The expression of mannose-ligand receptor is correlated with sperm morphology

The objective of the present study was to evaluate the association between the expression of sperm mannose-ligand receptors and sperm morphology. Sperm samples were obtained from 45 men, 30 fertile sperm donors and 15 infertile men. Sperm concentration, motility and morphology were evaluated and then incubated with control medium (Ham's F-10 + 1% HSA) for 4 h. Expression of mannose-ligand receptors was evaluated by mannosylated-BSA-FITC (subdivided into 3 patterns: I, for uncapacitated sperm; II, for capacitated; and III, for acrosome-reacted sperm). The mean (+/- SE) frequencies of sperm cells of the total sperm population that expressed patterns I, II, and III were 88 +/- 2.1%, 7 +/- 1.6%, and 5 +/- 0.8%, respectively, for fertile men, and 90 +/- 2.1%, 7 +/- 1.3%, and 3 +/- 0.5%, respectively, for infertile men. The rate of pattern III expression of mannose-ligand receptors was significantly higher in the fertile group compared to the infertile patients (p <.01). A poor but significant correlation was observed between the rate of pattern III and the percentage of normal-forms sperm cell in the ejaculate (r =.35, p =.018). Fertile sperm samples express more advanced patterns of mannose-ligand receptors compared to infertile men. This phenomenon is related to the morphology of human sperm cell in the ejaculate more than to any other basic sperm characteristics.     

Induction of the acrosome reaction in bull spermatozoa with plasmin

Proteolytic enzymes appear to have an essential role in multiple phases of mammalian fertilization. Plasmin, the active enzyme of the plasminogen activation system that stimulates fibrinolysis and proteolysis has a less well-documented role in reproduction. The current study was conducted to investigate the effect of the active protease, plasmin, on the ability of bovine sperm to undergo the acrosome reaction. Aliquots of freshly ejaculated bull sperm were incubated in capacitating conditions with 10 microg ml-1 of heparin for 4 h. Every 2 h an aliquot of spermatozoa was exposed to lysophosphatidylcholine (100 microg ml-1) or 0, 0.1, 1, 10 and 100 mU of plasmin to induce the acrosome reaction in capacitated spermatozoa. Plasmin increased the percentage of live acrosome reacted sperm after 4 h of incubation in the capacitation medium. Viability was not affected by any of the treatments. This study provides new information on bovine acrosome reaction during in vitro incubation with plasmin and indicates that this protease may participate in the proteolytic events that accompany fertilization.     

Epididymal epithelial cells cultured in vitro prolong the motility of bovine sperm

It is well known that the epididymis is an excellent environment to maintain sperm viability. Therefore, we used different sections of bovine epididymis (caput, corpus, and cauda) to develop epithelial cell culture monolayers to identify factors that will increase sperm survival in the freezing-thawing process. Each epididymal section was dissected and treated with collagenase to obtain epithelial cell clusters. The cells were cultured in RPMI-1640 medium with 10% serum at 38.5 degrees C. A confluent monolayer was obtained after 5-7 days in culture and preliminary characterization using cytokeratin antibody indicated that the cell culture contained 85%-95% of epithelial cells. These cellular cultures were tested for their ability to maintain motility of epididymal and frozen-thawed spermatozoa. Washed spermatozoa were added to obtain a final dilution of 1 x 10(6) spermatozoa/mL. The motility of frozen-thawed spermatozoa was also recorded after incubation in conditioned media. Our results show that cocultures of spermatozoa and epididymal cell monolayers for 24 and 48 hours were beneficial for maintaining epididymal and frozen-thawed sperm motility (36.0% and 20.4%) compared with spermatozoa cultured with fibroblast cells or in the absence of a cell monolayer (0%; P < .01). The conditioned medium provides favorable conditions for sperm motility. Results with conditioned medium on maintenance of frozen-thawed sperm motility suggest that epididymal cells in vitro secrete beneficial factors that prolong the sperm survival.     

