Characterization of seminal plasma proteins stabilizing the sperm viability in rainbow trout (Oncorhynchus mykiss)

In seminal plasma of the rainbow trout 12 proteins were detected by SDS-PAGE, ranging in their molecular weight from 135 to 16 kDa. Only those proteins with a molecular weight of 65, 54, 47 and 16 kDa occurred in all investigated seminal plasma samples. The 65 and the 54 kDa protein were found in highest quantities (34-45% of the total quantified protein content) followed by the 47 and the 16 kDa protein (6-7% of the total quantified protein content). The 65 and the 48 kDa protein were glycoproteins as they stained positively with Periodic-Acid-Schiff reagent (PAS) specific for carbohydrates as well as with Coomassie Blue. The 90 and 19 kDa protein were found in 82-91% of the investigated samples, all other proteins in lower frequencies of 36-73%. Seminal plasma contained no lipoprotein as staining with Sudan black B was negative. To find out which proteins positively affected the sperm viability (defined as sperm motility which could be activated) spermatozoa were incubated in sperm motility inhibiting saline solution containing different seminal plasma protein fractions. Sperm motility which could be activated after an incubation period of 48 h was highest in those fractions which shared the 54, 47, and the 16 kDa protein. When spermatozoa were incubated in untreated seminal plasma sperm viability was still higher than in the seminal plasma protein fractions indicating that other components of the seminal plasma positively affected sperm viability, too. The possible influence of seminal plasma proteins on sperm physiology is discussed.     

Separation of canine epididymal spermatozoa by Percoll gradient centrifugation

The objective was to characterize the separation of canine epididymal spermatozoa on a Percoll gradient. Epididymal spermatozoa were overlaid on a 45 and 90% discontinuous Percoll gradient and centrifuged at 700 x g for 20 min. The Percoll column was separated into six fractions (top to bottom, A-F) after centrifugation. Fractions A-C contained few spermatozoa. Spermatozoa with bent or folded tails and a large amount of granular debris were observed in Fraction B. Fraction D contained many nonmotile spermatozoa, erythrocytes and round epithelial cells. Spermatozoa in Fraction E had significantly lower motility than those in the initial layer. Spermatozoa in Fraction F had motility similar to those before separation. Fraction F contained 40.6% of the motile spermatozoa layered and 67.5% of all motile spermatozoa recovered. There was no significant difference between Fraction F and the initial layer in sperm membrane integrity. In the sperm-oocyte penetration assay, spermatozoa from Fraction F had a significantly higher penetration rate into the immature homologous oocytes than those from Fraction E. Although the recovery rate of the motile spermatozoa was low, the canine epididymal spermatozoa with motility, membrane integrity and penetrating capability could be separated by two-layer discontinuous Percoll gradient centrifugation.     

Seminal plasma proteins prolong the viability of rainbow trout (Oncorynchus mykiss) spermatozoa

Rainbow trout (Oncorhynchus mykiss) spermatozoa were incubated in artificial sperm motility inhibiting saline solution (SMIS), in SMIS containing seminal plasma proteins or in pure seminal plasma. In SMIS containing the total seminal plasma protein fraction or the <50 kDa protein fraction or in pure seminal plasma, significantly higher motility rates and swimming velocities could be activated than in SMIS without seminal plasma proteins and in SMIS containing the >50 kDa protein fraction. These preliminary results indicated that seminal plasma proteins have physiological functions in prolongation and stabilization of sperm viability when using sperm motility as viability index.     

