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Chemical investigation of the methanolic extract of Cornulaca monacantha (Amaranthaceae), an annual wild herb collected from North Sinai, Egypt, yielded a new isoflavone cornulacin 1 and five known compounds: N-trans-feruloyltyramine 2, N-trans-feruloyl-3'-methoxytyramine 3, N-trans-caffeoyl tyramine 4, Cannabisin F 5 and (2aS, 3aS) lyciumamide D 6. Using MTT assay, the isolated compounds were evaluated for their in vitro cytotoxicity against pancreatic (Panc1) and ovarian (A2780) cancer cell lines. Compounds 1, 2, 3, and 4 exhibited promising cytotoxic activity against the tested cells, among which compound 1 (IC50 of 2.1 ± 0.21 µM) was the most active one against A2780 cells, whereas compound 2 (IC50 of 3.4 ± 0.11 µM) was the most effective compound against Panc1 cells. Accordingly, compound 1 was further investigated for its apoptotic induction in A2780 cancer cells using Annexin V/PI staining. Compound 1 significantly stimulated apoptotic ovarian A2780 cancer cells by 45.9-fold and arrested cell proliferation in the S-phase. Such activity was mediated through the upregulation of proapoptotic genes Bax; P53; and caspase 3, 8, and 9 besides the downregulation of the Bcl-2 gene, the anti-apoptotic one. Furthermore, molecular docking investigation demonstrated the strong binding affinity of compound 1 with EGFR active sites, which validated its experimental EGFR enzyme inhibition activity.

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Polycystic ovary syndrome (PCOS) is a prevalent endocrinologic and gynecologic disorder that affects women of reproductive age; besides, insulin resistance (IR) occurs in 50-70 % of PCOS cases. Metformin (Met) is commonly prescribed for IR management; however, it does not affect IR with some gastrointestinal symptoms. Spirulina platensis (SP) is a blue-green alga that may increase insulin sensitivity. Therefore, our study aims to evaluate SP as an alternative treatment to Met for improving glucose homeostasis by assessing the expression of 11 crucial genes involved in the insulin signaling pathway. After induction of the PCOS model using dehydroepiandrosterone (DHEA) (60 mg/kg bwt) for 30 consecutive days, rats were allocated into six groups. Relative liver weight, glutamic pyruvic transaminase (GPT) serum levels, glutamic-oxaloacetic transaminase (GOT), and insulin were determined. Furthermore, the gene expression of Ins1, Irs1, Pik3ca, Prkcz, Foxo1, Srebf1, Ppargc1a, Pklr, Gk, G6pc, and Pepck in the rat's liver tissue was determined using qRT-PCR. Treatment of the PCOS control group with Met or SP revealed a decrease in all these parameters compared with the PCOS model. Additionally, we found a statistically significant difference in the expression of both the Gk and Prkcz genes. To summarize our study results, SP or Met supplementation to PCOS rats had almost the same effect on assessed relative liver weight, GOT, GPT, and insulin levels compared with PCOS control rats. If further studies confirm and detect more impact of SP on IR in PCOS, SP could be used instead of Met since the latter causes many side effects.

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Species may be prevented from interspecific hybridization by a number of different reproductive barriers that operate precopulatory and postcopulatory. In situation, when natural precopulatory reproductive barriers are affected by anthropogenic factors, postcopulatory reproductive barriers may be important for maintaining gametic isolation and hence preventing interspecific hybridization. This is highly topical in sturgeon (order Acipenseriformes) which exhibits remarkable ease of interspecific hybridization. The objectives of the present study were to evaluate the fertilization success of Acipenser ruthenus and Acipenser baerii spermatozoa under the interspecific competitive conditions and assessed, whether their spermatozoa tend to differentially fertilize eggs of conspecifics. We set up several in vitro fertilization experiments: (i) pooled eggs of both species were fertilized by sperm of each species separately; (ii) eggs of each species were fertilized by pooled sperm; (iii) pooled eggs were fertilized by pooled sperm and (iv) purebred and hybrid control groups. Using parental assignment by molecular markers, we found that when these species competed in pooled sperm, 78.9% of progeny were sired by A. ruthenus and 21.1% by A. baerii, demonstrating higher fertilization success for the former, irrespective of conspecificity of fertilized eggs. When pooled eggs were inseminated by A. ruthenus or A. baerii sperm separately, progeny almost equally comprised hybrid and purebred individuals. Hence, neither A. ruthenus nor A. baerii eggs showed a tendency to biased fertilization by spermatozoa of conspecific males. These findings together show that there may not be postcopulatory mechanisms preventing hybridization between A. ruthenus and A. baerii.

