In-vitro development of the fertilizing ability of hamster epididymal spermatozoa after co-culture with epithelium from the proximal cauda epididymidis

The epididymides of adult male hamsters were surgically ligated at the junction of the distal corpus and proximal cauda regions. After 3 days, spermatozoa recovered from the distal corpus displayed greater progressive motility and head to head agglutination in capacitating medium than did those from intact controls, but had low fertilizing ability (3% fertilization rate) in vitro or in vivo. When these spermatozoa were incubated for 6 h with epithelial cells from the proximal cauda epididymidis, previously cultured for 3 days, they maintained motility and exhibited a significant increase in fertilizing ability (30% and 29% in vitro and in vivo respectively). The fertilizing ability of distal corpus spermatozoa incubated with 3-day-old cultures without androgens, or 8-12-day-old epithelial cells with fibroblast overgrowth, or without epithelial cells, remained low (5%). Increase in sperm fertilizing ability was associated with increased sperm binding to the zona pellucida in vitro. These results demonstrate that, under suitable culture conditions, the final stages in the development of hamster sperm fertilizing ability can be achieved in vitro. Factors secreted by cultured epithelium from the proximal cauda epididymidis are implicated in this maturation process.     

Hormonal regulation of zona-binding ability and fertilizing ability of rat epididymal spermatozoa

Rat spermatozoa from the proximal caput, the proximal corpus, the middle corpus, and the distal cauda epididymidis were examined for their ability to bind to the zona pellucida after a 1-, 2.5-, or 4.5-h incubation at 34°C with rat eggs in cumulus. Caput spermatozoa did not bind to the zona after 1, 2.5, or 4.5 h of incubation. Corpus spermatozoa did bind to the zona, but the percentage of eggs with bound spermatozoa and number of bound spermatozoa per egg increased with the length of incubation. Cauda spermatozoa bound readily to the zona pellucida, and their zona binding ability did not change with longer incubations. It thus appears that rat spermatozoa gradually acquire the ability to bind to the zona pellucida in the corpus epididymidis. The zona-binding capacity of cold immobilized cauda spermatozoa, defined as the percentage of eggs with bound spermatozoa, increased with the number of spermatozoa incubated and reached a plateau characteristic of the endocrine status of the animal. After castration, zona-binding ability is progressively lost from day 3 until day 10 where it is nil. Testosterone supplementation maintains zona-binding ability to control levels. Similarly, fertilizing ability declines from day 5 after castration until day 10. Testosterone prevents this loss of fertilizing ability. It thus appears that the development of zona-binding ability during epididymal transit is, like the development of fertilizing ability, under androgen regulation. The close correlation between the onset of fertilizing ability and zona-binding ability during maturation, the loss of fertilizing ability and zona-binding ability after castration, and the recovery of both fertilizing ability and zona-binding ability with testosterone treatment suggests that the androgen-dependent development of zona-binding ability is an important component of the acquisition of sperm fertilizing ability during epididymal transit.     

Effect of different fractions of seminal plasma on the fertilizing ability of fowl spermatozoa stored in vitro.

This paper describes the effects of whole seminal plasma and of dialysed seminal plasma on the fertilizing ability of fowl spermatozoa stored for 24 h at 4 degrees C. The fertilizing ability of fowl semen diluted 1:1 with Beltsville Poultry Semen Extender and stored for 24 h at 4 degrees C was enhanced after replacement of the homologous seminal plasma by the diluent (89 versus 77% fertilization rate). Better results were obtained with seminal plasma dialysed against water before sperm storage to discard the less than 1 kDa or the less than 50 kDa fractions. It was concluded that low molecular weight seminal plasma fractions could damage the fertilizing ability of spermatozoa during storage at 4 degrees C, whereas high molecular weight fractions appeared to enhance fertilizing ability.     

