Effects of osmolality and ions on the motility of stripped and testicular sperm of freshwater-and seawater-acclimated tilapia, Oreochromis mossambicus

A significantly higher concentration of testicular spermatozoa was obtained from freshwater Oreochromis mossambicus (9·9×10⁹ spermatozoa ml⁻¹) than seawater O. mossambicus (4·6×10⁹ spermatozoa ml⁻¹). The mean osmolality of the urine of freshwater fish (78·5 mOsmol kg⁻¹) was significantly different from that of seawater fish (304·8 mOsmol kg⁻¹). The mean length of the mid-piece of the spermatozoa together with the tail was more variable in freshwater O. mossambicus (8·80±0·23μm) than in seawater specimens (8·27±0·18 μm). Stripped sperm of freshwater O. mossambicus was highly contaminated by urine which was a good activator of sperm motility in O. mossambicus held in both fresh and sea water. The osmolality for initiation of motility in freshwater O. mossambicus spermatozoa was from 0 to 333 mOsmol kg⁻¹ while for seawater O. mossambicus spermatozoa it was from 0 to 1022 mOsmol kg⁻¹. The optimum osmolality for motility was from 70 to 333 mOsmol kg⁻¹ for freshwater O. mossambicus spermatozoa and from 333 to 645 mOsmol kg⁻¹ for seawater fish. In freshwater O. mossambicus spermatozoa, the presence of 20 mM CaCl₂ increased the permissive osmolality of NaCl from 184 to 645 mOsmol kg⁻¹. For seawater O. mossambicus spermatozoa, solutions of NaCl devoid of CaCl₂ were unable initiate motility, but the addition of 1·5 to 30 mM CaCl₂ to the NaCl solution (0–934 mOsmol kg₁) had a full motility initiating effect.     

Effects of season and breed on sperm acrosin activity and semen quality of boars

Acrosin activity and semen quality (sperm concentration, ejaculate volume and number of spermatozoa) were assessed from March 1997 to March 1998 in semen of Large White, Pietrain and Duroc x Pietrain boars. Semen quality varied with season, including high production of spermatozoa in autumn and winter and low production in summer. Semen quality also differed across breeds. Acrosin activity of boar spermatozoa was not affected by breed (range 3.16-3.32 mU/10(6) spermatozoa), but exhibited distinct seasonal changes. Monthly changes in acrosin activity were parallel to changes in number of sperm in the ejaculate from November to March. On the other hand, dramatic changes in acrosin activity between July and October (range 1.85-4.59 mU/10(6) spermatozoa) were not paralleled by similar changes in number of ejaculated sperm. These fluctuations in acrosin activity may reflect either changes in sperm acrosin production or disturbances to sperm membranes, probably related to effects of high summer temperatures during spermatogenesis. Results confirmed seasonal and breed-related differences in boar semen quality characteristics.     

Hormonal induction and semen characteristics of tambaqui Colossoma macropomum

In the hatchery-bred tambaqui Colossoma macropomum, spontaneous semen release does not occur, and hand-stripping produces reduced semen volume. The goal of this work is to evaluate the effects of hormonal induction with carp pituitary extract (CPE) on both qualitative (visual aspect, pH, motility, viability and morphological abnormalities) and quantitative (volume, concentration and number of spermatozoa per ejaculate) traits of tambaqui semen. Eleven males were treated with CPE (induced), and 11 were left untreated as a control (non-induced). All analysed parameters except motility and percentage of viable spermatozoa presented significant differences (p < 0.05) between the induced and non-induced treatments. CPE induction resulted in a 25-fold increase in semen volume and a 10-fold increase in the number of spermatozoa collected. However, both sperm concentration and the frequency of sperm with morphological abnormalities (commonly detached heads or bent tails) were significantly lower in CPE-induced fish. The hormonal induction of tambaqui males with CPE is efficient and positively influences some qualitative and quantitative properties of semen. Additionally, semen collection via gentle abdominal massage occurs more readily in CPE-induced fish.     