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The present study examined the lipid composition of plasma membranes in carp sperm with different post-thaw motility. The approach adapted for carp sperm cryopreservation, which involves the selection of the most effective protocol for individual males by comparing two cryoprotective media, was applied to the cryopreservation procedure. Sperm motility prior to freezing was greater than 80% but decreased to 40% in one group and to 10% in another group following cryopreservation. Lipid content of fresh sperm in all groups was analysed by thin layer chromatography and gas chromatography, with significant differences in phospholipid content, cholesterol and free fatty acids detected between groups, whereas the cholesterol/phospholipid ratio was extremely similar between groups (0.52 ± 0.038 and 0.52 ± 0.022). Increasing concentrations of saturated fatty acids, monounsaturated acids and decreasing concentrations of polyunsaturated n-6 fatty acids were negatively correlated (P < 0.05) with post-thaw motility of the carp sperm.

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Artificial propagation of sturgeons is becoming increasingly important for recovery efforts as well as for commercial production. Sterlet Acipenser ruthenus is a common Eurasian sturgeon with a small body size and one of the fastest reproductive cycles among the sturgeons. The practical question being addressed in this study was how long fertilization of ovulated eggs can be delayed without substantially reducing the hatching rate, and an ancillary question is under what' temperature conditions do eggs retain good quality. Broodstock were injected with homogenized carp pituitary extract (CPE); ovulated eggs from three females were allocated to various treatment groups for temperature storage (control, 7°C, 11°C, 15°C and 19°C) until fertilized. Storage times at the regulated temperatures prior to fertilization were for 2.5, 5.0, 7.5 and 10.0 h. After the selected storage times in ovarian fluid, eggs were fertilized and transferred to incubation cages and then they were counted. Three replicates were allocated to each storage period and temperature. Hatched larvae were counted at 7-day post-fertilization. We found that sterlet eggs do not need to be fertilized immediately after collection. Reasonably good quality was retained for several hours if temperature conditions are fairly cool and stable. Eggs retained good quality when stored at 7°C and 11°C for up to 10 h with 54.1 ± 2.9 to 69.9 ± 7.9% hatching success, but egg quality was significantly reduced after 5-h storage at 19°C (p < 0.01) and 7.5-h storage at 15°C (p < 0.05) compared to cooler temperatures. Uniform temperatures between 7°C and 11°C can be considered as appropriate for storage of eggs in ovarian fluid for up to 10 h. This information can have practical application to routine hatchery practice for acipenserids, as well as for certain research protocols.

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Sturgeon spermatozoa maturation during their passage through the kidney is a prerequisite for initiation of motility. Samples of sterlet () testicular sperm (TS) were matured in vitro by incubation in seminal fluid (SF) or in SF supplemented with carbonyl cyanide -chlorophenyl hydrazone (CCCP; a respiration uncoupling agent). Sperm was diluted in activation medium (AM) containing 10 m Tris-HCl buffer (pH 8.5) and 0.25% Pluronic, and spermatozoon motility was assessed. Samples were taken and fixed in 3 perchloric acid at 3 points in the incubation process. Quantification of ATP, ADP, and creatine phosphate (CrP) was conducted using liquid chromatography/high-resolution mass spectrometry. We observed a significant decrease in CrP during artificial maturation of TS in SF. In contrast, ATP and ADP were not significantly affected. Addition of CCCP to SF halted maturation and led to significantly lower CrP whereas ADP significantly increased and ATP was unaffected. Dilution of matured and immature TS with AM led to a significant decrease of ATP and CrP and an increase of ADP compared with their levels before dilution, although immature TS were not motile. Energy dependency of TS maturation in sturgeon was confirmed, which suggests that mitochondrial oxidative phosphorylation is needed for maturation of sturgeon TS.

