Individual differences in capacitation of bull spermatozoa by heparin in vitro: relationship to fertility

Several parameters of motility and integrity of frozenthawed spermatozoa are compared with the ability of selected motile, intact spermatozoa to acrosome reaction induced by 0.005% hyamin. Between semen donors there exist distinct individual differences; however only the induced acrosome reaction after heparin treatment showed a significant correlation with fertility. For 12 bulls the nonreturn rates from 334 to 559 services correlated significantly with induced acrosome reaction (r = 0.607). The in vitro fertilization of 53 to 93 tubal bovine oocytes with frozen-thawed spermatozoa from five bulls yielded a correlation of r = 0.621 with the rate of induced acrosome reaction. The different capacity levels of heparin-treated spermatozoa to undergo acrosome reaction appears to correspond to the varying intensity and kinetics of heparinmediated head-to-head aggregation of motile cells. The applied functional parameters could be used for the selection of bulls with low fertility in artificial insemination programs, and for spermatozoa donors for in vitro fertilization.     

Role of sperm motility and acrosome integrity in the filtration of bovine semen

In this study, the role of sperm motility and acrosome integrity in filtration of bovine semen was investigated. In Experiment 1, the treatment of semen with formaldehyde, hyperosmotic buffer, heating and direct freezing immobilized the spermatozoa completely but their acrosomal status varied significantly (P < 0.01). The immotile spermatozoa, of any kind, did not pass through the Sephadex ion-exchange column at room temperature. In Experiment 2, semen samples possessing different percentages of immobilized spermatozoa (0, 50, 75 and 100%) were filtered through the Sephadex ion-exchange column. The immotile/dead spermatozoa were removed proportionately to their number in the semen by Sephadex ion-exchange column. The type and number of immotile spermatozoa in semen had no effect (P > 0.05) on the post-filtration recovery rate of motile spermatozoa. Filtered spermatozoa exhibited higher (P < 0.01) motility (> 90%), progressive motility (> 70%) and normal acrosomes (> 95%) than non-filtered spermatozoa. In conclusion, sperm motility seems to be more important than acrosome integrity for semen filtration, and the Sephadex ion-exchange column can remove the known quantities of different kinds dead/immotile spermatozoa.     

Effect of platelet activating factor on the motility and acrosome reaction of buffalo (Bubalus bubalis ) spermatozoa

Platelet activating factor (PAF; 1-0-alkyl-2 acetyl-sn-glycerol-3 phosphocholine) has been shown to have a wide range of biological activities. In this study, PAF was used to induce acrosome reactions in fresh as well as frozen-thawed buffalo spermatozoa at different incubation periods and PAF levels. As the period of incubation increased, there was a gradual decrease in motility and increase in acrosome reaction in both fresh and frozen-thawed spermatozoa. With increasing PAF levels, the motility of fresh spermatozoa decreased and acrosome reaction increased whereas in frozen-thawed semen, motility remained almost constant, and the increase in acrosome reaction was not pronounced. Differences in motility and acrosome reaction among different bulls, types of semen, periods of incubation and PAF levels were significant (P < 0.01). A PAF level of 100 microM and an incubation period of 15 min were found to be optimum for inducing acrosome reaction in buffalo spermatozoa, since at this combination acrosome reaction increased significantly (P < 0.01) over that of the control without much loss of motility.     

Assay of frozen boar semen with Sephadex filtration

The suitability of filtration of frozen boar semen through Sephadex G-15-120 as a viability assay was investigated. Semen thawed on a hot plate at 38 degrees C was counted with a Coulter Counter before and after filtering through Sephadex columns with 0.1 M sodium citrate as flushing medium. More spermatozoa passed through the column when the temperature of the flushing medium was elevated from room temperature to 37 degrees C and added with 5 mM caffeine (12.3 vs. 22.8% p<0.01). The use of caffeine and 37 degrees C flushings produced a filtrate containing spermatozoa with 89+/-4% motility and 97.6+/-1.6% normal acrosome ridges with the use of frozen semen from 5 different boars. The repeatability was +/-7.3%. The unfiltered samples were judged to contain 45 +/- 8% motile spermatozoa and 66.6 +/- 7.1% spermatozoa with normal acrosome ridges. Filtering of frozen boar semen through Sephadex is proposed as a rapid, objective assay combining the benefits of differential counting of normal acrosome ridges and motility determination.     