Effects of different surfactants on motility and DNA integrity of brown trout (Salmo trutta fario) and common carp (Cyprinus carpio) spermatozoa

In this study we evaluated effects of surfactants on motility parameters and DNA integrity of spermatozoa of freshwater teleost fish. Common carp (Cyprinus carpio) and brown trout (Salmo trutta fario) spermatozoa were exposed to either sodium dodecyl sulphate (SDS, anionic surfactant) or octoxynol 9 ( Triton X-100, nonionic surfactant). Both surfactants added at activation caused a decrease in sperm motility characteristics measured by computer-assisted sperm analysis (CASA). Intraspecific differences in speed and trajectory of movement were detected. Triton X-100 and SDS when added to non activated sperm were also effective in the decrease of sperm motility and caused an increase of DNA fragmentation. Our results suggest that not only sperm motility apparatus but also DNA are targets for surfactant action. Therefore any exposure of spermatozoa to surfactants, in aquaculture conditions or natural environment, would have a negative impact on fish reproduction.     

Fundamental aspects of sperm cryobiology: the importance of species and individual differences

While semen cryopreservation is successfully used for a few species, application to other species can be problematic. Here, I argue that species differences in female tract anatomy, subtle differences in sperm transport mechanisms, ability to time inseminations and deliver spermatozoa effectively are powerful determinants of fertility with cryopreserved spermatozoa. Poor sperm survival represents one major aspects of the problem and determining biophysical characteristics of the sperm plasma membrane is an established approach to solving it. However, this approach is unable to account for the consistent differences in post-cryopreservation sperm quality between individual males, an effect that is recognized is many species although only documented in a few. Searching for genetic differences between these individuals might offer a genomically-based direction in semen cryopreservation research. Cryopreservation of spermatozoa and spermatogenic cells for intracytoplasmic sperm injection has been developed primarily to deliver an intact genome and presents a very different set of technical problems.     

Effects of media,fertilization technique,extender, straw volume,and sperm to egg ratio on hatchability of cyprinid embryos,using cryopreserved semen

To enable cryopreservation of fish semen to become an efficient, routine technique, much more detailed information is required. Therefore, the present study was conducted to investigate various fertilization techniques and media, straw volumes as well as optimal semen volume for cryopreservation. The bleak (Chalcalburnus chalcalburnus) was used as the main model for investigation. Using frozen-thawed semen the fertilization rate was similar up to the morula stage, independent of the fertilization technique. Thereafter, in egg batches fertilized using the wet technique most embryo development stopped. In egg batches fertilized using the dry technique, embryonic development proceeded normally. For cryopreserved semen full activation of sperm motility was obtained at ratios of fertilization media (hatchery water and all tested types of saline solutions) to semen of 10:1. Sperm motility rate was much higher in the saline solutions than in water. In contrast hatching rates were higher when water was used as fertilization medium. Therefore, the requirements necessary for optimal sperm motility and optimal sperm-egg contact were different and so for these parameters optimal levels could not be achieved. When adjusting the freezing and thawing conditions 0.5ml straws as well as larger straws (1.2ml) proved suitable for cryopreservation of cyprinid semen. The highest fertilization rates were obtained with sperm to egg ratios of (1.3-2.5) x 10(6):1 and were 77-92% of fresh semen control. This was also similar for Ch. nasus, R. meidingerii, B. barbatus and C. carpio and suggests that the cryopreservation requirements of spermatozoa are not species specific.     

Haematological assessment of the effects of the anaesthetic MS 222 in natural and neutralized form in three freshwater fish species: intraspecies differences

The effects of different concentrations of natural MS 222 and neutralized MS 222 were studied on the haematology of Cyprinus carpio, Sarotherodon mossambicus and Salmo gairdneri . As judged from the results, MS 222, which is acidic in nature, produced haemo-concentration effects in all species studied, being least in the trout followed by carp and Sarotherodon mossambicus . These differences are ascribed to acid-base regulatory functions and metabolic activities of the fish species investigated. The use of neutralized MS 222 improved the haematological profiles markedly and resulted in stabilization of acid-base parameters and red blood cells sizes and numbers. Haemoconcentration effects, however, still persisted. Trout were found to be more susceptible to the stress of MS 222 anaesthesia than carp and Sarotherodon mossambicus .     