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The role of environmental ion composition and osmolality in calcium ion (Ca(2+) ) signalling of spermatozoa activation over the course of the spawning period of brook trout Salvelinus fontinalis was investigated. Motility at the end of spawning was low (mean ± s.d. of 5 ± 2% motile spermatozoa with curvilinear velocity of 25 ± 8 µm s(-1) ). Addition of 10 mM Ca(2+) to the activation medium resulted in values similar to those recorded during the middle of the spawning period (mean ± s.d. of 95 ± 6% motile spermatozoa with curvilinear velocity of 130 ± 15 µm s(-1) ).

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Sturgeon spermatozoa are immotile in the testis and acquire the potential for motility after contact with urine in Wolffian duct. The present study tested if in vitro incubation of testicular sperm in seminal fluid from Wolffian duct sperm leads to the acquisition of sperm fertilization ability. Sterlet sperm was taken from the testes, matured in vitro and cryopreserved. The fertility and motility of cryopreserved semen were tested. Matured testicular sperm showed freeze-thaw survival rates similar to Wolffian duct sperm, which is commonly used in sturgeon artificial propagation. Matured testicular sperm and Wolffian duct sperm post-thaw motility rate and curvilinear velocity were not significantly different, while duration of matured testicular sperm motility was significantly shorter than that of Wolffian duct sperm. Development rates of embryos obtained with post-thaw matured testicular sperm and Wolffian duct sperm were not significantly different. In vitro maturation of sterlet testicular sperm can potentially be useful in sperm cryobanking.

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We applied comparative genomic hybridization (CGH) and genomic in situ hybridization (GISH) to examine genomes of artificially produced sturgeon hybrids between sterlet, Acipenser ruthenus female (∼120 chromosomes) or Russian sturgeon, A. gueldenstaedtii female (∼240 chromosomes) and a spontaneous triploid Siberian sturgeon A. baerii male (∼360 chromosomes), respectively. The ploidy levels of progenies were analyzed by karyotyping and flow cytometry. We found that the species-specific regions were surprisingly identifiable only on some micro- and small(er) macrochromosomes in hybrid metaphases. We hypothesize that these distinguishable regions are represented by species-specific repetitive sequences driven by more dynamic molecular evolutionary mechanisms. On larger chromosomes, GISH faintly visualized only blocks of pericentromeric and telomeric repetitive sequences, remaining regions were equally shared by both parental species. We concluded that the interspecies hybridization producing viable and even fertile progeny is enabled by the fact that genomes of the species involved are likely divergent at the level of the repetitive sequences only and probably highly conserved in the coding sequences. These small differences of coding sequences are in concordance with previous estimations of relatedness of examined species producing artificial as well as natural hybrids. CGH and GISH represent a challenge in sturgeon cytogenetics as a valuable though technically not simple tool to discriminate chromosomes of parental species in hybrids. The potentials and drawbacks of CGH and GISH application in sturgeons are discussed and further experimental possibilities are proposed.

