Composition of seminal plasma and ovarian fluid in Ide Leuciscus idus and Northern pike Esox lucius.

Seminal plasma (SP) and ovarian fluid (OF) plays an important role as storage media to prevent the activation of gametes both in vivo and under artificial conditions. The objectives of this study were to quantify gamete biochemistry and explore correlations among quantitative characteristics of SP, OF and sperm performance traits of Ide Leuciscus idus and Northern pike Esox lucius. Generally, Na+ , K+ and Cl- were found to be the most dominating ions, although concentrations of K+ were higher in SP, while Na+ and Cl- concentrations were higher in OF for both species. Several significant correlations among the biochemical properties such as total protein, glucose, osmolality, cholesterol, K+ , Ca2+ , Cl- and Mg2+ were observed for SP and OF. Total protein content of Ide SP was positively correlated with sperm activity traits (r ≥ .89, p ≤ .05), while K+ concentration was negatively correlated with sperm traits (r ≥ -.89, p ≤ .05). Moreover, Ca2+ concentration in Northern pike SP was positively correlated with the percentage of sperm motility (r = . 98, p < .01). In conclusion, these results can be used to better understand the biochemistry of SP and OF, improve methods for short- and long-term storage of gametes and standardize fertilization protocols.

Lipid composition in common carp (Cyprinus carpio) sperm possessing different cryoresistance.

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.

Effects of Temperature on In Vitro Short-Term Storage of Sterlet Sturgeon (Acipenser Ruthenus) Ova.

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.

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.

Motility and fertilization ability of sterlet Acipenser ruthenus testicular sperm after cryopreservation.

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.

Calcium ion supplementation increases brook trout Salvelinus fontinalis spermatozoa activation at the end of the spawning season.

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) ).

Cryopreservation of common carp (Cyprinus carpio L.) sperm induces protein phosphorylation in tyrosine and threonine residues.

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.

Motility of sturgeon spermatozoa can sustain successive activations episodes.

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.

Comparison of protein fractions in seminal plasma from multiple sperm collections in sterlet (Acipenser ruthenus).

Seminal plasma of sterlet Acipenser ruthenus was evaluated using comparative proteomics to characterize its protein fractions and to determine any influence of multiple sperm collections on these proteins. An experimental group of fish was used, in which sperm was collected three times at 5 h intervals. Protein fractions of seminal plasma were determined by SDS-gel electrophoresis (SDS-PAGE) and two-dimensional electrophoresis high-resolution gels (2D). At all stripping times, five protein bands with molecular weights of 93, 53, 48, 33 and 28 kDa were identified using SDS-PAGE. No significant differences (p > 0.05) in relative mass of protein bands among collections were observed. At the third collection, 20 protein spots were detected from the two-dimensional gels, compared to 17 found at the first and second collections. Ten protein spots, from the third stripping, were analysed. Screening of these spots by mass spectrometric analysis showed positive results for spot 10. Direct comparison across public databases revealed sequence similarity with two hypothetical proteins, MCAG_00854 and IscW_ISCW011489. Differences in the seminal plasma protein fractions were found at the third stripping compared to the first two. It is hypothesized that these extra proteins after the third collection could be involved in some step of intracellular mechanism which is responsible for regulating of spermatozoa motility. However, protein identification revealed no significant distinction for any protein spot and protein sequences available in public databases. These results highlighted the need for a complete genome sequences for sturgeons.

Different swimming behaviors of sterlet (Acipenser ruthenus) spermatozoa close to solid and free surfaces.

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.

Spermatozoa motility and variation in the seminal plasma proteome of Eurasian perch (Perca fluviatilis) during the reproductive season.

This study evaluated physiological and functional sperm parameters and the seminal plasma proteome of Eurasian perch (Perca fluviatilis) over the course of their reproductive season. Spermatozoa velocity (169.56 ± 6.53 to 158.5 ± 7.4 µm sec(-1)), percent motility (95.89 ± 4.28% to 89.55 ± 4.5%), and osmolality of seminal plasma (290 ± 5 to 297 ± 12 mOsmol kg(-1)) remained stable throughout the reproductive season. Milt volume and protein concentration of seminal plasma gradually increased and reached the highest values late in the reproductive period. Spermatozoa concentration peaked in the mid-reproductive season (66.90 ± 13 × 10(9) spermatozoa ml(-1)) and decreased towards the end (54 ± 10 × 10(9) spermatozoa ml(-1)). A proteomic analysis of seminal plasma using two-dimensional polyacrylamide gel electrophoresis revealed 10 protein spots significantly altered over the course of the reproductive season. Subsequent protein characterization suggested that time in the reproductive season predominantly affected proteins involved in membrane trafficking, organization, cell motility, and oxido-reductase activity. This study provides new data on physiological properties of sperm and protein patterns of seminal plasma over the course of the reproductive season that should be considered in the development of methods for artificial reproduction of perch.

Regulation of spermatozoa motility in response to cations in Russian sturgeon Acipenser gueldenstaedtii.

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.

Evaluation of spermiation indices with multiple sperm collections in endangered sterlet (Acipenser ruthenus).