Relationship between the movement characteristics of human spermatozoa and their ability to penetrate cervical mucus and zona-free hamster oocytes

In a group of normospermic donors exhibiting hamster oocyte penetration scores of 0-100%, multiple regression analysis indicated that only 20% of the variation in fertilizing potential could be explained by differences in the movement characteristics of the spermatozoa following incubation in vitro. When the movement characteristics of the spermatozoa in semen were considered this figure was reduced to 6.8% as a result of significant differences in the motility patterns exhibited by the seminal and post-incubation sperm populations. A much closer relationship was observed between the movement characteristics of human spermatozoa in semen and their ability to penetrate cervical mucus. When differences in motile sperm densities were taken into account, 76% of the variation in cervical mucus penetration could be accounted for by the existence of linear correlations with certain aspects of sperm movement (multiple R = 0.874). Of the various attributes of sperm motility measured (linear velocity of progression, frequency of rotation, amplitude of sperm head displacement, % rolling and % yawing), a failure to exhibit an adequate amplitude of lateral sperm head displacement was consistently found to be the most significant factor determining the success of sperm-cervical mucus interaction (R2 = 0.53).     

A Comparative Study on the Chemical Composition of Plasma from the Cauda Epididymidis,Semen Fractions,and Whole Semen in Boars

A comparative study was carried out on the chemical composition of plasma from the cauda epididymidis, semen fractions, and whole semen of boars. A total of 22 boars were used in this study. The boars, which ranged in age from 8 to 14 months, were of Swedish Landrace and Swedish Yorkshire breed. All boars used presented a normal semen picture. A dummy sow and an artificial vagina were employed for semen collection. The semen was collected as whole semen and as semen fractions in 10 nil volumes. The contents of the cauda epididymidis was removed post mortem. The following parameters were investigated: sperm concentration, dry weight of spermatozoa and of seminal plasma, osmotic pressure, sodium, potassium, chloride, inorganic phosphorus, calcium, magnesium, total protein, GOT, GPT, and alkaline phosphatase in seminal plasma. Paper electrophoresis was carried out on seminal plasma. Tlxe results of the analysis are summarized in Tables 1–6. The sperm concentration was approximately 3.2 mill./mm3 in the cauda epididymidis, 1 mill./mm3 in the sperm-richest fraction (II) and 0.25 mill./mm3 in whole semen. The dry weight (expressed in per cent dry matter) of spermatozoa was highest in the cauda epididymidis (25.47 %), showing a tendency to decreasing in semen fractions I—IV and was lowest in whole semen (15.29 %). The per cent dry weight in plasma was higher in the cauda epididymidis (4.56 %) than in semen fraction I (2.20 %). In semen fractions I—IV the per cent dry weight rose from 2.20 (U to 4.51 % and reached the level of approximately 3.80 % in the sperm-free fractions V—VII. The osmotic pressure was significantly higher in the cauda epidi-dymal plasma than in the whole seminal plasma or the seminal plasma fractions. The same phenomenon was observed in a boar where the cauda epididymal content was collected in vivo from a patent established fistula. There appears to be a connection between the per cent dry weight of spermatozoa and the osmotic pressure, which means that the per cent dry weight of the cauda epididymal spermatozoa decreases when mixed with the accessory gland secretions, which have a lower osmotic pressure. The fall in per cent dry weights is thought to be caused by an intake of water. The amount of sodium, chloride and magnesium was higher in ejaculated seminal plasma than in cauda epididymal plasma. The reverse was true for inorganic phosphorus and potassium. Moreover the sperm-free fractions contained more sodium, chlorides and magnesium than the sperm-containing fractions, while the concentration of potassium and inorganic phosphorus was comparatively higher in the sperm-containing fractions. A connection is apparent between sperm concentration and the potassium, inorganic phosphorus and magnesium levels. Statistical analysis of the values of chloride and magnesium revealed significant differences between individual boars for most of the semen fractions. The concentration of plasma proteins in the cauda epididymidis was approximately the same as in whole semen and in the semen fractions except for fraction I, which contained a relatively low concentration. As regards total protein there were significant differences between individual boars in most of the semen fractions as well. The paper electrophoretic pattern of epididymal plasma was different from that of semen plasma. Thus there were three or four distinct components in the cauda epididymidis numbered 1, 2, 3, and 4, and three distinct components in whole seminal plasma numbered 3, 4, and 5, while the sperm-richest semen fractions contained four components (2, 3, 4, and 5) and the others three components, namely 3, 4, and 5. The level of GOT was high in the cautlu cpiflidymill contents (99.1 i. u./ml) compared with that for whole seminal plasma (99.1 i.u/ml). In semen fractions there was a clear positive correlation between the level of GOT and the sperm concentration. The GPT concentration wis as a whole low and. in contrast to GOT. somewhat higher in the sperm-free fractions than in the sperm-containing fractions. The concentration of alkaline phosphatase was very high in cauda epididymal plasma (31,463 i. u./ml) as well as in the sperm-rich fractions (e.g. 7,096 i. u./ml in fraction II). Preliminary investigation has moreover revealed a very low alkaline phosphatase concentration in seminal plasma of vasectomized boars, which condition suggests thai the main origin for alkaline phosphatase in boars is the testis and epididymis.     