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The effect of cryopreservation on the protein phosphorylation/dephosphorylation pattern of common carp (Cyprinus carpio) sperm is described. Sperm was diluted in dimethyl sulfoxide (DMSO) and ethylene glycol (EG)-based extenders, followed by equilibration, freezing, and thawing. Proteins extracted from fresh and cryopreserved spermatozoa were separated on SDS-PAGE and two-dimensional gel electrophoresis, blotted on polyvinylidene difluoride membrane, and treated with anti-phosphotyrosine, anti-phosphothreonine, or anti-phosphoserine antibodies. For the subsequent protein identification we used matrix-associated laser desorption/ionization time-of-flight mass spectrometry. The results demonstrated that cryopreservation with either DMSO or EG extender significantly altered the phosphorylation state of sperm proteins on tyrosine or threonine residues. A dramatic decrease in tyrosine phosphorylation was detected in the cryopreservation procedures with DMSO extender. Endoplasmin, transketolase, and S-adenosylhomocysteine hydrolase were identified as proteins that play a key role in cellular stress responses and oxidation and/or reduction reactions. Results indicate that the phosphorylation and/or dephosphorylation modifications of sperm proteins that occur during cryopreservation could stimulate a series of biochemical effects interfering with spermatozoa function and leading to a loss of motility and fertilization ability. Our findings indicated that use of EG extender provided superior protein preservation during sperm storage.

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Here we report for the first time the possibility of sequential sperm motility activation in sturgeon (sterlet, Acipenser ruthenus), a fish with external fertilization, through changes either in osmolality (global solute concentration) or in the Ca(2+) concentration of the medium surrounding the spermatozoa. Sperm motility was initiated in any of three solutions containing buffer and sucrose at 80, or 40 or 10mM (called S80, S40, S10, respectively); S80 is hypertonic relative to sterlet seminal fluid, while S40 is isotonic and S10 is hypotonic. After cessation of sperm movement at the end of this first motility period, a second and then a third, subsequent motile phase were observed. The second motility period was induced at cessation of motility in S80 by imposing a two-fold decrease in osmolality. After arrest of motility in this half-diluted S80, a third motility period could be initiated by addition of CaCl2 to 1mM final concentration. At the end of a first motility period in either S40 or S10, subsequent motility re-activation episodes were achieved only by addition of 1mM CaCl2. Depending on conditions in which sperm samples were activated, significant differences in curvilinear velocity, percent motile spermatozoa, motility duration time, and specific external features of spermatozoa flagella were observed. Altogether, these observations on the ability of sturgeon spermatozoa to sustain sequential activation episodes by experimental adjustment of their environmental conditions represent a potent model for deeper investigations on the sperm motility activation mechanisms.

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Polyploidization has played an important role in vertebrate evolution. Acipenseridae bring clear examples of polyploidy ancestry and, also, polyploidization seems to be an ongoing process in these fishes. In the present study, the genetic origin of six triploid specimens morphologically determined as Acipenser ruthenus from commercial aquaculture was analyzed using a combination of mitochondrial and nuclear markers. A further five successive statistical analyses including median joining of mitochondrial DNA control region sequences, principal coordinate analysis (PCA), factorial correspondence analysis (FCA), STRUCTURE assignation, and NewHybrids status determination for microsatellite data were applied for the clarification of the origin of one extra chromosome set added in these triploids genomes. Although interspecific hybridization had been suggested as a source of these triploids, the statistical analyses showed that the investigated triploids originate from autotriploidization rather than from interspecific hybridization. Therefore, we conclude that a combination of molecular markers with suitable statistical analyses should be used to verify the origin of unusual ploidy level. Evidently, such an approach is critically essential in aquaculture, where interspecific hybridization is very common and usually detected by changes in ploidy levels only.