Selection of a high quality subpopulation of frozen thawed bovine spermatozoa by filtration in a Sephadex column. An assessment of functional effects of progesterone

It is accepted that cryopreservation exerts deleterious effects on functional characteristics of mammalian spermatozoa. Conventional procedures for processing frozen-thawed gametes, such as centrifugation, produce additional damage. In the present work, we investigated the efficacy of processing bovine cryopreserved semen by filtration in a Sephadex column (SF group) or by washing by centrifugation (100 g, 10 min, twice) (W group); the results obtained from both procedures were compared to untreated samples (C group). The effects of in vitro addition of progesterone (10 μM, 20 min) upon sperm functional activity were studied also. The evaluated sperm parameters were concentration, motility (progressive or non progressive cells), viability and acrosome reaction. They were measured at time 0 (immediately after processing) or after 4 h incubation in capacitating conditions. Sperm concentration was (× 10⁻⁶): 37.5 ± 5.4 in C, 8.3 ± 2.1 in W and 12.5 ± 2.9 in SF. The percentages of motile, progressive, viable or acrosome intact gametes were significantly higher in SF than in W or in C. in SF group, after 4 h incubation in capacitating conditions, progesterone increased significantly the population of acrosome reacted cells whereas this parameter was not modified when the cells were incubated in absence of heparin. Motility and viability were not modified by the hormone. We conclude that Sephadex filtration method is an adequate tool to obtain a subpopulation of spermatozoa with superior quality, as assessed by motility, viability and acrosomal integrity; besides, our results strongly support that, as in other species, progesterone would be a physiological inductor of acrosome reaction in bovine.     

In vitro fertilizing capacity of frozen-thawed boar semen

We describe a porcine semen cryopreservation technique and assess the in vitro fertilizing capacity of the frozen-thawed spermatozoa. The thawed spermatozoa did not lose the physiological properties of motility, viability, and acrosome reaction or capacity to fertilize in vitro. Immediately after thawing, the spermatozoa showed 51% mean motility, 60% viability, and 5% induced acrosome reaction. After 2.5 h of incubation in TALP medium, the spermatozoa exhibited 61% motility, 63% viability and 40% induced acrosome reaction. The average in vitro fertilization capacity of thawed spermatozoa was 68% compared with that of spermatozoa from fresh semen (85%). The percentage of polyspermy was highly variable, with frozen-thawed samples ranging from 0 to 28% and fresh samples from 0 to 30%. The results obtained with frozen semen from 5 boars of different breeds did not show considerable variation. This suggests that the freezing-thawing technique is reproducible and adequate for in vitro fertilization.     

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.     

Penetration of bovine follicular oocytes by frozen-thawed spermatozoa in the presence of caffeine and heparin

Bovine follicular oocytes matured in culture were inseminated with frozen-thawed spermatozoa which were either preincubated for 5-5.5 h or not preincubated in a medium with caffeine (5 mM) and heparin (10 micrograms/ml). When the oocytes with cumulus and corona cells were inseminated, spermatozoa started to penetrate oocytes 3 h later regardless of whether spermatozoa were preincubated or not. However, a significantly higher proportion of oocytes was penetrated by preincubated than non-preincubated spermatozoa. When the oocytes were freed from cumulus and corona cells, penetration was observed to start 1 h after insemination and there were no differences in penetration rates 1-5 h after insemination between preincubated and non-preincubated spermatozoa. This study demonstrates that capacitation and the acrosome reaction of bovine spermatozoa can be induced within 1 h in a medium containing both caffeine and heparin when denuded oocytes are inseminated.     