Optimal physicochemical conditions for the manipulation and short-term preservation of koala (Phascolarctos cinereus) spermatozoa

Protocols for the successful manipulation and preservation of semen in a given species depend upon a fundamental knowledge of how spermatozoa respond to the physicochemical conditions of the extension media; methods developed for the preservation of eutherian spermatozoa may not necessarily be suitable for marsupial semen. The aim of this study was to investigate the effects on koala sperm motility of serial dilution, changes in temperature, diluent pH and osmolality to establish the optimal physicochemical conditions for short-term semen storage. This study showed that electroejaculated koala semen diluted 1∶1 (v/v) with PBS frequently coagulated after incubation at 35 degrees C, but that further dilution and incubation resulted in a corresponding increase in the percentage of spermatozoa swimming in a non-linear trajectory. The effect of rapid temperature change on the motility of koala spermatozoa was investigated by exposing semen, initially diluted at 35 degrees C, to temperatures of 45, 25, 15 and 5 degrees C. Although sperm motility was reduced after incubation at 45 degrees C, a rapid decrease in temperature of up to 20 degrees C did not result in a significant reduction in sperm motility. However, contrary to evidence in other marsupials, there was a small but significant decrease in sperm motility after rapid cooling of diluted semen from 35 to 5 degrees C. The effects of diluent pH and osmolality on the motility of koala spermatozoa were investigated. These experiments indicated that diluents for koala sperm manipulation should buffer in a pH range of 7-8 and have an osmolality of approximately 300 mmol kg(-1). The final experiment compared the relative effectiveness of Tris-citrate buffer (1% glucose) and PBS to maintain koala sperm motility over a range of incubation temperatures (5-35 degrees C) for up to 8 days. Reduction in sperm motility was directly related to temperature, and motility was sustained for the longest duration when stored at 5 degrees C. The Tris-citrate buffer solution was superior to PBS as a preservation diluent at all temperatures, and koala spermatozoa remained motile even after 42 days storage at 5 degrees C. Spermatozoa diluted in PBS (with Ca(2+) or Mg(2+)) and cooled to 5 degrees C showed evidence of an unusual motility pattern, similar to that of hyperactivated eutherian spermatozoa. This study showed that koala spermatozoa respond to different physicochemical conditions associated with short-term liquid storage in essentially the same way as the spermatozoa of eutherian mammals, although koala spermatozoa appear to be more tolerant of rapid temperature shock. The results of this study can be used to make informed selections with regard to appropriate diluent composition and improved short-term sperm preservation protocols and represent the first such database for any species of marsupial.     

Association of heat shock protein 70 with semen quality in boars

This study attempted to clarify the relationship between the levels of 70kDa heat shock protein (HSP70) and semen quality in boars. Semen samples from 29 (13 Duroc, 9 Landrace, and 7 Yorkshire) boars (mean age=25.2+/-2.2 months) were examined. Three to four ejaculates per boar, collected during cool and hot seasons, were evaluated in terms of the sperm concentration, sperm motility, percentage of normal and abnormal sperm, as well as percentage of sperm with proximal and distal plasma droplets. Significant seasonal and breed differences in semen quality were observed. Experimental results indicate that the semen quality of Landrace boars was better than those of Yorkshire and Duroc boars (P<0.05) and semen quality declined significantly during the hot season (P<0.05). One-dimensional SDS-PAGE analysis of spermatozoa proteins indicated that protein profiles did not significantly differ between seasons and among breeds. Both constitutive and stress-inducible form of HSP70 were detected in boar spermatozoa by Western blot analysis. The level of HSP70, which revealed no difference among breeds within a season, was significantly lower during the hot season in all the three breeds (P<0.05). Although there appeared to be low correlation coefficients between the level of HSP70 and semen quality traits, the semen quality tended to decline significantly in samples with a lower level of HSP70. Results in this study suggest that the levels of HSP70 in boar spermatozoa are significantly lower during the hot season and might be associated with semen quality.     