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Spermatozoa tend to swim near surfaces. Such attraction toward surface vicinity was approximated by the force-dipole theoretical approach and hydrodynamic modeling, but the physical parameters of surfaces have not usually been included in these models and their effect on sperm mobility remains unknown. In spermatozoa, changes in wave parameters, together with rotation around their longitudinal axis and circling appear when movement takes place close to surfaces. Here we show, by analysis of microscopy images (including high-speed video), a strong influence of the liquid-solid interface on sterlet spermatozoa motility characteristics compared with motility near the liquid-gas interface. Sperm cells swam at 16% lower velocity near a liquid-solid interface, rotating at a stable frequency of 25 Hz, each 180° rotation corresponding to one beat cycle and circling clockwise (when observed from top). In case of spermatozoa close to a water-air interface, rotation and circling were sporadic and irregular. Sterlet spermatozoa movement near a surface affects their velocity and possibly causes rotation. These behaviors are highly dependent on the level of suppleness of the interface, as has been previously predicted by modeling. Our results enhance the understanding of how surfaces influence fish spermatozoa motility. These insights on the effects of surfaces on fish spermatozoa motility imply that widely used methods rating sperm motility, such as computer-assisted sperm analysis, might lead to erroneous results. Further study of sperm motility near surfaces is urgently needed to correct our rating methods and better understand sperm behavior in natural conditions. Improved evaluation of sperm motility behavior near surfaces could be used to determine physical properties of aquatic interfaces with various surfaces composed of different materials.

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The aim of this study was to investigate the response of Russian sturgeon (Acipenser gueldenstaedtii) sperm to external cations (Na(+), K(+), Ca(2+), and Mg(2+)) and their susceptibility on the induction of motility and swimming behavior. An in vitro spermatozoa motility assay was used by a computer-aided Motion-Analysis system. Sperm motility was inhibited by 60 mM NaCl (~140 mOsm/kg) and 0.7 mM KCl solutions (~ 21.4 mOsm/kg). The Ca(2+) and Mg(2+) ions were not able to inhibit spermatozoa motility. By contrast, Na(+) within a limited concentration range (between 45 and 55 mm) was able to reverse the inhibitory effect of K(+) at the critical concentration (0.7 mM). Ca(2+) and Mg(2+) were also able to reverse the K(+)-mediated spermatozoa motility restriction at concentrations starting at 0.01 and 0.1 mM, respectively. These results provide evidence for the role of K(+) in suppressing spermatozoa motility, and suggest that Ca(2+), Mg(2+), and possibly Na(+) trigger motility in Russian sturgeon sperm.

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The present study shows the roles of osmolality, calcium (Ca(2+))-potassium (K(+)) antagonist and Ca(2+) in sperm activation and flagellar beating of a sturgeon species, sterlet (Acipenser ruthenus). Sperm motility was activated at hypoosmolality relative to seminal plasma and suppressed at 175 mOsmol kg(-1). Sperm activation was totally suppressed by 0.35mM K(+), but Ca(2+) could fully reverse K(+) inhibitory effect at Ca(2+): K(+) ratio of 0.25. Neither EGTA (a chelator of Ca(2+) ions) nor nifedipine (a Ca(2+) channel blocker) prevented sperm activation. But, sperm motility and velocity were significantly decreased by EGTA, nifedipine and an inhibitor for Ca(2+)/calmodulin activated phosphodiesterase (w-7) that suggest role of Ca(2+) signaling after triggering sperm activation through hypoosmolality. Symmetric flagellar beating was also turned to asymmetric after activation in w-7, which is an evidence for modulation of Ca(2+)-binding proteins activity. Sturgeon sperm, similar to salmonids, is immotile in seminal plasma due to high K(+) concentrations, but the mechanism of sperm activation seems to be closer to other fish species where osmolality prohibits sperm activation in seminal plasma. In these species, hypoosmolality is the primary signal for sperm Ca(2+)-dependent signaling of axonemal beating.