Effects of lipid peroxide formation in fowl semen on sperm motility, ATP content and fertilizing ability

The ability of samples of semen from individual male fowl to form the products of lipid peroxidation during 5 h aerobic incubation at 40 degrees C varied between 0 and 8 nmol malonaldehyde/10(9) spermatozoa. Formation of higher concentrations of malonaldehyde was associated with a partial or complete loss of fertilizing ability whilst the fertilizing ability of samples producing low or negligible concentrations of malonaldehyde remained unimpaired. The semen of birds which showed a tendency to form high concentrations of malonaldehyde was not readily identifiable as abnormal by assessment of sperm motility, morphology or ATP content. Nor was the loss of fertilizing ability during aerobic incubation associated with an obvious change in these characteristics.     

In vitro capacitation of boar ejaculated spermatozoa: Effect of conditioned media prepared from preincubated sperm suspension

The fertilizing ability of boar ejaculated spermatozoa was examined in vitro after prcincubation at a concentration of 2.5 × 10⁸/ml for 4 hr in several conditioned media (CM). For preparation of CM, boar spermatozoa were incubated in a modified Krebs-Ringer bicarbonate solution (TYH) at concentrations of 20 to 40 × 10⁸/ml for several hours up to 4 hr; then their supernatant fluids were collected by centrifugation. When boar ejaculated spermatozoa were preincubated in TYH alone, 14.1% of oocytes were penetrated by them as we reported previously. On the other hand, preincubating them with CM, their fertilizing ability was elevated according as the incubation time of CM preparation was lengthened. The fertilization rate reached 75.0%, using 4 hr-incubated CM for the preincubation medium. The effect of CM was not deteriorated by heat treatments (56°C, 30 min, or 100°C, 5 min). The components of CM were separated at a molecular weight of 25,000 by ultrafiltration, and high fertilization rate (69.8%) was obtained when low molecular weight fraction was used for the preincubation medium. Sperm extracts prepared from directly frozen-thawed sperm suspension and 0.1–10 mM of taurine or hypotaurine had no effect on the fertilizing ability of boar spermatozoa. These results suggest that substances stimulating boar sperm capacitation were accumulated from viable spermatozoa into the medium during incubation and that the effective substances were heat-stable and of low molecular weight and were not taurine and hypotaurine.     

Inhibition of adenosine-metabolizing enzymes modulates mouse sperm fertilizing ability: A changing role for endogenously generated adenosine during capacitation

The effect of inhibiting adenosine-metabolizing enzymes on sperm fertilizing ability was studied to investigate a possible role for endogenously generated adenosine in the regulation of capacitation. The compounds used have been shown to be effective inhibitors of the relevant enzymes in similarly incubated mouse sperm suspensions. Inhibition of 5′-nucleotidase activity with α,β-methylene adenosine 5′-diphosphate (AMPCP), to reduce available endogenous adenosine, caused a dose-dependent inhibition of the fertilizing ability of partially capacitated spermatozoa, which was significant with 100 and 250 μM AMPCP. Conversely, inhibition of adenosine deaminase with 100 nM coformycin, to increase available endogenous adenosine, promoted the fertilizing ability of partially capacitated spermatozoa when the fertilization rate of control suspensions was low. However, coformycin had no effect on sperm suspensions with moderate fertilizing ability, and it inhibited fertilizing ability when added to capacitated spermatozoa. These data are consistent with a promotion of the early stages of capacitation by endogenously generated adenosine and suggest that sensitivity to adenosine changes as capacitation proceeds. Because the majority of adenosine-metabolizing enzyme activity resides in or is directed toward the extracellular compartment in such suspensions, these effects of adenosine may be mediated at the outer surface of the cell. By interacting with receptors on adenylate cyclase, externally produced adenosine could modulate intracellular levels of cyclic adenosine monophosphate (cAMP), thereby influencing fertilizing ability.     