双荧光探针检测冷冻对精子质膜完整性的影响

目的:检测精液冷冻对山羊精子质膜完整性的影响。方法:用假阴道法采集3只公山羊精液,经冷冻-解冻处理后,检测精子畸形率和顶体完整率,用碘化丙锭结合羟化荧光素双荧光探针标记,检测和比较精液冷冻对山羊精子质膜完整性的影响。结果:冷冻明显降低了精子的活动率[(74.43±13.78)%vs(46.25±2.69)%,P<0.01]和质膜完整率[(64.26±7.03)%vs(6.27±2.90)%,P<0.01];显著降低了顶体完整率[(80.77±10.70)%vs(58.42±18.05)%,P<0.05]。结论:冷冻使精子活动率和顶体完整率降低的同时导致精子质膜严重破损。     

己酮可可碱、孕酮和A23187对人精子甘露糖受体表达的影响

人精子经ＢＷＷ获能５～６小时后，分别用己酮可可碱（ＰＦ）、孕酮（Ｐ）和Ａ２３１８７三种已知的获能及（或）顶体反应促进剂处理１小时，对实验和对照组精子用异硫氰酸荧光素标记的甘露糖化牛血清白蛋白（Ｍ ＦＩＴＣ ＢＳＡ）作探针标记精子甘露糖受体（ＭＲ）。结果表明，获能促进剂ＰＦ并不促进ＭＲ表达而Ｐ和Ａ２３１８７则有显著促进ＭＲ表达的作用。Ｐ对ＭＲＩＩ型的促表达作用略强于对ＩＩ型，而Ａ２３１８７则对ＩＩ型的促进显著强于Ｉ型。认为ＭＲ的表达是一种依赖于钙离子的细胞生物学过程，并与顶体反应有密切关系。推测ＭＲ可能具有介导精子与卵膜融合的作用。     

采用冷冻精子与新鲜精子进行卵胞浆内单精子注射的结果比较

目的　比较冷冻精子与新鲜精子进行卵胞浆内单精子注射 ( ICSI)助孕技术的治疗效果。　方法　对 1 6 1对不育夫妇进行 1 6 3个辅助生殖技术治疗周期 ,其中采用冷冻精子 47个周期 ,比较了冷冻精子组与新鲜精子组 1 1 6个周期的受精率、卵裂率、A级胚胎率与临床妊娠率。　结果　冷冻精子组 (组 I)的受精率为 77.6 %、卵裂率为 92 .9%、A级胚胎率为 6 5.4%、临床妊娠率为 45.5% ;新鲜精液严重异常组 (组 II)分别为 54 .4%、94.0 %、45.7%及 2 5.0 % ;新鲜精液轻、中度异常组 (组 III)分别为 73 .5%、92 .5%、46 .8%及 2 9.3 %。组 I的受精率、A级胚胎率和临床妊娠率明显高于组 II( P<0 .0 5) ;与组 III比较无显著性差异 ( P>0 .0 5)。　结论　患者本身的精子质量直接影响 ICSI的受精率 ,精子冷冻复苏处理不影响 ICSI的受精率、卵裂率和优质胚胎率     

冻储时间对冷冻精子复苏率的影响

目的 :探讨冻储时间对冷冻精子复苏率的影响 ,以期找到最佳复苏时间。　方法 :取 88份正常供精者标本进行精液常规分析 ,精液用程序降温仪通过三步降温法冻存后置 - 196℃液氮中冻储 ,88份冻存标本随机分成 5组 ,分别于冻存后的 1d(Ⅰ组 ,n =14 )、7d(Ⅱ组 ,n =2 4 )、30d(Ⅲ组 ,n =19)、180d(Ⅳ组 ,n =18)、30 0d(Ⅴ组 ,n =12 )取出 ,37℃水浴复苏 ,再次进行精液常规分析。计算冷冻复苏率。　结果 :5组间的复苏率差异均无显著性 (P均 >0 .0 5 ) ,冻储时间与复苏率之间无相关性 (r=- 0 .0 5 ,P >0 .0 5 )。　结论 :程序降温仪冻存精液标本置 - 196℃液氮后 2 4h即可复苏分析 ,便于人类精子库耐冻实验批量筛查     

Loss of acid phosphatase from rat spermatozoa as a method for assessing the acrosome reaction

A method is presented for evaluating the extent of the acrosome reaction by measuring the release of acrosomal acid phosphatase from rat spermatozoa during incubation under capacitating conditions. Treatment of spermatozoa with lysophosphatidylcholine or Triton X-100 released the acid phosphatase from the sperm cell. Using this enzymatic method we could not detect an alteration in enzyme activity following 5 h incubation under capacitating conditions. The effect of in vitro capacitation for 5 h in the absence or presence of heparin or ionophore A23187 was studied. Incubation in the presence of heparin (10 micrograms ml-1) caused a 32% increase in enzyme activity. After exposure of the spermatozoa to ionophore A23187 (0.5 microM) 16% increase of enzyme activity could be detected.     