Influence of seminal plasma on the fecundity of chicken spermatozoa

This study was undertaken to investigate the influence of seminal plasma on the fecundity of chicken sperm. Sperm diluted with either incubated seminal plasma (5 or 37 degrees C for 24 h) or seminal plasma from incubated whole semen (5 or 37 degrees C for 24 h) had lower fertility levels and motility scores than sperm diluted in either fresh seminal plasma or a synthetic diluent. The number of sperm with damaged membranes increased with seminal plasma derived from 37 degrees C incubation. The depressive effect of incubated seminal plasma on semen fertility was eliminated by microfiltering .(0.22 mum) the seminal plasma either before or after incubation. Filtration of seminal plasma was only effective in eliminating the depressive effect on sperm motility when filtering was done after incubation. Filtration of seminal plasma reduced the percentage of damaged sperm in all treatments. It can be concluded that there are factors in seminal plasma that are deleterious to the fecundity of chicken spermatozoa and they may be derived from degenerating sperm and/or various fluids, cells and debris collected with the semen during manual semen collection.     

Motility and penetration competence of frozen-thawed miniature pig spermatozoa are substantially altered by exposure to seminal plasma before freezing

The objective was to determine if exposure of spermatozoa to seminal plasma before freezing decreases its freezability, assessed by percentage motile cells (using computer-assisted semen analysis) and in vitro penetration ability (using in vitro fertilization and chlortetracycline fluorescence assessment). Ejaculated spermatozoa from miniature pigs were washed by centrifugation within 20 min after collection, then incubated in seminal plasma or modified Hulsenberg VIII diluents (mHM). When the spermatozoa were cryopreserved, spermatozoa incubated in seminal plasma before freezing had significantly lower post-thaw motility than spermatozoa incubated in mHM. The incubation of spermatozoa in seminal plasma also significantly prevented frozen-thawed spermatozoa from penetrating the oocytes. The second experiment, using unfrozen spermatozoa, was to determine if the incubation of spermatozoa with seminal plasma reduced penetration ability before freezing, resulting in a significantly lower penetration rate after freezing (compared with spermatozoa incubated without seminal plasma). The penetration competence of unfrozen spermatozoa was significantly decreased by incubation in seminal plasma, but no difference in motility was observed between spermatozoa exposed to seminal plasma versus mHM. We concluded that ejaculated seminal plasma contained some factor(s) that modified the sperm before freezing and reduced the freezability and post-thaw penetration competence of spermatozoa.     

Effect of seminal plasma on the cryopreservation of equine spermatozoa

Seminal plasma is generally removed from equine spermatozoa prior to cryopreservation. Two experiments were designed to determine if adding seminal plasma back to spermatozoa, prior to cryopreservation, would benefit the spermatozoa. Experiment 1 determined if different concentrations of seminal plasma affected post-thaw sperm motility, viability and acrosomal integrity of frozen/thawed stallion spermatozoa. Semen was washed through 15% Percoll to remove seminal plasma and spermatozoa resuspended to 350 x 10(6)sperm/mL in a clear Hepes buffered diluent containing either 0, 5, 10, 20, 40 or 80% seminal plasma for 15 min, prior to being diluted to a final concentration of 50 x 10(6)sperm/mL in a Lactose-EDTA freezing diluent and cryopreserved. Sperm motility was analyzed at 10 and 90 min after thawing, while sperm viability and acrosomal integrity were analyzed 20 min after thawing. Seminal plasma did not affect sperm motility, viability or acrosomal integrity (P>0.05). Experiment 2 tested the main affects of seminal plasma level (5 or 20%), incubation temperature (5 or 20 degrees C) and incubation time (2, 4 or 6 h) prior to cryopreservation. In this experiment, spermatozoa were incubated with 5 or 20% seminal plasma for up to 6h at either 5 or 20 degrees C prior to cryopreservation in a skim milk, egg yolk freezing extender. Samples cooled immediately to 5 degrees C, prior to freezing had higher percentages of progressively motile spermatozoa than treatments incubated at 20 degrees C (31 versus 25%, respectively; P<0.05), when analyzed 10 min after thawing. At 90 min post-thaw, total motility was higher for samples incubated at 5 degrees C (42%) compared to 20 degrees C (35%; P<0.05). In addition, samples containing 5% seminal plasma had higher percentages of total and progressively motile spermatozoa (45 and 15%) than samples exposed to 20% seminal plasma (33 and 9%; P<0.05). In conclusion, although the short-term exposure of sperm to seminal plasma had no significant effect on the motility of cryopreserved equine spermatozoa, prolonged exposure to seminal plasma, prior to cryopreservation, was deleterious.     