Decrease of fertilizing ability of mouse spermatozoa after freezing and thawing is related to cellular injury

In general, the fertilizing ability of cryopreserved mouse spermatozoa is less than that of fresh spermatozoa. This ability is especially low in C57BL/6, the main strain used for the production of transgenic mice. To solve this problem, the relationship between cell damage and fertilizing ability in cryopreserved mouse spermatozoa was examined in this study. Sperm motility analysis revealed no significant difference among the motilities of cryopreserved C57BL/6J, BALB/cA, and DBA/2N sperm (67.6%, 43.4%, and 60.0%, respectively) after thawing. However, the results of in vitro fertilization (IVF), scanning electron microscopy (SEM), and transmission electron microscopy (TEM) showed a strong correlation between the frequency of aberrant spermatozoa (FAS) and fertilization rates (FR; C57BL/6J: FAS, 83.7%; FR, 17.0%; BALB/cA: FAS, 67.2%; FR, 24.2%; and DBA/2N: FAS, 10.2%; FR, 93.6%), and damage to spermatozoa was localized particularly in the acrosome of the head and mitochondria.     

Effects of cold storage on plasma membrane, DNA integrity and fertilizing ability of feline testicular spermatozoa

This study examined the effects of cold storage on plasma membrane, DNA integrity, and fertilizing ability of domestic cat spermatozoa. Intact cat testes were stored at 4°C in Dulbecco's phosphate buffered saline (DPBS) for 7 days. Membrane integrity (experiment 1) and DNA integrity (experiment 2) of extracted spermatozoa were assessed over time during storage. Testicular spermatozoa were also tested for their fertilizing ability via intracytoplasmic sperm injection (ICSI) in term of gamete activation and early embryonic development at 18 h (experiment 3). The membrane integrity of testicular spermatozoa was well preserved in DPBS for 4 days compared to non-preserved control (Day 0) (P<0.05). The incidence of testicular sperm DNA fragmentation was <1% after 7 days of cold storage and was not significantly affected by the duration of cold storage (P>0.05). Finally, testicular spermatozoa could form pronuclei and sustain embryo development following ICSI regardless of the storage time (P>0.05). In conclusion, cat testicular spermatozoa can be preserved at 4°C for up to 7 days without severely compromising of plasma membrane and DNA integrity while retaining a normal fertilizing ability.     

Stabilizing rôle of epididymal plasma in relation to the capacitation time of boar spermatozoa

Functional aspects of the maturation of epididymal spermatozoa have been examined by means of surgical insemination of two types of sperm suspension directly into the oviducts. Suspensions were prepared by macerating tissue from the upper corpus region of the epididymis, and cell-free plasma was prepared from the contents of the cauda epididymidis. Each comparison of the fertilizing ability of the two sperm suspensions was made within the same animal, known numbers of upper corpus spermatozoa in either medium TCM 199 or caudal plasma being instilled into separate oviducts close to the time of ovulation.Activated eggs were recovered from 11 of 12 inseminated animals some 4–6 h later, but within the intervals examined there was a distinct difference in the fertilizing ability of the two types of sperm suspension; 87% of the eggs were activated by upper corpus spermatozoa in TCM 199 compared with 9% of the eggs exposed to similar spermatozoa suspended in caudal plasma. Furthermore, the fertilization process was invariably more advanced when eggs had been activated by the upper corpus spermatozoa suspended in TCM 199, and the number of spermatozoa on or in the zona pellucida was likewise consistently higher with such sperm suspensions. The rôle of the factor(s) in cauda epididymal plasma contributing to the observed delay in fertilizing ability is discussed in the context of sperm transport and capacitation after natural mating.     

Prolonged motility of fowl spermatozoa in vitro due to a low molecular weight factor(s) released from cultured embryonic cells

Fowl spermatozoa were incubated at 41°C in a supernatant removed from a 4-day culture medium of embryonic chick skeletal muscle cells. Their motility, as assessed at room temperature (20–25°C), was maintained for 48 h. Fertilizing ability was also retained for at least 24 h. In contrast, spermatozoa incubated in the fresh culture medium lost their motility and fertilizing ability rapidly. A filtrate of the 4-day culture medium, obtained by passing the fluid through an Amicon PM-10 ultrafiltration membrane, prolonged the motility of spermatozoa. These results suggested that a low molecular weight factor(s) (mol. wt. < 10 000) supplied by the cultured cells effectively prolonged the motility of spermatozoa.     

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.     