A thrombelastographic study of the effect of stress on the blood coagulation in Cyprinus carpio (Cyprinidae) and Oreochromis mossambicus (Cichlidae)

The normal blood coagulation in Cyprinus carpio and Oreochromis mossambicus was investigated by thrombelastography and standard thrombelastographs were obtained for each species. The effect of stress on blood coagulation was thereafter determined. The thrombelastograms indicated hypercoagulability under stress conditions. A prominent observation is the differences in the elasticity of the clot under various conditions. Fibrinolysis occurred in all cases, except in the case of Cyprinus carpio after severe agitation for 30 min. Relevant values for various parameters are given.     

Apoptotic-like changes in the spermatozoa of fresh and stored boar semen and the quality of embryos produced in vivo

The aim of this study was to determine the apoptotic-like changes in the spermatozoa of fresh and stored boar semen and to investigate the relationship between this phenomenon and the quality of embryos produced in vivo. The experiments were divided into two series. In the first series, ten ejaculates were collected from five boars, which were crossbreeds of the Polish Landrace and Large White breeds. The semen was stored as a liquid until Day A (the day on which sperm motility decreased to 30%). Three fluorescence methods were used to evaluate semen quality: an assay to assess the early changes in sperm membrane integrity using the fluorophore YO-PRO-1, an assay for phosphatidylserine (PS) translocation across the plasma membrane using fluorescein-labeled annexin-V and the mitochondrial-specific probe JC-1 (5,5',6,6'-tetrachloro-1,1',3,3' tetraethylbenzimidazolylcarbocyanine iodide) for measuring changes in mitochondrial membrane potential. Our results showed that liquid preservation of boar semen causes apoptotic-like changes in the sperm, and a significant increase in both: apoptotic sperm (YO-PRO-1(+)/PI(-)) and early apoptotic sperm (annexin-V(+)/PI(-)) were observed between Day 0 (fresh semen) and Day A only in semen from three of the five boars. In the second series of experiments, the semen from boar nos. 1, 2, and 3 was selected for insemination of superovulated gilts. The fertilizing capacity of fresh and stored semen with different levels of apoptotic spermatozoa was measured based on the morphology and the number of cells of embryos that were obtained after insemination with this semen. Our studies indicated no significant differences in the fertilization rate of gilts after insemination with fresh and stored semen with increased levels of apoptotic spermatozoa. After insemination with stored semen, a significantly greater number of degenerated embryos were observed, but the morphologically normal blastocysts obtained after insemination with either fresh or stored semen had a similar number of nuclei.     

Effects of cold shock treatment on angiotensin-converting enzyme activity and on semen characteristics in roosters and bulls

Angiotensin-converting enzyme (ACE) activity has been determined in the semen of certain avian and mammalian species as well as its release during cold shock. The maximum and minimum levels of this enzyme were found in mammalian spermatozoa and in seminal plasma, respectively. It was found that ACE activity in mammalian spermatozoa was more pronounced than in the seminal plasma, whereas in the avian species a revers pattern was observed. However, there were no significant differences in ACE activity in spermatozoa and seminal plasma between layer and broiler strains of avian species. By contrast, ACE activity in the spermatozoa and seminal plasma of buffalo bulls was significantly higher (P/ 0.01) than in cattle bulls. Cold shock did not significantly alter semen characteristics in avian species, while a significant (P/ 0.01) decrease in sperm live counts and motility as well as a corresponding increase in morphological abnormalities were observed in the spermatozoa of cattle and buffalo bulls due to cold shock.     

Dialysis of boar semen prior to freezing-thawing: its effects on post-thaw sperm characteristics