Use of single-layer centrifugation with Androcoll-C to enhance sperm quality in frozen-thawed dog semen

The aim of this study was to investigate whether single-layer centrifugation (SLC) with Androcoll-C could select good quality spermatozoa, including those with specific motility patterns, from doses of frozen dog semen. Semen from five dogs was collected and cryopreserved following a standard protocol. After thawing, the semen samples were divided in two aliquots, one of which was used as a control and the other one processed by SLC. Assessment of sperm motility (assessed by computer-assisted semen analysis), morphology (Diff-Quick staining), viability (dual staining with propidium iodine/acridine orange), and acrosome integrity (dual staining with propidium iodine/isothiocyanate-labeled peanut [Arachis hypogaea] agglutinin) were performed on aliquots of fresh semen, frozen-thawed control samples, and frozen-thawed SLC-treated preparations. A multivariate clustering procedure separated 57,577 motile spermatozoa into three subpopulations (sP): sP1 consisted of poorly active and nonprogressive spermatozoa (48.8%), sP2 consisted of moderately slow but progressive spermatozoa (13.3%), and sP3 consisted of highly active and/or progressive spermatozoa (37.8%). SLC with Androcoll-C yielded sperm suspensions with improved motility, viability, and acrosome integrity (P < 0.01). The frozen-thawed SLC-treated samples were enriched in sP3, representing 38.5% of the sperm population. Likewise, sP2 was more frequently observed after SLC, but not significantly so. From these results, we concluded that for dog semen samples selected by SLC with Androcoll-C after thawing, the sperm quality parameters, including motility patterns, are better than in frozen-thawed control samples.     

Reactive oxygen species requirements for bovine sperm capacitation and acrosome reaction

Sperm capacitation is necessary for the fertilization of oocytes. During capacitation intracellular and membrane changes occur, that culminate with an exocytotic event called the acrosome reaction. The aim of this work was to study the participation of the superoxide anion (O2-.) and of hydrogen peroxide (H2O2) in the capacitation process and acrosome reaction in spermatozoa from cryopreserved bovine semen. Samples were capacitated with heparin or treated with the xanthine-xanthine oxidase-catalase system (X-XO-C) for the production of O2-. The percentage of capacitated spermatozoa was determined using the chlortetracycline (CTC) technique, by means of epifluorescence microscopy. Addition of X-XO-C to the incubation medium significantly induced capacitation (P < 0.05), but there were no differences with samples incubated with heparin. When the medium contained heparin or the X-XO-C, addition of superoxide dismutase (SOD, 0.5 mg/mL) significantly inhibited capacitation (P < 0.05). In samples treated with heparin and with diverse concentrations of H2O2 (10, 25, 50 and 250 microM) in the incubation medium, the percentage of capacitated spermatozoa was significantly reduced (P < 0.05); however, acrosome reaction was produced at concentrations of 10 and 25 microM H2O2. At concentrations greater than 25 microM H2O2 a deleterious effect was observed on sperm motility. From these results it may be inferred that O2-. is required in the capacitation process and that H2O2 may participate as an inductor of the acrosome reaction in spermatozoa from cryopreserved bovine semen.     

Motile sperm subpopulations in frozen-thawed dog semen: Changes after incubation in capacitating conditions and relationship with sperm survival after osmotic stress