Both taurine and albumin support mouse sperm motility and fertilizing ability in vitro but there is no obligatory requirement for taurine

When mouse spermatozoa were washed immediately upon release from the epididymis, preincubated for up to 120 min in PVA-containing, albumin-free medium and assessed for their ability to fertilize cumulus-intact eggs in vitro, they were poorly fertile in comparison with their unwashed counterparts in the same medium. Fertilizing ability could be significantly improved by introducing taurine or albumin or by washing a second time at the end of preincubation. The most effective treatment was provided by the continuous presence of low concentrations (0.05-0.1 mg/ml) of BSA, similar to the amount of albumin detected in the supernatants removed during washing. There was no evidence that acrosome loss was inhibited by washing; rather, it was enhanced by the removal of a surface component which inhibits the acrosome reaction. The presence of taurine did not further increase this response. Motility, reduced in washed suspensions, was improved by the presence of taurine or albumin and experimental results suggest that this was a major factor in the improvement of fertilizing ability after introduction of these compounds. Although taurine, hypotaurine and albumin were all found in the sperm washings and thus would be present in unwashed, fertile samples, low concentrations of albumin were able to maintain full fertilizing ability. Therefore, unlike hamster spermatozoa, mouse spermatozoa would not appear to have an obligatory requirement for a motility stimulating factor such as taurine.     

Effect on turkey spermatozoa of a frothy fluid derived from the cloaca of a male turkey

The influence on turkey spermatozoa of a frothy fluid derived from the cloacal region of a male turkey was investigated. The frothy fluid was collected from the turkey tom during mounting, and semen for the experiment was obtained from the ductus deferens removed after necropsy. Spermatozoa diluted with frothy fluid were examined for motility, viability, and fertilizing capacity and compared with semen diluted with phosphate buffer or undiluted ductal semen. The life-span of spermatozoa suspended in frothy fluid was slightly prolonged during in vitro storage as compared with the undiluted semen or the semen diluted with phosphate buffer; however, a rapid increase of the number of deformed spermatozoa during storage was observed in the semen diluted with frothy fluid. The fertilizing ability of spermatozoa was not influenced by the dilution with frothy fluid when the diluted spermatozoa were inseminated intravaginally immediately after the dilution. On the contrary, when spermatozoa suspended in frothy fluid were preserved at 0 C for 24 h, their fertilizing capacity decreased drastically, probably due to the increased number of abnormal spermatozoa during in vitro preservation.     

Effect of alpha-tocopherol on plasma testosterone and plasminogen activator activity or inhibition in ram spermatozoa

The objective of this study was to evaluate the effect of alpha-tocopherol on blood testosterone and specific proteolytic enzymes in spermatozoa and seminal plasma, with final aim the increase of sperm fertilizing ability with a nutritional supplement. The effect of alpha-tocopherol supplementation on testosterone parameters (mean value, basal level, peak number, mean concentration value during peaks, peak amplitude, peak duration) and plasminogen activator activity (PAA), plasminogen activator inhibition (PAI) and plasmin inhibition (PI) of spermatozoa and seminal plasma was studied in the ram during autumn (estrous period for the sheep in Greece) and spring (anestrous period). Treated animals showed a marked increase in serum alpha-tocopherol. Testosterone parameters were not affected by the alpha-tocopherol in either autumn or spring, however, the spermatozoal PAA and PAI (anti-tPA) were increased in the spring but not in autumn. These enzymes and their inhibitors are normally increased in autumn (the breeding season) and low in spring. If PAA can improve the fertilizing ability of spermatozoa in the spring, our finding may mean that a nutritional supplement, such as alpha-tocopherol, could provide rams for an accelerated onset of the breeding season in the ewe.     