Low-molecular weight components of the seminal plasma have a detrimental effect on sperm function. The present study was undertaken to evaluate the effect of the removal of low-molecular weight components by dialysis on sperm characteristics prior to and after freezing. Semen, collected from 5 boars, was extended in Kortowo-3 extender (K-3, Poland) and cooled for 3h (control non-dialysis) or dialyzed for 5h in semi-permeable dialysis bags of 12-14kDa molecular weight cut-off prior to freezing. The semen samples were diluted in lactose-hen egg yolk-glycerol extender (lactose-HEY-G) or lactose-lyophilized lipoprotein fractions-glycerol extender (lactose-LPFo-G), packaged into aluminum tubes and frozen in a controlled programmable freezer. Pre-frozen and frozen-thawed spermatozoa were evaluated for motility, plasma membrane (SYBR-14 and propidium iodide) and acrosome integrity, mitochondrial function (Rhodamine 123) and ATP content. The results of the study showed that dialysis significantly improved the sperm characteristics prior to freezing. Dialysis enhanced (P<0.05) post-thaw sperm motility, plasma membrane integrity and mitochondrial function, but had no significant effect (P>0.05) on recovery of spermatozoa with intact acrosomes. Furthermore, dialyzed spermatozoa exhibited higher (P<0.05) ATP content compared with the control after freezing-thawing. Consistent inter-boar variability was detected mainly in dialyzed semen following freezing-thawing. These results indicated that the improvement in sperm quality characteristics prior to freezing and the post-thaw sperm recovery were due to the removal of low-molecular weight components from the seminal plasma. It can be suggested that dialysis is effective in improving the post-thaw quality of boar spermatozoa and has also great practical importance in improving the protocols for cryopreservation of semen. Dialysis may also contribute to a better understanding of different mechanisms underlying cryo-induced damage to boar spermatozoa.     

Detection of lipid peroxidation in frozen-thawed avian spermatozoa using C(11)-BODIPY(581/591)

The aim of this study was to perform flow cytometric analysis of C11-BODIPY581/591 oxidation in fowl and geese sperm as a marker for membrane lipid peroxidation (LPO) and to establish if the cryopreservation process would make sperm membranes more susceptible to oxidative stress. The experiment was carried out on 10 meat type line Flex roosters and 10 White Koluda® geese. The semen was collected two times a week, by dorso-abdominal massage method and pooled from 10 individuals of each species. Fowl semen samples were subjected to cryopreservation using the “pellet” method and Dimethylacetamide (DMA) as a cryoprotectant. Geese semen samples were cryopreserved in plastic straws in a programmable freezing unit with Dimethyloformamide (DMF) as the cryoprotectant. A fluorescent lipid probe C11-BODIPY581/591 provided with two double bonds that are oxidized during their contact with ROS, was used for the purpose of the assessment of the LPO in freshly diluted semen samples and frozen-thawed semen samples. This probe changes its color according to its state (non peroxidized: red; peroxidized: green). Flow cytometric analysis was used to monitor these changes. The White Koluda® geese fresh semen had a higher level of LPO than the Flex fresh semen (P > 0.01). The cryopreservation of fowl semen significantly (P > 0.01) increased the percentage of live and dead spermatozoa with lipid peroxidation. In frozen-thawed semen of White Koluda® geese the percentage of live spermatozoa with LPO significantly decreased (P > 0.05) whereas significantly (P > 0.01) higher level of dead cells with LPO was observed. There were significant differences between the two studied species. After thawing, the percentage of live and dead spermatozoa with lipid peroxidation was higher in fowl semen than in geese semen (P > 0.01). In conclusion, our data clearly indicate the existence of species specific differences in susceptibility of spermatozoa to the oxidation of PUFAs in the cell membranes, where such oxidation is caused by cryopreservation. This study shows that avian spermatozoa are vulnerable to radicals and frozenthawed sperm have higher level of LPO than fresh sperm. According to our observation, fowl semen is more susceptible to LPO than geese semen.     

Haematological assessment of the effects of the anaesthetic MS 222 in natural and neutralized form in three fresh water fish species: haemoglobin electrophoresis, ATP levels and corpuscular fragility curves

The effects of MS 222 and neutralized MS 222 anaesthesia on haemoglobin electrophoresis, erythrocyte adenosine triphosphate (ATP) levels and corpuscular fragility curves were studied in Cyprinus carpio, Sarotherodon mossambicus and Salmo gairdneri . Haemoglobin electrophoresis showed no significant intra-species differences in the percentage composition of the various fractions for any concentration of MS 222 and neutralized MS 222 used. Significant interspecies differences were, however, still observed. ATP levels showed intra and interspecies differences ascribed to the response of the fish species to MS 222-induced stress and not to actual changes in erythrocyte ATP concentrations. Differences were also observed in corpuscular fragility curves for all three species when using MS 222 or neutralized MS 222 compared to curves obtained without the use of the anaesthetic, but the mechanisms involved are not clear.     