In this study we investigated the changes that in vitro incubation under capacitating conditions could induce on the motile sperm subpopulations present in frozen-thawed dog semen samples. In addition, cryopreserved dog spermatozoa were exposed to CCM (canine capacitating medium) solutions of 300, 150, 100 and 75mOsm and the proportions of live spermatozoa with swollen tails were recorded (HOST+). Finally, frozen-thawed dog semen samples were submitted to a second cycle of freezing and thawing and the overall sperm motility, as well as the motile sperm subpopulations structure, was determined. Cryopreserved dog semen samples were structured in four sperm subpopulations with different motility characteristics: Subpopulation (Sp) 1 contained moderately rapid and progressive spermatozoa (25.2±8.5%), Sp 2 included poorly motile and non progressive sperm (15.3±8.1%), Sp 3 was represented by moderately slow non progressive sperm (14.9±5.9%), and Sp 4 contained the most rapid and progressive sperm (20.8±14.7%). After 3h of incubation under capacitating conditions, percentages of spermatozoa assigned to Sp 2 (6.1±3.4%) and 3 (4.9±2.8%) significantly decreased, whereas those assigned to Sp 1 (17.0±11.2%) and 4 (16.2±12.8%) did not significantly change. Significant correlations were found between percentages of HOST+, for the 3 osmolarities tested, and percentages of spermatozoa included in Sp 1 and 4 after 3h of incubation in capacitating conditions or in Sp 4 after double freezing and thawing. These results indicated that subpopulations with the most rapid and progressive sperm seemed to be highly resistant to in vitro incubation in capacitating conditions and to osmotic stress, suggesting they are likely to be the source of the fertilizing population.     

Effect of filtration on post-thaw quality of bull semen

Semen from 4 Holstein bulls was diluted in 4 different extenders, filtered with Sephadex ion-exchange column, and frozen in liquid nitrogen. Sperm motility, progressive motility, path velocity, progressive velocity and the percentage of normal acrosomes of filtered and nonfiltered semen were recorded before and after freezing. Semen characteristics were significantly influenced by extender, filtration and freezing. Before and after freezing, motility measurements and the percentage of normal acrosomes were higher (P < 0.001) in filtered than in nonfiltered spermatozoa. Post-thaw recovery rate of motile spermatozoa was higher in filtered semen than nonfiltered (68 vs 39%, P < 0.0001). The reduction in motility, progressive motility and the percentage of normal acrosomes during freezing and thawing processes were significantly lower (P < 0.0001) in filtered semen (34, 34 and 4%, respectively) than nonfiltered (59, 54 and 15%, respectively). Post-thaw viability of spermatozoa was significantly affected by extender, filtration and time (P < 0.0001). Immediate (0 h) post-thaw motility of nonfiltered semen (29%) was similar to 4-h post-thaw motility of filtered semen (25%; P > 0.05). In conclusion, bull spermatozoa recovered by Sephadex ion-exchange filtration showed better post-thaw viability.     

Separation of motile spermatozoa from frozen-thawed buffalo semen: swim-up vs filtration procedures

Three experiments were conducted to maximize the recovery rate of motile spermatozoa from frozen-thawed buffalo semen. In Experiment 1, the swim-up of motile spermatozoa was performed in the presence or absence of HEPES in TALP medium and CO2 in the environment. The recovery rate of motile spermatozoa in TALP medium (control), TALP + HEPES + CO2, TALP + HEPES and TALP + CO2 was 15, 18, 12 and 10%, respectively (P > 0.05), with sperm motility at 87, 89, 90 and 90%, respectively (P > 0.05). In Experiment 2, the pH of TALP medium was adjusted to 7.0, 7.5, 8.0, 8.5 and 9.0, and swim-up procedure was performed in the presence of HEPES and CO2. The recovery rate of motile spermatozoa at different pH was 14, 20, 24, 27 and 16%, respectively (P < 0.05). Motility of separated spermatozoa was 88, 91, 90, 89 and 90%, respectively (P > 0.05). In Experiment 3, the efficiency of ion-exchange filtration and Swim-up procedure in separating motile spermatozoa from frozen-thawed buffalo semen was compared. The recovery rate of motile spermatozoa was 95% in filtration procedure and 33% in swim-up procedure (P < 0.005). In all experiments, normal acrosomes did not vary due to treatments (P > 0.05). In conclusion, HEPES and CO2 had no significant effect on swim-up of buffalo spermatozoa. The pH 8.5 of TALP improved the recovery rate of motile spermatozoa in swim-up procedure. The ion-exchange filtration was found superior to swim-up procedure in harvesting maximum number of motile spermatozoa from frozen-thawed buffalo semen (95 vs 33%; P < 0.001).     