This study investigated the effects of multiple collections of sperm on endangered sterlet (Acipenser ruthenus) sperm functional parameters [spermatozoa motility and curvilinear velocity (VCL)] as well as on protein concentration and osmolality of seminal plasma. The average sperm volume and mean spermatozoa concentration per male were significantly altered with multiple collections. On the other hand, no significant effect of multiple collections on protein concentration of seminal plasma was observed. In all experimental groups, moderate impact of sequential collection on osmolality (p < 0.05) of seminal plasma was observed. Ninety to 100% of motile spermatozoa were observed at 15 s after activation, with an average VCL of 181.12 ± 19.10 µm/s. After 90 s, average VCL decreased to 130 ± 26 µm/s. Motility was maintained for up to 4 min. The maximum percentage of motile spermatozoa was observed after the third collection of sperm. The spermatozoa VCL increased significantly with subsequent collections. The results of this study provide new data on the effects of multiple collections on quantitative and qualitative parameters of sperm in sterlet. The data confirmed that the sequential stripping has no negative effect on the percentage of motility and spermatozoa velocity. This should be beneficial for the development of sterlet aquaculture programs.

Roles of osmolality, calcium - Potassium antagonist and calcium in activation and flagellar beating pattern of sturgeon sperm.

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.

Spermatozoa concentration, seminal plasma composition and their physiological relationship in the endangered stellate sturgeon (Acipenser stellatus) and Russian sturgeon (Acipenser gueldenstaedtii ).

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.

Ice-age endurance: the effects of cryopreservation on proteins of sperm of common carp, Cyprinus carpio L.

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.

Relationships between reproductive characteristics in male Vimba vimba L. and the effects of osmolality on sperm motility.

In Vimba vimba, GSI, sperm volume, and spermatozoa concentration range from 3.4-7.4 %, 0.1-1.1 ml, and 13.3-34.8 x 10(9) spz ml(-1), respectively. Gonad mass (r = 0.90) and sperm volume (r = 0.35) significantly correlated with weight of males. Significant correlation was also found between gonad mass and length of males (r = 0.85). Sperm motility (r = 0.99) and velocity (r = 098) significantly decreased after activation in Tris-HCl 20 mM, pH 8.5. Osmolality of the seminal plasma was 273.2 mOsmol kg(-1). Sperm motility and velocity were significantly affected by the osmolality of the activation medium (P < 0.01). Hyper-osmolality compared to seminal plasma osmolality totally suppressed the sperm activation. At 15 s post-activation, the sperm motility significantly decreased at 240 mOsmol kg(-1) in KCl or NaCl media. The highest sperm motility and velocity (at 60 s post-activation) were observed at 200 mOsmol kg(-1) in NaCl, KCl, or sucrose media. In all treatments, the tip of the flagellum of spermatozoa became curled into a loop shape after activation of sperm in distilled water containing 20 mM Tris-HCl, pH 8.5 that shortened the flagellum.

Freeze-thawing as the factor of spontaneous activation of spermatozoa motility in common carp (Cyprinus carpio L.).

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.

Dynamics of ATP and movement in Eurasian perch (Perca fluviatilis L.) sperm in conditions of decreasing osmolality.

Repetitive activation of perch (Perca fluviatilis L.) sperm motility was investigated in this study. The first phase of sperm motility activation was initiated by dilution in a 260 mM glucose solution (75% motility). The second phase of motility was achieved by adding water to previously activated sperm, so that the glucose concentration dropped to 220 mM (24% motility). Finally, the third phase was obtained by further addition of water (down to 90 mM glucose) to the activated sperm suspension (15% motility). Parallel measurements of sperm ATP content were also made. The median value for nonactivated sperm was 43.9 nmol ATP/10(9) spermatozoa. The ATP concentration decreased significantly from 35 to 7 nmol ATP/10(9) spermatozoa after successive activations of motility in the above glucose solutions. Sperm velocity ranged in value from 25 to 330 microm/sec at 10 sec postactivation, from 10 to 290 microm/sec at 30 sec, and from 0 to 200 microm/sec at 45 sec. A model postulating several classes in the population of spermatozoa is developed, tentatively accounting for such successive activation. Possible further application of multiple sperm activation is discussed.

Preliminary studies on cryopreservation of common tench (Tinca tinca) embryos (work in progress).

Vitrification could provide a promising tool for the cryopreservation of fish embryos. However, to achieve cryopreservation using vitrification, chilling sensitivity and cryoprotectants toxicity were determined using tench embryos at four developmental stages (11, 17, 23 and 29 h). Embryos treated with alcalase (2 ml/998 ml, 2 min at 22 degrees C) were exposed to chilling with/without warming. Other embryos were exposed to methanol and glycerol at the concentration of 10% and 20% for periods of 20 min. At last, embryos were incubated at special incubator cages where hatching rates were counted. Regarding chilling sensitivity and exposure to chilling followed by warming, the hatching rates of embryos decreased significantly (p < 0.001) after exposure to 0 degrees C at all developmental stages except the 29-h stage compared with the controls. The embryo stage most sensitive to chilling was 11-h stage. The 29-h stage exhibited the least sensitivity to low temperature while 17-h and 23-h stages were intermediate in their sensitivity to chilling. The toxicity of methanol increased significantly (p < 0.001) with developmental stage for 11, 17 and 23-h stages. The highest hatching rates of tench embryos were obtained with 29-h embryos using various concentrations of methanol. The hatching rates of tench embryos exposed to glycerol concentrations were approximately similar to those embryos exposed to methanol concentrations except for 11-h embryos that showed no hatching. Unfortunately, we could not obtain living embryos in any of the conditions examined after vitrification. In conclusion, it was quite difficult to vitrify the tench embryos during this study using various vitrifying solutions and the method reported by Chen & Tian (2005) and further studies are needed to achieve successful cryopreservation.