Improvement of Sperm Motility of Sex-Reversed Male Rainbow Trout, Oncorhynchus mykiss, by Incubation in High-pH Artificial Seminal Plasma

Changes in the motility time of spermatozoa collected from the testes and the sperm duct of normal and sex-reversed male (XX) rainbow trout in physiological balanced salt solution were examined after incubation in artificial seminal plasmas of various pHs. Although untreated spermatozoa from the sperm duct retained motility for 60–90 s in the balanced salt solution, the spermatozoa collected from the testes were immotile. During the incubation in artificial seminal plasma of pH 7.0, the spermatozoa from the sperm duct hardly moved, similar to the testicular spermatozoa in the balanced salt solution. By suspending and incubating the testicular spermatozoa in artificial seminal plasma of pH 9.9 for 2 h at 4°C, the percentage of motile spermatozoa increased from 0–5% to 80%. The spermatozoa remained motile for at least 2 min after long-term incubation (12 h). When the full-sib eggs were inseminated with untreated testicular spermatozoa or testicular sperm treated for 2 h at high pH, the percentage survival increased from 5.5% to 53.8% at the eyed stage due to the high-pH treatment. The incubation of the spermatozoa in high-pH artificial seminal plasma improved the motility of the spermatozoa from the testes of the sex-reversed male that had lost its sperm duct. By this treatment, it is possible to markedly increase the mass production efficiency of all-female or all-female triploid sterile progenies.     

Induction and enhancement of progressive motility in hamster caput epididymal spermatozoa

Progressive motility was induced in hamster caput epididymal spermatozoa incubated in Tyrodes medium containing 50 mM theophylline, 1.0% Fraction V bovine serum albumin, and 15% (v/v) heat-treated human seminal plasma. Under these induction conditions, however, the maximum percent of caput spermatozoa exhibiting progressive motility (21%) and the time during which motility was sustained (120 min) were significantly less (p less than 0.05) than that of controls from the cauda epididymidis. Moreover, in contrast to caudal spermatozoa, the majority of the induced caput spermatozoa exhibited some degree of flagellar bending at the neck or midpiece. In subsequent experiments the procedure for motility induction was modified to achieve levels of motility in caput spermatozoa equivalent to those observed for caudal spermatozoa. The addition of 5 microM diamide, a sulfhydryl oxidant, to the induction medium prevented the flagellar angularity observed in induced caput sperm preparations. The percentage of caput spermatozoa induced to progressive motility was increased to levels characteristic of caudal spermatozoa (48%) by the addition of hamster caudal epididymal fluid (CEF) to the induction medium. Finally, the viability of the induced caput spermatozoa was significantly enhanced (p less than 0.05) by the removal of Fraction V albumin from the induction medium. In the presence of CEF and in the absence of albumin, 50% of the caput spermatozoa acquired progressive motility and sustained this motility for 4 h. Moreover, when fatty acid-free, charcoal-extracted albumin instead of Fraction V albumin was utilized in the induction procedure, a maximum of 43% of the caput spermatozoa acquired progressive motility and maintained this motility for 4 h, suggesting that the decreased sperm viability observed in the presence of Fraction V albumin was due to a contaminant of albumin, possibly fatty acids. The studies described herein demonstrate for the first time that immature quiescent caput epididymal spermatozoa can be induced to acquire progressive and sustained motility equivalent to that observed in mature caudal epididymal spermatozoa.     