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Comparative studies of ionic composition, osmolality, protein concentration and pH of seminal plasma along with spermatozoa concentrations were carried out in stellate sturgeon, Acipenser stellatus, and Russian sturgeon, Acipenser gueldenstaedtii. Analysis of A. gueldenstaedtii sperm showed significantly higher concentrations of Na(+) (34.58 ± 4.61 mm), Ca²(+) (0.35 ± 0.12 mm), Mg²(+) (0.70 ± 0.25 mm), Cl(-) (13.50 ± 4.04 mm) and proteins (0.60 ± 0.29 mg/ml) in the seminal plasma than did seminal plasma of A. stellatus: Na(+) (20.08 ± 10.75 mm), Ca²(+) (0.28 ± 0.06 mm), Mg²(+) (0.29 ± 0.05 mm), Cl(-) (7.50 ± 3.00 mm) and 0.30 ± 0.11 mg/ml proteins. Significantly higher concentration of K(+) (5.42 ± 1.06 mm) was observed in A. stellatus compared to A. gueldenstaedtii K(+) (2.29 ± 0.50 mm). Concentration of Na(+) was positively correlated with osmolality (r = 0.819), levels of Cl(-) (r = 0.922) and Mg²(+) (r = 0.727) and pH (r = 0.848). The concentration of Mg²(+) was positively correlated with protein concentration (r = 0.774), Na(+) (r = 0.727), Cl(-) (r = 0.872) and Ca²(+) (r = 0.801). A positive relationship was also found between concentration of K(+) and spermatozoa concentration (r = 0.709). Results revealed strong inter-species differences in several parameters. The data should be useful for artificial fertilization and for cryopreservation of sturgeon sperm.

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Damage to spermatozoa during cryopreservation is regarded as a major obstacle to the expansion of sperm storage technology. The authors used two-dimensional polyacrylamide gel electrophoresis and matrix-associated laser desorption/ionization time-of-flight mass spectrometry to explore whether the protein profile of common carp (Cyprinus carpio) spermatozoa is affected by cryopreservation. Fourteen protein spots were significantly altered following cryopreservation. Eleven of these were identified: three as specific membrane proteins (N-ethylmaleimide-sensitive fusion protein attachment protein alpha, cofilin 2, and annexin A4) involved in membrane trafficking, organization, and cell movement; six as cytoplasmic enzymes (S-Adenosylhomocysteine hydrolase, Si:dkey-180p18.9 protein, lactate dehydrogenase B, phosphoglycerate kinase 1, transaldolase 1, and esterase D/formylglutathione hydrolase) involved in cell metabolism, oxidoreductase activity, and signal transduction; and two as transferrin variant C and F. Based on these findings, the authors hypothesize that transferrin in cryopreserved sperm may protect spermatozoa against oxidative damage during the freeze-thaw process. Cryopreservation caused changes in spermatozoa protein profiles that may lead to decreased spermatozoa velocity, motility, and fertilization success, and to subsequent ova hatching rate.

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In the present study, we investigated the possibility of spontaneous carp spermatozoa activation by freeze-thawing. To evaluate this, the parameters of spermatozoa motility percentage, velocity, ATP content level and fertility rate of sperm were used. The motility and velocity of spermatozoa activated by freeze-thawing were characterized by motile spermatozoa with a median value of 16% and a velocity of 98 microm/s. In addition, the motility and velocity of sperm from the thawed samples were significantly lower than in the control (median value of 100% for sperm motility and 175 microm/s for sperm velocity). Furthermore, a spontaneously activated spermatozoa motility terminated within five minutes post-thaw time. After freeze-thawing the ATP level significantly decreased with post-thaw time (46 nmol ATP/10(9) and 10 nmol ATP/10(9) at 25s and 10 min after thawing, respectively). Fertility of spermatozoa was not significantly affected within 10 min post-thaw. On the other hand, the fertility of frozen-thawed sperm was significantly lower if compared to fresh sperm. We conclude that the freeze-thawing procedure spontaneously activated spermatozoa motility in common carp. However, this activation did not negatively affect the fertility of frozen-thawed sperm.