Molitity and acrosomal changes in ionophore-treated bovine spermatozoa and their relationship with in vitro penetration of zone-free hamster oocytes

Frozen-thawed spermatozoa from Friesian bulls held at stud in Ireland were used to assess the effect of ionophore on motility, acrosome reaction and heterologous in vitro fertilization. Bovine spermatozoa penetrated zone-free hamster oocytes following treatment with calcium ionophore in the absence of bovine serum albumin (BSA) and in the presence of 10 mM caffeine. Sperm velocity was stimulated in concentrations of caffeine <2.5 mM following dilution with medium containing BSA. Sperm attachment to the plasmalemma showed no association with penetration rates of zona-free hamster oocytes. Penetrated oocytes in regimens with >0.1 mM ionophore did not progress through Meiosis II. Increasing concentrations of ionophore induced the acrosome reaction more rapidly, although this was associated with reduced motility. Hyperactive motility was observed in calcium ionophore-treated spermatozoa which were capable of penetrating zona-free hamster oocytes. Sperm velocity remained unchanged. whereas the track:vector ratio, a measurement of curvilinear movement, was reduced. This work may have important implications for the assessment of bovine fertility and cytogenetic analysis of bovine sperm.     

Atrial natriuretic peptide induces an acrosome reaction in giant panda spermatozoa and enhances their penetration of salt-stored porcine oocytes

Atrial natriuretic peptide (ANP) is a vasodilator peptide primarily produced in the heart. Locally synthesized ANP has been found in reproductive tissues of various mammals and humans, and plays an important role in rat oocyte maturation and human sperm function. The objective of the present study was to determine the effects of ANP on the function (acrosome reaction and zona penetration) of giant panda spermatozoa. In fresh and frozen-thawed spermatozoa that had been preincubated for 2.5h, treatment with ANP (for 60 min) significantly increased the proportion of acrosome-reacted spermatozoa; maximal response (an acrosome reaction in 18.3 and 21.8% of fresh and frozen-thawed spermatozoa, respectively) was detected at 1 nM ANP. Treatment with C-ANP-(4-23), an analogue of ANP and specific binder to natriuretic peptide receptors-C (NPRC), had no significant effect on the acrosome reaction. However, the cyclic guanosine 5'-monophosphate (cGMP)-dependent protein kinase (PKG) inhibitor KT5823 completely abolished the effect of ANP on acrosome reaction. The effects of ANP, caffeine and heparin on frozen-thawed sperm function were studied by insemination of porcine salt-stored oocytes in a modified Tris-buffered medium (mTBM). The presence of ANP, caffeine or heparin in the insemination medium resulted in a higher proportion (P < 0.05) of oocytes with spermatozoa in the zona and perivitelline space (PVS), and a higher average number of spermatozoa/oocyte (P < 0.05) in the zona and PVS. However, in the absence of ANP, caffeine and heparin, there were no oocytes with a spermatozoon in the PVS. There were no differences among ANP, caffeine or heparin treatments for the proportion of oocytes penetrated or average number of spermatozoa/oocyte in the zona and PVS. In conclusion, we inferred that ANP induced the acrosome reaction of preincubated giant panda spermatozoa by a PKG pathway. Furthermore, ANP enhanced the penetrability of porcine salt-stored oocytes by frozen-thawed giant panda spermatozoa.     