Correlation between the motility of frozen–thawed epididymal spermatozoa and the outcome of intracytoplasmic sperm injection

The purpose of this study was to investigate if the outcome of ICSI was influenced by epididymal sperm motility in frozen-thawed specimens. A total of 18 ICSI treatment cycles using spermatozoa retrieved by microsurgical epididymal sperm aspiration (MESA) were analysed retrospectively. Cryopreservation of epididymal spermatozoa was performed when enough epididymal aspirates were collected. Sixty-nine out of 126 oocytes injected with spermatozoa retrieved by MESA were fertilized, giving a fertilization rate of 54.8%. Out of 18 embryo transfer cycles, 6 (33.3%) achieved pregnancies. Fresh epididymal spermatozoa were used in 5 cycles while frozen-thawed epididymal spermatozoa were used in 13 cycles for ICSI. The fertilization rates were 68.6% (35/51) in the former group and 45.3% (34/75) in the latter group, respectively. There was a significant difference between the two groups (p < 0.05). In ICSI treatments using fresh epididymal spermatozoa, the cells used for injection were all motile. However, motile epididymal spermatozoa could be used in only five ICSI treatment cycles after freeze-thawing. In 6 cycles, only immotile sperm were used for injection of frozen-thawed spermatozoa. The fertilization rate in each group was 68.4% (13/19) and 31.6% (12/38), respectively. There was a significant difference between these groups (p < 0.01). These results indicate that the outcome of ICSI was influenced by sperm motility in frozen-thawed epididymal specimens. When no sperm motility could be recovered after freeze-thawing even with chemical treatments, consideration should be given to retrieving fresh epididymal spermatozoa again to achieve a better fertilization rate in such patients.     

Effect of procaine, pentoxifylline and trolox on capacitation and hyperactivation of stallion spermatozoa

Reasons for low in vitro fertilisation rates in the horse include the difficulties in inducing capacitation and/or hyperactivation of stallion spermatozoa. The aim of this study was to analyse the effect of noncapacitating and capacitating modified Whitten's (MW) and modified Tyrode's medium (MT) and treatment with procaine (5 mmol), pentoxifylline (3.5 mmol) and trolox (120 mmol) on motility (CASA), capacitation, acrosomal status, viability and mitochondrial membrane potential of stallion spermatozoa (n = 4). While there was no influence of MW and MT on sperm motility, a significant increase in the percentage of viable-capacitated spermatozoa was observed after incubation in capacitating MW (P < 0.05). Pentoxifylline showed no significant effect on the motility pattern but increased the proportion of live-capacitated spermatozoa (P < 0.05). Trolox had no detectable effect on either capacitation or hyperactivation. Procaine was the only agent that induced hyperactivation in terms of a reduced proportion of progressively motile spermatozoa, straight line velocity, straightness, linearity and beat-cross frequency and an increase in the amplitude of lateral head displacement (P < 0.05). The combination of capacitating Whitten's medium and procaine showed the best results for the induction of capacitation and hyperactivation in stallion spermatozoa; this was possible even after short-term incubation.     

Does seminal plasma PSP-I/PSP-II spermadhesin modulate the ability of boar spermatozoa to penetrate homologous oocytes in vitro?