Stimulation of sperm motility and oxygen consumption of fowl spermatozoa by a low molecular weight fraction of seminal plasma

Washed fowl spermatozoa were incubated in a phosphate buffer containing various concentrations of fowl seminal plasma at 41 degrees C, normal body temperature, and the motility and oxygen consumption of spermatozoa were determined. Immediately after the incubation, spermatozoa showed good motility in the various diluents. However, with concentrations of seminal plasma at or below 20%, spermatozoa quickly became immotile. In contrast, at concentrations higher than 40% seminal plasma, spermatozoa were motile even after 15 min. As the concentration of seminal plasma was increased, oxygen consumption of spermatozoa also increased. A filtrate of the seminal plasma, obtained by passing the fluid through an Amicon YM-2 ultra-filtration membrane (Mr less than 1000), also stimulated the motility and oxygen consumption of spermatozoa. These results suggest that some low molecular weight factor(s) in fowl seminal plasma stimulated motility and oxygen consumption of fowl spermatozoa at 41 degrees C. A physiological role of this factor(s) may be to assist passage of spermatozoa through the vagina after natural mating.     

The influence of antioxidant, cholesterol and seminal plasma on the in vitro quality of sorted and non-sorted ram spermatozoa

In an effort to improve the number of functional spermatozoa following sex-sorting and cryopreservation, the effects on in vitro sperm characteristics of the additives: (i) catalase (pre-sorting); (ii) cholesterol-loaded cyclodextrins (CLCs; pre-sorting); and (iii) seminal plasma (post-thawing) were investigated. For all experiments, spermatozoa (three males, n=3 ejaculates/male) were processed using a high speed flow cytometer before cryopreservation, thawing and incubation for 6h. Catalase had no effect (P>0.05) on post-thaw motility characteristics (as measured by CASA) of sex-sorted ram spermatozoa, but pre-sort addition of CLCs reduced (P<0.05) sperm quality after post-thaw incubation for 0 h (motility), 3h (motility, average path velocity, viability and acrosome integrity) and 6h (motility, average path and curvilinear velocity, straightness, linearity, viability and acrosome integrity). Seminal plasma had a differential effect (P<0.001) on sex-sorted and non-sorted spermatozoa. Post-thaw supplementation of increasing levels of seminal plasma caused all motility characteristics of sex-sorted, frozen-thawed spermatozoa to decline (P<0.05); conversely, non-sorted, frozen-thawed spermatozoa exhibited improvements (P<0.05) in motility, viability, acrosome integrity and mitochondrial respiration. In summary, incorporation of catalase, CLCs and seminal plasma into the sorting protocol failed to improve post-thaw sperm quality and, consequently efficiency of sex-sorting of ram spermatozoa. The paradoxical effect of seminal plasma supplementation on the in vitro characteristics of ram spermatozoa provides further evidence that sex-sorting by flow cytometry produces a selected population of cells with different functions compared with non-sorted spermatozoa.     

In vitro fertilization of in vitro matured pig oocytes: effects of boar and ejaculate fraction

Ejaculates from 3 young boars were collected on 4 occasions as a series of separate 15-ml fractions. The contribution of different fractions of these ejaculates to observed variability in the quality of the semen when used for IVF was then determined. On the basis of sperm concentration, 3 fractions representing the first peak concentration (Fraction 1), the lowest sperm concentration after Fraction 1 (Fraction 2), and the second peak concentration (Fraction 3) were selected for analysis in vitro. Oocyte-cumulus-granulosa cell complexes were obtained by dissection from slaughterhouse ovaries. In vitro matured oocytes were randomly assigned for fertilization by the 3 semen samples from each boar. Sperm concentration was the same in all the samples during prefertilization incubation, while the final concentration for fertilization was 5 x 10(5) sperm/ml. Data were analysed using ANOVA for a split-plot design. In the presence of fraction effects, Student-Newman-Keuls (SNK) test was used for multiple comparison of treatment means. Oocyte penetration rates differed among fractions (P = 0.001) and varied from 69 to 100% (mean 95.7%) for Fraction 1, from 0 to 100% (mean 53.3%) for Fraction 2, and from 50to 100% (mean 89.9%) for Fraction 3. There were also differences in male pronuclear formation rate (P = 0.028; mean 27.6, 9.3 and 16.4% for Fractions 1, 2 and 3, respectively); in the rate of polyspermy (P = 0.0001; mean 92.3, 31.9 and 76.3% for Fractions 1, 2 and 3, respectively); and in the number of penetrated spermatozoa per oocyte P = 0.002; mean 5.58, 1.94 and 4.07 for Fractions 1, 2, and 3, respectively). The first peak concentration of semen (Fraction 1) showed superiority in fertilizing ability and less variability in penetration rate from replicate to replicate compared with the other 2 fractions. By multiple comparison, Boar 1 showed higher rates of penetration (P < 0.05), male pronuclear formation (P < 0.05) and polyspermy (P < 0.05) than the other 2 boars. There was no fraction-by-boar interaction. The IVM-IVF system adopted proved to be a promising method for boar semen evaluation.     