Preparation and recovery of frozen-thawed bovine spermatozoa via various sperm selection techniques employed in assisted reproductive technologies

A number of semen manipulative techniques are currently available to remove the undesirable spermatozoa, debris and other factors and to increase sperm quality. The use of motility stimulants such as caffeine or others could optimize the recovery and quality of frozen-thawed spermatozoa processed by a variety of sperm selection techniques. Frozen-thawed specimens from 5 bulls were slowly diluted and washed with Ham's F-10 medium containing 3% BSA (w/v) and 0 or 2 mM caffeine. Aliquots containing approximately 50 x 10(6) total sperm cells were used for conventional sperm wash, swim-up, Percoll density gradient centrifugation (80, 70, 55 and 40% Percoll gradients) and Sephadex (SpermPrep I) filtration. Quantitative and qualitative characteristics of selected spermatozoa included: total sperm (x 10(6)), percentage and grade (0 to 4) of motility, percentage of spermatozoa with coiled tails and response to the hypoosmotic swelling (HOS) test (percentage of swollen spermatozoa). When compared to washed specimens, fewer spermatozoa were recovered via the swim-up, Percoll and SpermPrep I filtration methods. Quantitative and qualitative characteristics of these spermatozoa improved further after processing with Ham's F-10 containing 2 mM caffeine, followed by selection via the various techniques. Enhancement of sperm motility, in conjunction with the most appropriate sperm selection technique, represents an efficient method for the recovery of spermatozoa with improved qualitative characteristics.     

Glass wool filtration of bull cryopreserved semen: a rapid and effective method to obtain a high percentage of functional sperm

Frozen-thawed bull sperm are widely used in assisted reproductive technologies, but cryopreservation negatively affects semen quality. Several sperm selection techniques have been developed to separate motile sperm from non-motile cells. The aim of the present study was to evaluate the effectiveness of the glass wool column filtration to select functional sperm from frozen-thawed bull semen samples. Frozen semen from six Holstein bulls was thawed and filtered through a glass wool column, followed by assessment of routine and functional sperm parameters. In a set of experiments, sperm aliquots were also processed by swim up to compare both selection methods. Samples recovered in the glass wool filtrate had high percentages of viable (94 ± 3%, mean ± SD), progressively motile (89 ± 4%), acrosome-intact (98 ± 1%), and non-capacitated (80 ± 10%) sperm; these values were higher (P < 0.05) than those obtained after performing the swim up procedure. Moreover, the glass wool filtration yielded 67 ± 19% motile cells, in comparison with 18 ± 8% obtained with swim up (P < 0.05), calculated as the concentration of progressively motile cells selected relative to their concentration in the sample before the selection procedure. Glass wool-filtered sperm were able to undergo capacitation-related events, based on the increase in the percentage of cells classified as capacitated by CTC staining (B-pattern) after incubation with heparin (50 ± 5%) in comparison with control conditions with no heparin (17 ± 4%) or heparin + glucose (16 ± 2%; P < 0.05). Moreover, they underwent acrosomal exocytosis in response to pharmacologic (calcium ionophore A23187 and lysophosphatidylcholine) and physiological (follicular fluid) stimuli, and they fertilized in vitro matured cumulus-oocyte complexes and denuded oocytes (two-cell embryos: 72 ± 4% and 52 ± 6%, respectively). We conclude that glass wool filtration is a low-cost, simple, and highly effective procedure to select functionally competent sperm for reproductive technologies in the bull, which may be useful for other domestic and farm animals, as well as for endangered species.     

Effect of methylxanthines on motility and fertility of frozen-thawed goat semen

We studied the effects of 2 methylxanthines (caffeine and theophylline) at different concentrations on goat sperm motility and live spermatozoa and on the percentage of acrosomal damage and fertility. Altogether, 144 semen samples collected from 12 bucks (3 each from Black Bengal and Beetal, and 6 from cross-breds) were diluted in TRIS extender, divided into 5 equal fractions; then caffeine and theophylline were added at 2 concentrations (2 and 5 mM) in different fractions. These samples were frozen in liquid nitrogen vapor, thawed at 37 degrees C for 15 sec, and evaluated for motility and other semen attributes. Addition of caffeine and theophylline had a stimulatory effect on goat spermatozoa. It was further observed that the effect of these agents was concentration-dependent, with 2 mM caffeine and 5 mM theophylline yielding the best results in respect to the percentage of motility in all 3 breeds of goats tested. Among the two methylxanthines used, caffeine was found to be the more effective in Improving motility than theophylline. There was no significant effect on the percentages of live spermatozoa and acrosomal damage due to the addition of these 2 methylxanthines to the extender. Fertility rates with Tris + 2 mM caffeine (60.20 %) and with Tris + 5 mM theophylline (58.88 %) extended semen were apparently higher than those with the Tris-diluted semen (50.0 %), although these differences were not significant.     