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The objectives of the present study were to characterize sperm volume and density, seminal plasma indices (ionic contents and osmolality) and to study the effects of dilution ratio, ions and osmolality on sperm motility parameters (percentage of motile sperm and sperm velocity) in farmed European perch (Perca fluviatilis L.). The means of sperm volume (ml), sperm density (x10(9)spermml(-1)) and total number of sperm (volumexdensity) per fish were 2.75+/-0.51, 29.19+/-3.15 and 82.19+/-15.26. The seminal plasma osmolality (mOsmkg(-1)), sodium, chloride, potassium and calcium ions concentrations (mM) were measured to be 298.07+/-5.09, 130.97+/-2.19, 106.75+/-2.37, 10.70+/-0.61 and 2.41+/-0.09, respectively. At 15s post-activation of stripped sperm, the percentage of motile sperm (%) and sperm velocity (mums(-1)) were 91.90+/-1.27 and 115.54+/-1.25, respectively, and decreased significantly following sperm activation (P<0.05). The optimal sperm motility was observed when the sperm was prediluted in immobilizing solution (IS) at a ratio 1:50. Prediluted sperm showed the maximum velocity when activated in 2.5mM Ca(2+), 50mM K(+) and sucrose with osmolality 100mOsmkg(-1). Neither Ca(2+) nor K(+) showed a significant effect on the percentage of motile sperm at 15s post-activation. Osmolality higher than 200mOsmkg(-1) significantly decreased the percentage of motile sperm, while osmolality of 300mOsmkg(-1) or above totally suppressed sperm motility.

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The aim of the present study was to elaborate cryopreservation methods for ex situ conservation of tench. Success of cryopreservation was tested during two series of experiments. The first set of experiments studied the effects of two types of cryoprotectants (DMSO and a combination of DMSO with propanediol at ratio 1:1) at concentrations of 8 and 10% and three different equilibration times in two different immobilization solutions (IS) (Kurokura 180 and Kurokura) before freezing (0.0, 2.0 and 4.0h after T(0)). The K4 cooling programme was used to freeze 1ml of cryoextended sperm using 1.8ml cryotubes. Main monitored parameter was hatching rate after using of cryopreserved sperm. The second set of experiments studied the volume effect of 0.5, 1 and 5ml straws and compared these with 1.8ml cryotubes as well as the effect of the cooling programme (K4 and L1). Following the results of the first study, a combination of DMSO and propanediol (ratio 1:1) at concentration of 10% was added to extended sperm in Kurokura 180 IS. Main monitored parameter was hatching rate after using cryopreserved sperm, supplementary parameters were sperm velocity and motility percentage assessed at 10s post-activation. Sperm was collected directly into IS and stored at 4 degrees C for 2.5h. Thereafter were sperm samples pooled, equlibred in IS (first set of experiments) or directly mixed with cryoprotectants (DMSO or a mixture of DMSO with propanediol at ratio 1:1) and transferred to 1.8ml cryotubes or straws (0.5, 1 and 5ml). Then the cryotubes/straws were directly transferred to pre-programmed PLANER Kryo 10 series III and cooled using two different cooling programmes including a slow cooling programme (a) named K4 (from +4 to -9 degrees C at a rate of 4 degrees Cmin(-1) and then from -9 to -80 degrees C at a rate of 11 degrees Cmin(-1)) and a rapid cooling programme (b) named L1 (directly from +4 to -80 degrees C at a rate of 20 degrees Cmin(-1)). Both slow (K4) and rapid (L1) cooled samples were held 6min at -80 degrees C. Finally, samples were transferred into liquid N(2). The frozen spermatozoa were thawed in a water bath (40 degrees C) according to the frozen volume and checked for fertilization and hatching rates. Percentage of sperm motility and sperm velocity were measured using video recorded frames. ANOVA showed a significant influence of frozen and fresh sperm in all treatments. The hatching rates of 33.8% were obtained when sperm was equilibrated for 0h before freezing in IS of Kurokura 180 and frozen with a 10% of mixture 1:1 of DMSO and propanediol into straws of 5ml and cooled using program L1. The velocity of frozen-thawed spermatozoa ranged from 31 to 46microms(-1) and in post-thawed sperm was not significantly different according to frozen sperm volume, but a higher velocity was obtained when sperm was fast frozen using programme L1. A large volume of frozen sperm could reveal the best procedure for freezing, but also for simulating methods of artificial propagation for future practical use of frozen tench sperm at a large scale.

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