Isolation of a population of intact, highly-motile porcine spermatozoa using a discontinuous bovine serum albumin gradient

This study was conducted to determine the efficacy of isolating intact, highly-motile porcine spermatozoa using a discontinuous bovine serum albumin (BSA) gradient. Either 0.25, 0.5, 1, 2.5 or 5 x 10(9) spermatozoa extended to 26 ml with Kiev extender were layered on a discontinuous BSA gradient (4% BSA over 10% BSA) contained within a 500-ml separatory funnel. After 1 h of sperm migration at 23 degrees C, four 30-ml aliquots designated Fractions 1, 2, 3, 4 and Fraction T were collected from the bottom of the funnel. Fraction 4 was the bottom fraction and Fraction T was the remnant of the applied sample. For all concentrations of applied spermatozoa, the percentage of progressively motile spermatozoa was greater in Fraction 4 than in Fractions T or 1 (P<0.05). Fraction 4 contained a greater (P < 0.05) proportion of spermatozoa with normal apical ridge acrosomes than Fraction T. Regardless of the concentration of spermatozoa applied, Fraction 4 contained more than 90% of progressively motile spermatozoa and spermatozoa possessing normal apical ridge acrosomes. The percentage of applied cells recovered in Fraction 4 decreased as the concentration of spermatozoa applied to the gradient increased.     

Chemical composition of seminal and ovarian fluids of chinook salmon (Oncorhynchus tshawytscha) and their effects on sperm motility traits

The relationships between the compositions of ovarian, seminal fluids and sperm function are not well known in teleostean fish species. The objective of the present study was to determine the concentration of the major inorganic ions (Na(+), K(+), Ca(2+), Mg, Cl(-)), osmolality, and pH of ovarian and seminal fluid of sexually mature chinook salmon (Oncorhynchus tshawytscha), and to determine if the composition of these fluids influences sperm motility traits (swimming speed, duration of forward mobility, swimming path trajectory, and percent motility). Cation concentrations and osmolality were significantly different in the two fluids. The ionic composition of ovarian fluid differed among individual females, and also among samples collected at different times through the spawning season. Carbonate and bicarbonate were the principal buffer ions in ovarian fluid, and its viscosity was considerably greater than that of water and was shear-dependent. The duration of forward motility (longevity) of spermatozoa, swimming speed, percent motility, and path trajectory were measured using milt from 10 males activated in the ovarian fluid from 7 females whose ion concentrations were known. No significant correlations were observed between the composition of the seminal fluid and sperm traits. However, in ovarian fluid, sperm longevity was negatively correlated with variation in [Ca(2+)] and [Mg(2+)], while percent motility increased with increasing [Mg(2+)]. These observations provide a possible chemical basis for cryptic female mate choice whereby female ovarian fluid differentially influences the behaviour of sperm from different males, and thus their fertilization success.     

