Emphasis Type="Italic">Cyprinus carpio</Emphasis>: optimization of activation solution

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.     

Extenders and cryoprotectants for cooling and freezing of piracanjuba (Brycon orbignyanus) semen, an endangered Brazilian teleost fish

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.     

Effect of two commercial preparations containing different GnRH analogues with dopamine antagonists on barbel Barbus barbus (L.) sperm quantity and quality

The effectiveness of applying Ovaprim [(D-Arg⁶, Pro⁹NEt)-sGnRH + domperidone] and Ovopel [(D-Ala⁶, Pro⁹NEt)-mGnRH + metoclopramide] to male barbel Barbus barbus (L.) 6, 12 and 24 h after hormonal stimulation was analyzed. The control group (Control) during each time interval was stimulated with 0.9 % NaCl. Milt was collected from seven fish only once (n = 7) for Ovopel, Ovaprim and Control group in order to determine total volume of milt, volume of milt per kg of body weight, sperm concentration, total sperm production, seminal plasma osmotic pressure, pH of milt and pH of seminal plasma. Woynarovich’s solution (68 mM NaCl + 50 mM urea) with the addition of 0.5 % BSA (pH 7.7; 181 mOsm kg^?1) was used as the activating liquid. Selected parameters of sperm motility (MOT %) and progressively motile sperm (%), curvilinear velocity (VCL, μm s^?1), straight-line velocity (μm s^?1), movement linearity (%), wobbling index (%), amplitude of lateral head displacement (μm) and beat cross frequency (Hz) were determined using the Computer-assisted sperm analysis system. A time of 6 h proved to be too short to obtain milt from barbel following hormonal stimulation with Ovaprim and Ovopel. Extending the time to 12 h, however, resulted in 100 % spermiation in males, regardless of hormonal preparation used for stimulation. The stimulation of spermiation in barbel is best performed using Ovopel 12 h upon application. Extending the latency period to 24 h following the application of this preparation results in a significant decrease in the volume of milt obtained, sperm count and motility parameters, including MOT and VCL, which may influence sperm fertilization ability.     

Dietary n‐3/n‐6 ratio affects the biochemical composition of Eurasian perch (Perca fluviatilis) semen but not indicators of sperm quality

In general, the effects of dietary fatty acids (FA) on sperm quality have received less attention than egg quality, and were never studied in perch. This study investigated the effects of dietary FAs on the quality and chemical composition of sperm in Eurasian perch (Perca fluviatilis). Two experimental diets containing 16% lipids and 45% proteins were compared. The n‐3/n‐6 ratios tested were 0.2 for diet 1 (D1) and 7.0 for diet 2 (D2). No significant effects of the n‐3/n‐6 ratio were observed on the sperm characteristics, either in terms of the sperm volume (around 1.2 mL) and density, spermatozoa motility (94%) and velocity, or the sperm osmolality. All these parameters corresponded to semen of good quality in Eurasian perch. Interestingly, both the FA composition and the lipid class profile of the semen were correlated to the tested diet. However, basal levels of certain highly unsaturated FAs such as eicosapentaenoic acid, 20:5 n‐3 and docosahexaenoic acid, 22:6 n‐3, were maintained in the sperm irrespective of the diet tested. Perch semen was characterized by high levels of cholesterol, phosphatidylethanolamine and phosphatidylcholine. In conclusion, the dietary n‐3/n‐6 ratio affects the lipid composition of perch semen but not the indicators of sperm quality.     

Sperm motility and seminal plasma characteristics in Barbus sharpeyi (Günther, 1874)

Spermatozoa concentration, ionic composition, osmolality, glucose and total protein contents of seminal plasma and sperm motility were determined in Barbus sharpeyi (Cyprinidae, Teleosotei). Spermatozoa concentration ranged from 9.77 to 20.20 × 10⁹ spermatozoa mL^?1. Osmolality (mOsmol kg^?1) and ionic contents (mM L^?1) of the seminal plasma were 274.5±9.0, 70.0±3.4 Na⁺, 28.8±0.9 K⁺, 101.7±3.1 Cl^?, 0.9±0.1 Mg²⁺ and 2.1±0.1 Ca²⁺ respectively. Total protein and glucose were 5.3±0.2 g L^?1 and 76.7±4.3 mM L^?1 respectively. Sperm motility was initiated in a hypo‐osmotic condition, composed of either an ionic (KCl or NaCl) or a non‐ionic (sucrose) activation medium. Duration of sperm motility was very short: <2 min after activation in distilled water. Percentage of motile spermatozoa was significantly higher in an activation medium containing NaCl compared with that of distilled water. An activating medium containing NaCl or KCl higher than 150 mM or sucrose higher than 275 mM totally inhibited the activation of sperm motility. Immediately after sperm activation, wave(s) propagated along the flagellum, but waves were restricted to the proximal part of the flagellum (close to the head) at 1 min post activation. Studied characteristics in the present study were compared with those of other cyprinids for understanding inter‐species differences.     

金乌贼纳精囊组织结构及其精子的储存和利用

为揭示金乌贼精子进入纳精囊及产卵过程中的精子利用方式,丰富金乌贼繁殖生物学研究内容,本研究利用实验生态学和组织切片技术,检测了交配后不同时间段雌性口膜表面精子囊和纳精囊中精子数量的变化,观察分析了雌性金乌贼纳精囊的组织结构。结果显示,金乌贼纳精囊位于繁殖期雌性个体口膜腹面的突起处,共1对。纳精囊开口于口膜内表面,通过一根中央管连通整个纳精囊。中央管内壁含有大量褶皱和纤毛。在中央管两端,有12~20个储精小囊与之相连。储精小囊四周具有发达的环肌,其中储存有大量精子,并且大部分精子头部均朝向腔室内壁。完成一次交配后,雌性金乌贼对精子囊和纳精囊中精子的利用可以分为三个阶段,主要利用精子囊中的精子(交配后1~2 d);由利用精子囊中的精子向纳精囊中的精子过渡(交配后2~3 d);主要利用纳精囊中的精子(交配后3 d以上)。研究表明,从精子囊释放出的精子进入雌性口膜表面的褶皱中,通过自身运动到达纳精囊。进入纳精囊的精子通过自身运动及中央管内壁纤毛的摆动进入储精小囊,其中大部分精子头部朝向储精小囊内壁有规律地分布。在产卵过程中,雌性优先利用精子囊中的精子,而在精子囊中精子不足时,纳精囊通过肌肉收缩以及纤毛摆动将其中的精子逐渐释放出来,卵子在雌性口膜附近完成体外受精。