Influence of caffeine on movement characteristics, fertilizing capacity and ability to penetrate cervical mucus of human spermatozoa

When added to frozen-thawed human semen, the 3 doses of caffeine tested (2, 5 and 10 mM) induced a significant increase in the percentage of motile spermatozoa but did not influence the quality of movement. Considerable variability was noted between samples in their responsiveness to caffeine which, at the 5 and 10 mM doses, was significantly correlated with the degree of motility lost during cryostorage. Caffeine treatment of frozen-thawed human spermatozoa also increased the number of spermatozoa penetrating cervical mucus in unit time, by increasing the frequency rather than the success of collisions between spermatozoa and the cervical mucus interface. When caffeine-stimulated spermatozoa were washed free of seminal plasma containing this compound they were no longer at an advantage with respect to their motility or fertilizing ability. When 2 mM-caffeine was added to washed suspensions of capacitated spermatozoa it failed to stimulate motility but did significantly enhance the fertilizing ability of the spermatozoa, indicating a possible clinical role for this compound in in-vitro fertilization therapy.     

Effect of a partial deletion of Y chromosome on in vitro fertilizing ability of mouse spermatozoa.

The effect of a partial deletion of Y chromosome on sperm fertilizing ability was investigated through an in vitro fertilization technique. Epididymal spermatozoa of a congenic line, B10.BR-Ydel, which is characterized by a high incidence of abnormal spermatozoa, revealed a significantly lower in vitro fertilization rate (22%) than that (79%) of its control strain (B10.BR/SgSn), which has a normal-sized Y chromosome. Incidence of capacitated spermatozoa as determined by chlortetracycline fluorescence assay was significantly lower in B10.BR-Ydel than in B10.BR/SgSn spermatozoa. The fertilization rate was significantly improved when B10.BR-Ydel spermatozoa were separated from the supernatant of sperm suspension by Percoll gradient centrifugation. A reconstitution experiment revealed that the B10.BR-Ydel spermatozoa were more sensitive to the inhibitory effect of the supernatant than B10.BR/SgSn spermatozoa. Spermatozoa from F1 (C57BL/6N male x B10.BR-Ydel female) males showed higher fertilization rates than those from F1 (B10.BR.Ydel male x C57BL/6N female) males. These observations suggest that not only the morphology but also the fertilizing ability of spermatozoa is directly related to partial deletion of Y chromosome.     

Quality characteristics and fertilizing ability of ram sperm subpopulations separated by partition in an aqueous two-phase system

Centrifugal countercurrent distribution (CCCD) in an aqueous two-phase system (TPS) is a resolute technique revealing sperm heterogeneity and for the estimation of the fertilizing potential of a given semen sample. However, separated sperm subpopulations have never been tested for their fertilizing ability yet. Here, we have compared sperm quality parameters and the fertilizing ability of sperm subpopulations separated by the CCCD process from ram semen samples maintained at 20°C or cooled down to 5°C. Total and progressive sperm motility was evaluated by computer-assisted analysis using a CASA system and membrane integrity was evaluated by flow cytometry by staining with CFDA/PI. The capacitation state, staining with chlortetracycline, and apoptosis-related markers, such as phosphatidylserine (PS) translocation detected with Annexin V, and DNA damage detected by the TUNEL assay, were determined by fluorescence microscopy. Additionally, the fertilizing ability of the fractionated subpopulations was comparative assessed by zona binding assay (ZBA). CCCD analysis revealed that the number of spermatozoa displaying membrane and DNA alterations was higher in samples chilled at 5°C than at 20°C, which can be reflected in the displacement to the left of the CCCD profiles. The spermatozoa located in the central and right chambers (more hydrophobic) presented higher values (P<0.01) of membrane integrity, lower PS translocation (P<0.05) and DNA damage (P<0.001) than those in the left part of the profile, where apoptotic markers were significantly increased and the proportion of viable non-capacitated sperm was reduced. We have developed a new protocol to recover spermatozoa from the CCCD fractions and we proved that these differences were related with the fertilizing ability determined by ZBA, because we found that the number of spermatozoa attached per oocyte was significantly higher for spermatozoa recovered from the central and right chambers, in both types of samples. This is the first time, to our knowledge that sperm recovered from a two-phase partition procedure are used for fertilization assays. These results open up new possibilities for using specific subpopulations of sperm for artificial insemination or in vitro fertilization, not only regarding better sperm quality but also certain characteristics such as subpopulations enriched in spermatozoa bearing X or Y chromosome that we have already isolated or any other feature.     