The effect of seminal plasma on alpaca sperm function

In order to advance the development of assisted reproductive technologies in alpacas and other Camelids, the objective of this study was to explore the role of seminal plasma concentration on motility and functional integrity of alpaca sperm. Sixteen male alpacas > 3 y of age were used. In Experiment 1, epididymal sperm were incubated for 0 to 6 h in 0, 10, 25, 50, or 100% seminal plasma and motility was assessed. In Experiment 2, epididymal sperm were incubated in 0, 10, or 100% seminal plasma for 3 h and motility, acrosome integrity and DNA integrity were assessed. In Experiment 3, ejaculated sperm were incubated in 10, 25, 50, or 100% seminal plasma for 0 to 6 h and motility assessed. In Experiment 4, ejaculated sperm were incubated in 10 or 100% seminal plasma for 3 h and motility, acrosome integrity, DNA integrity, and viability were assessed. Epididymal and ejaculated sperm maintained motility longer when incubated in the presence of 10% seminal plasma compared to 0, 25, 50, or 100% seminal plasma (P < 0.001). The mean ± SEM percentage of epididymal sperm with intact acrosomes was less (P < 0.001) in samples incubated in 0% seminal plasma (39.4 ± 3.73) compared to 10% (75.3 ± 1.20) or 100% (77.4 ± 0.90) within 1 h after incubation. However, DNA integrity of ejaculated and epididymal sperm was not significantly affected by seminal plasma concentration. The mean viability of ejaculated sperm was reduced in the presence of 100 (12.7 ± 2.33) compared to 10% (36.2 ± 4.68) seminal plasma (P < 0.001) within 1 h of incubation. We concluded that alpaca semen should be diluted to a final concentration of 10% seminal plasma to prolong motility, preserve acrosome integrity, and maintain viability of sperm.     

The effect of sulfhydryl oxidation on the morphology of immature hamster epididymal spermatozoa induced to acquire motility in vitro

Immotile spermatozoa from the caput epididymidis become progressively motile when incubated in medium containing theophylline, seminal plasma, and albumin. We previously reported that under these incubation conditions the spermatozoa induced to acquire motility exhibited a marked flagellar angularity, with the sperm head or midpiece bent 90-180 degrees towards the tail. In addition, we demonstrated that sperm flagellar bending did not occur when the sulfhydryl oxidant diamide was added to the motility induction medium. In the present study, we examined further the effect of sulfhydryl oxidation on the morphology and sulfhydryl content of immature caput spermatozoa induced to acquire motility in vitro. We found that flagellar bending was prevented and sperm flagellar straightness was maintained in a dose-dependent manner by diamide. Moreover, flow cytometric analysis of caput sperm sulfhydryls using the sulfhydryl reagent monobromobimane (mBBr) revealed that 1) diamide oxidizes caput sperm sulfhydryls, and 2) less than 15% of the total reactive sperm sulfhydryls were oxidized at diamide concentrations capable of preventing sperm angulation. Sodium tetrathionate (NaTT), another sulfhydryl oxidant, and hamster cauda epididymal fluid (CEF) containing sulfhydryl oxidase enzyme activity also maintained flagellar straightness in induced caput spermatozoa and oxidized sperm sulfhydryls. The flagellar straightness in caput spermatozoa treated with sulfhydryl oxidants, however, was temporary; with extended incubation, diamide- or CEF-treated spermatozoa exhibited flagellar bending. Additional studies showed that the flagellar straightness observed in sulfhydryl-oxidized spermatozoa was sustained when nitrofurantoin, an inhibitor of glutathione reductase, was included in the induction medium. Flow cytometric analysis of nitrofurantoin-treated spermatozoa showed that nitrofurantoin maintained the sperm disulfides formed by diamide and prevented the reduction of sperm disulfides back to sulfhydryls. Taken together, these studies demonstrate the significance of sulfhydryl oxidation in maintaining the morphology of immature caput epididymal spermatozoa induced to acquire motility in vitro and suggest that sulfhydryl oxidation may be important in the development of motility during sperm epididymal maturation in vivo.     

Sperm metabolism of the telost fishes Chalcalburnus chalcoides and Oncorhynchus mykiss and its relation to motility and viability.