Post-coital sperm recovery and cryopreservation in the Sumatran rhinoceros (Dicerorhinus sumatrensis) and application to gamete rescue in the African black rhinoceros (Diceros bicornis)

Sumatran rhinoceros (Dicerorhinus sumatrensis) sperm samples were collected from a post-copulatory female and characterized to determine their potential for sperm preservation and future use in artificial insemination. Five samples of acceptable quality from one male were used to compare the effect of two cryoprotectants (glycerol and dimethyl sulfoxide (DMSO)) and two post-thaw protocols (untreated and glass wool column) on sperm quality. The percentage of motile spermatozoa, sperm motility index (0-100) and sperm morphology were evaluated subjectively, and viability and acrosomal status were assessed using fluorescent markers. Evaluations of frozen-thawed spermatozoa were performed over a 6 h incubation interval. Post-coital semen samples (n = 5; 104.0 +/- 9.1 ml; 2.5 +/- 0.8 x 10(9) total spermatozoa; mean +/- SEM) exhibited a sperm motility index of 56.7 +/- 3.3, and contained 40.2 +/- 6.3%, 72.0 +/- 3.2% and 79.8 +/- 6.5% normal, viable and acrosome-intact spermatozoa, respectively. Glycerol and DMSO were equally effective as cryoprotectants and, regardless of post-thaw protocol, samples retained greater than 80% of all pre-freeze characteristic values. Processing semen samples through glass wool yielded higher quality samples, but only half the total number of motile spermatozoa compared with untreated samples. High values for pre-freeze sperm characteristics were also maintained after cryopreservation of epididymal spermatozoa from one black rhinoceros (Diceros bicornis) using the same protocol. In summary, Sumatran rhinoceros spermatozoa of moderate quality can be collected from post-copulatory females. Rhinoceros sperm samples show only slight reductions in quality after cryopreservation and thawing and have potential for use in artificial insemination.     

Effects of platelet activating factor on the motility and acrosome reaction of bovine spermatozoa

The effects of platelet activating factor (PAF) on motility and the acrosome reaction of ejaculated bull spermatozoa were evaluated. Washed spermatozoa (30 x 10(6)/ml) were incubated (39 degrees C) for up to 2 h with 10 to 200 muM PAF in a modified Tyrode's solution (pH 7.4) containing 3 mg/ml bovine serum albumin. Sperm motility was evaluated subjectively and by computer-assisted semen analysis. Percent acrosome-reacted spermatozoa was quantified microscopically from fixed smears following Giemsa staining. Percent fertilization by PAF-treated spermatozoa was determined using in vitro-matured bovine ova. Percent sperm motility decreased with >/= 50 muM PAF, while the rate of motility loss increased with PAF concentration (P<0.001). Percent acrosome reactions increased with PAF concentration during incubation (P<0.001). Acrosomal loss was rapid and complete with 200 muM PAF. At concentrations between 80 to 120 muM PAF, bull spermatozoa underwent acrosome reactions without a rapid loss of motility and penetrated in vitro-matured bovine ova at a rate comparable to that of heparin-capacitated spermatozoa (68 versus 54%, respectively). Incubation of bull spermatozoa with 10 to 50 muM PAF for 45 min had no effect on percent progressive motility, sperm velocity or other motility parameters. These results indicate that PAF can be used to induce acrosome reactions in bull spermatozoa and to promote in vitro fertilization of bovine ova. Under the conditions used in this study, PAF did not stimulate bovine sperm motility.