Effects of vitamin E on fowl semen storage at 4 degrees C

Vitamin E was assayed for either in chicken spermatozoa or seminal plasma. Effects of vitamin E on the motility and fertilizing ability of chicken semen stored for 24 hours at 4 degrees C were also studied. A mean of 0.25 mug vitamin E 10 (9) cells was found in spermatozoa and 0.074 mug in seminal plasma. When the medium for in vitro storage of semen was supplemented with vitamin E the motility of spermatozoa was not affected. However, vitamin E improved the fertilizing ability of semen stored for 24 hours at 4 degrees C, especially at the dose of 8 mug/ml of semen diluent.     

Cryopreservation of mouse spermatozoa in the presence of raffinose and glycerol

When mouse epididymal spermatozoa were rapidly frozen in two steps (37 to -70 degrees C for solid CO2 and -70 to -196 degrees C for liquid nitrogen) as pellets, 18% raffinose provided the greatest protection to ICR mouse spermatozoa against cold-shock; sperm motility and fertilizing ability were 43% and 22.4%, respectively. A small proportion of spermatozoa frozen with 10% sucrose was motile but incapable of fertilizing ovulated oocytes. Glycerol and dimethylsulphoxide were less effective at any concentration examined. However, the fertilizing ability of frozen-thawed ICR spermatozoa was significantly improved (35.5%) by addition of glycerol (1.75% final concentration) to medium containing 18% raffinose. Spermatozoa from one outbred (ddY) and 5 inbred (C57BL/6N, C3H/HeN, DBA/2N, BALB/c and kk) strains of mice were successfully frozen in the presence of 18% raffinose and 1.75% glycerol, although the fertilization rates of frozen-thawed spermatozoa varied among strains (13% for C57BL/6N to 64% for DBA/2N). A small fraction of mouse eggs resulting from fertilization by frozen-thawed spermatozoa developed normally in vitro (37% in C57BL/6N to 71% in ICR) to the blastocyst stage and in vivo (19% for C57BL/6N spermatozoa and ddY oocytes) to Day 18 of gestation.     

Effect of cholesterol and phosphatidylcholine of different chain length on acrosome breakdown and fertilizing capacity of amphibian spermatozoa

The effect of different lipids on the fertilizing capacity of Bufo arenarum spermatozoa and on acrosome breakdown of Leptodactylus chaquensis spermatozoa was studied. Sonicated vesicles of egg yolk phosphatidylcholine (1 mM) were as effective as vesicles of egg yolk phosphatidylcholine:cholesterol (molar ratio 1:0.9) in inhibiting the fertilizing capacity of Bufo arenarum spermatozoa. This suggests that cholesterol depletion from the spermatozoa was not the cause of the fertility loss. Bufo arenarum spermatozoa were incubated with phosphatidylcholines with even chain length from 6 to 18 carbons. At a concentration of 0.01 mM, didecanoyl-phosphatidylcholine reduced fertilizing capacity to 10% in a few minutes and to 0% within 60 minutes. Didodecanoyl-phosphatidylcholine required 2 hours to reduce fertility to 10% and 4 hours to cause a 100% loss of fertilizing capacity. A concentration of didecanoyl-phosphatidylcholine as low as 5 × 10^?4 mM caused a more than 95% fertility loss in less than five minutes. At a concentration of 0.1 mM, didecanoyl-phosphatidylcholine induced complete acrosome breakdown in Leptodactylus chaquensis spermatozoa in 15 minutes, whereas didodecyl-phospatidylcholine required 2 hours. At a concentration 100-fold lower didecanoyl-phosphatidylcholine induced complete acrosome breakdown in 2 hours. Electron microscopic observations in both species showed loss of acrosome caused by the action of the didecanoyl-phosphatidylcholine. Longer chain phosphatidylcholines exerted an inhibitory effect on Bufo arenarum spermatozoa fertilizing capacity at a higher concentration when in a vesicular form.