In the teleost fish Chalcalburnus chalcoides (Cyprinidae) the influence of metabolic inhibitors, substrates, coenzymes, and oxygen concentrations on spermatozoal parameters during motility and during immotile incubation was studied, the respiration rate was characterized, representative metabolite levels were measured, and the results were compared with Oncorhynchus mykiss (Salmonidae). In Chalcalburnus chalcoides the sperm motility rate, the average path swimming velocity, the motility duration, and the viability of immotile semen were significantly reduced in the presence of inhibitors of respiration (potassium cyanide, 2.4-dinitrophenol, atractyloside). Anaerobic conditions (<1 mg O(2)/liter) and inhibition of the tricarboxylic acid cycle by malonate and >7.5 mmol/liter succinate had similar effects on the sperm motility parameters and on the viability of immotile spermatozoa. Pyruvate and coenzyme A (an acyl-group carrier during oxidative carboxylation of pyruvate) prolonged the duration of sperm motility and the viability of immotile incubated spermatozoa, and also increased the spermatozoal respiration rate. Glucose levels significantly decreased during motility and during immotile storage and, under anaerobic conditions, the levels of lactate increased indicating that pyruvate derived from glycolysis. The respiration rate and the glycolytic rate significantly increased during motility. Therefore oxidative phosphorylation, tricarboxylic acid cycle, and aerobic glycolysis are central energy-supplying pathways for spermatozoa of Chalcalburnus chalcoides. The stimulatory effect of pyruvate and coenzyme A indicated that glycolysis is a rate-controlling pathway. Similar results were obtained for Oncorhynchus mykiss with the only exception that the stimulatory effect of coenzyme A was more significant than the stimulatory effect of pyruvate. When the sperm motility-activating saline solutions were optimized in aspects of energy supply, ionic composition, and osmolality, about 50% of the motile spermatozoa swam progressively (>20 mm/sec) for about 3 min in Chalcalburnus chalcoides and in Oncorhynchus mykiss. About 20% swam progressively for >2 hr in Chalcalburnus chalcoides and for >30 min in Oncorhynchus mykiss. J. Exp. Zool. 284:454-465, 1999.     

Influence of seminal plasma on fresh and post-thaw parameters of stallion epididymal spermatozoa

Fresh and post-thaw parameters (motility, morphology and viability) of stallion epididymal spermatozoa that have been and have not been exposed to seminal plasma were evaluated, and directly compared to fresh and post-thaw parameters of ejaculated spermatozoa. Six sperm categories of each stallion (n=4) were evaluated for motility, morphology and viability. These categories were fresh ejaculated spermatozoa (Fr-E), fresh epididymal spermatozoa that had been exposed to seminal plasma (Fr-SP+), fresh epididymal spermatozoa that had never been exposed to seminal plasma (Fr-SP-), frozen-thawed ejaculated spermatozoa (Cr-E), frozen-thawed epididymal spermatozoa that had been exposed to seminal plasma prior to freezing (Cr-SP+) and frozen-thawed epididymal spermatozoa that had never been exposed to seminal plasma (Cr-SP-). Results show that seminal plasma stimulates initial motility of fresh epididymal stallion spermatozoa while this difference in progressive motility is no longer present post-thaw; and that progressive motility of fresh or frozen-thawed ejaculated stallion spermatozoa is not always a good indicator for post-thaw progressive motility of epididymal spermatozoa. This study shows that seminal plasma has a positive influence on the incidence of overall sperm defects, midpiece reflexes and distal cytoplasmic droplets in frozen-thawed stallion epididymal spermatozoa while the occurance of midpiece reflexes is likely to be linked to distal cytoplasmic droplets. Furthermore, seminal plasma does not have an influence on viability of fresh and frozen-thawed morphologically normal epididymal spermatozoa. We recommend the retrograde flushing technique using seminal plasma as flushing medium to harvest and freeze stallion epididymal spermatozoa.     