Sperm motility patterns and metabolism in Catalonian donkey semen

The Sperm-Class Analyzer detected four subpopulations of spermatozoa with different motility characteristics in the ejaculate of the Catalonian donkey. Significant differences (P < 0.001) in the distribution of these subpopulations, as well as in total sperm number and percentage total motility, were seen in the diluted semen of four sampled donkeys. All the ejaculates evaluated showed excellent semen quality characteristics; the sperm they contained was more rapid than horse sperm. Principal components analysis showed sperm l-lactate production to be a good predictor of semen condition. This, plus the characteristics of the motility patterns of the different sperm subpopulations, provides an excellent overall indicator of semen quality.     

Decrease in glutathione content in boar sperm after cryopreservation. Effect of the addition of reduced glutathione to the freezing and thawing extenders

Although glutathione content in boar spermatozoa has been previously reported, the effect of reduced glutathione (GSH) on semen parameters and the fertilizing ability of boar spermatozoa after cryopreservation has never been evaluated. In this study, GSH content was determined in ejaculated boar spermatozoa before and after cryopreservation. Semen samples were centrifuged and GSH content in the resulting pellet monitored spectrophotometrically. The fertilizing ability of frozen-thawed boar sperm was also tested in vitro by incubating sperm with in vitro matured oocytes obtained from gilts. GSH content in fresh semen was 3.84 +/- 0.21 nM GSH/10(8) sperm. Following semen cryopreservation, there was a 32% decrease in GSH content (P < 0.0001). There were significant differences in sperm GSH content between different boars and after various preservation protocols (P = 0.0102 ). The effect of addition of GSH to the freezing and thawing extenders was also evaluated. Addition of 5 mM GSH to the freezing extender did not have a significant effect on standard semen parameters or sperm fertilizing ability after thawing. In contrast, when GSH was added to the thawing extender, a dose-dependent tendency to increase in sperm fertilizing ability was observed, although no differences were observed in standard semen parameters. In summary, (i) there was a loss in GSH content after cryopreservation of boar semen; (ii) addition of GSH to the freezing extender did not result in any improvement in either standard semen parameters or sperm fertilizing ability; and (iii) addition of GSH to the thawing extender resulted in a significant increase in sperm fertilizing ability. Nevertheless, future studies must conclude if this is the case for all boars. Furthermore, since addition of GSH to the thawing extender did not result in an improvement in standard semen parameters, this suggests that during the thawing process, GSH prevents damage of a sperm property that is critical in the fertilization process but that is not measured in the routine semen analysis.     

Seasonal changes in semen quality and freezability in the Warmblood stallion

The objective of this study was to investigate seasonal changes in stallion semen quality and to determine the best time for semen cryopreservation. Experiments were performed using 10 Warmblood stallions from the National Stud Farm in Avenches (Switzerland). Ejaculates were collected and frozen every other week during 1 year from January to December 1999. Volume, concentration, and motility, and the number of morphologically normal sperm and sperm with major defects (abnormal heads, acrosome defects, nuclear vacuoles, proximal droplets, abnormal midpieces) were evaluated. For all frozen-thawed semen samples motility as well as viability (SYBR-14/PI) was tested, and the hypoosmotic swelling test (HOS) was performed. To analyze seasonal differences 4 periods of 3 months each were defined: autumn (September, October, November), winter (December, January, February), spring (March, April, May) and summer (June, July, August). During the 1 year experiment all semen quality parameters showed a clear seasonal pattern. The volume, total sperm count and motility in fresh semen were significantly higher (P<0.05) in summer than in winter, while sperm concentration was significantly lower in summer compared to the other seasons. Regarding morphology, normal sperm was significantly lower (P<0.05) in summer than at any other time of the year and higher values (P<0.05) were found for major defects in summer than in spring and autumn. In frozen-thawed semen motility was significantly (P<0.05) improved in autumn when compared to spring and summer. Viability was lowest in summer and differed significantly (P<0.05) from other seasons. The HOS test revealed significantly more (P<0.05) membrane damaged spermatozoa in winter than in spring, summer and autumn. Our results demonstrate that in our climatic conditions clear seasonal differences occur in semen quality of fresh and frozen-thawed semen and that cryopreservation of stallion semen should preferably be performed in autumn.