Effects of exposure of epididymal boar spermatozoa to seminal plasma on the binding of zona pellucida proteins during in vitro capacitation

The purpose of the investigation was to determine whether seminal plasma plays a role in the increase during in vitro capacitation of the number of boar spermatozoa with enhanced binding of zona pellucida proteins. Ejaculated spermatozoa and spermatozoa collected from the caudae epididymides of boars were incubated at 39 degrees C in a Tyrode's IVF medium. During incubation, the zona binding ability of individual spermatozoa was assessed with fluorescein-conjugated solubilized zona pellucida proteins (FITC-sZP), using a flow cytometer. Propidium iodide (PI) was included to simultaneously monitor cell viability. During incubation of ejaculated spermatozoa, a percentage of the spermatozoa expressed enhanced binding of FITC-sZP. The percentage of viable spermatozoa with enhanced binding reached a maximum of 37% (S.D.=8, averaged over five boars) after 2-3 h. In epididymal sperm, a similar maximum was observed after incubation in vitro, but a longer time of incubation was needed (6 h). Also, the rate of cell death of epididymal sperm was much lower than that of ejaculated sperm. When epididymal spermatozoa was exposed to seminal plasma in vitro, the time needed to reach a maximal percentage of viable spermatozoa with enhanced FITC-sZP binding was similar to that in ejaculated semen. However, the rate of cell death was still much lower than in ejaculated sperm. We concluded that the binding sites on the sperm surface that are involved in the increased binding of zona proteins during incubation under IVF conditions were not derived from the seminal plasma. The cellular processes leading to the increased binding capacity were accelerated by exposure of the sperm to seminal plasma.     

Antioxidant supplementation in vitro improves boar sperm motility and mitochondrial membrane potential after cryopreservation of different fractions of the ejaculate

Antioxidant supplementation during cooling was assayed to improve the motility of frozen-thawed (FT) boar spermatozoa from two different fractions of the ejaculate, the first component of the sperm-rich fraction (Fraction I) and the rest of the bulk ejaculate (Fraction II). Using a split-sample design, addition of two different concentrations (100 and 200 microMl(-1)) of the water-soluble Vitamin E analogue Trolox (6-hydroxy -2,5,7,8-tetramethylchroman -2-carboxylic acid) was evaluated for an effect on sperm motility (measured both subjectively and by means of a computer assisted motility assessment (CASA)), and on mitochondrial membrane potential using flow cytometry after cell-loading with JC-1. The effect of the Vitamin E analogue was clearly dose-dependent and varied with the fraction of the ejaculate considered. Motility was significantly higher in Trolox-treated spermatozoa (200 microm), from either ejaculate fraction, albeit the effect was more evident in spermatozoa from Fraction II (P<0.05) for any Trolox-concentration. Antioxidant supplementation resulted, also dose-dependent, in a higher number of spermatozoa showing high mitochondrial activity as assessed by the JC-1 staining, in both ejaculate fractions. In the present trial, exogenous Trolox positively affected post-thaw sperm viability (as motility and mitochondrial membrane potential) in both fractions of the ejaculate. The magnitude of the effect appeared, however, to be dependent of the fraction of the ejaculate considered.     

ATP Content and Sperm Motility of Extended Bovine Semen under Different Storage Conditions

The effects of incubation temperature (+20°C vs +35°C) and media type on the ATP content and motility of spermatozoa were determined in fresh bovine semen in order to develop a method for assaying post-thaw quality. Semen was obtained from 3 bulls at 2 occasions. The spermatozoa were washed using a Ficoll-containing medium before being resuspended in each of 4 different media (I. 0.9 % NaCl; II. Trisbuffer solution; III. seminal plasma; IV. seminal plasma + Tris-buffer solution) and incubated for 6 h. The least-squares means for ATP content were higher (p ≤ 0.05) at +20°C than +35°C for all media except no. I. By contrast, the least-squares means for sperm motility were higher (p ≤ 0.05) at +35°C than at +20°C in media II and III. A decrease over time in ATP content and motility at both temperatures was also observed. The single most important factor responsible for changes in ATP content and sperm motility was the temperature and the medium, respectively.