Low concentration (0.15 mg per million of spermatozoa) of seminal plasma-derived PSP-I/PSP-II spermadhesin heterodimer is able to preserve the viability of highly extended boar spermatozoa. Whether spermatozoa also keep their fertilizing capacity is not yet known. The present study evaluated the effect of exposing freshly extended and frozen-thawed boar spermatozoa (10 million/mL) to PSP-I/PSP-II (1.5 mg/mL) for 30 or 120 minutes on sperm characteristics and the outcome of in vitro penetration of immature (IM) and in vitro matured (IVM) homologous oocytes, aiming to identify this spermadhesin as a suitable modulator for sperm-handling protocols. Although exposure to the heterodimer improved sperm viability and motility without increasing the levels of sperm acrosome exocytosis in both freshly extended and frozen-thawed spermatozoa, this pretreatment did not affect sperm penetration rates or sperm numbers per oocyte when pretreated fresh spermatozoa were coincubated with IM or IVM oocytes compared with controls. When cryopreserved spermatozoa were tested, however, on IVM oocytes, already a 30-minute preincubation exposure to PSP-I/PSP-II showed a significant blocking effect on penetration rate (from 90% to 32%, P < .05) and on mean sperm numbers per oocyte (2.9 to 1.6, P < .05). To disclose the nature of this paradox, frozen-thawed spermatozoa were cleansed (by centrifugation in saline bovine serum albumin or through Percoll density gradient separation) and the procedure repeated. Oocyte penetration (but not number of spermatozoa per oocyte) increased (P < .05) when spermatozoa were cleansed with Percoll compared with either washed or unwashed controls (53% vs 13% vs 31%, respectively). In addition, the percentages of polyspermic oocytes remained lower than control (38.5% vs 68.7%, respectively; P < .05). In conclusion, the results confirm that exposure of fresh or frozen-thawed boar spermatozoa to a low dose of seminal PSP-I/PSP-II spermadhesin preserves sperm viability and motility in vitro. Although there was no obvious influence of the heterodimer on the capability of freshly extended boar spermatozoa to penetrate homologous oocytes (either IM or IVM), PSP-I/PSP-II exerted a deleterious effect when frozen-thawed spermatozoa were used to penetrate IVM oocytes. Such an effect of cryopreservation seems to a certain extent reversible, since cleansing of the sperm surface decreased, at least partially, this blocking effect, increasing both penetration and the monospermic rates.     

Characterization of hyperactivated motility by human spermatozoa during capacitation: comparison of fertile and oligozoospermic sperm populations

Suspensions of capacitating human spermatozoa were analyzed for potential hyperactivated movements using videomicrographic methods. Analysis was carried out on aliquots of 22 sperm suspensions, which were proved fertile several hours later during human in vitro fertilization. After approximately 3 h of capacitation, 22.1% of the fertile spermatozoa displayed motility patterns designated as hyperactivated. Over 80% of these hyperactivated spermatozoa moved with a wide-amplitude, two-dimensional whiplash pattern, displaying marked lateral displacement of the head. Only 8.4% of capacitating spermatozoa from oligozoospermic patients showed these hyperactivated movements. The incidence of hyperactivated movements by fertile and oligozoospermic spermatozoa could be significantly increased after exposure to various motility stimulants. The clinical significance of hyperactivation as a functional assay of fertilizing capacity is discussed.     

In vitro incubation of human spermatozoa promotes reactive oxygen species generation and DNA fragmentation

The aim of this study was to investigate the oxidative process associated with sperm capacitation and its impact on DNA fragmentation and sperm function. Redox activity and lipid peroxidation were analysed in human spermatozoa after 3, 6 and 22 h of incubation in Ham′s F10 medium plus bovine albumin at 37° and 5% CO₂ for capacitation. DNA status, tyrosine phosphorylation pattern and induced acrosome reaction were evaluated after capacitating conditions. At 22 h of incubation, there was a significant (P < 0.05) increase in oxygen‐free radicals and lipid peroxidation, with no effect on sperm viability. There also was a significant (P < 0.001) increase in fragmented DNA in capacitated spermatozoa compared to semen values with higher rates being found after the occurrence of the induced acrosome reaction. Protein tyrosine phosphorylation pattern confirms that capacitation took place in parallel with the occurrence of DNA fragmentation. These results indicate that when spermatozoa are incubated for several hours (22 h), a common practice in assisted reproductive techniques, an increase in oxidative sperm metabolism and in the proportion of fragmented DNA should be expected. However, there was no effect on any of the other functional parameters associated with sperm fertilising capacity.     

Effects of thawing procedure on postthawed in vitro viability and in vivo fertility of red deer epididymal spermatozoa cryopreserved at -196 degrees C

In this study, we have determined the effects of individual factor and thawing procedure on in vitro viability and in vivo fertility of frozen-thawed red deer epididymal spermatozoa. The spermatozoa that were collected from 13 Iberian deer stags were diluted at room temperature in a Triladyl-20% egg yolk medium and frozen in nitrogen vapors. In the first experimental series, sperm samples were collected from 10 mature stags. For thawing, the frozen straws were subjected to 3 different procedures: I (37 degrees C for 20 seconds), II (60 degrees C for 8 seconds) and III (70 degrees C for 5 seconds). Sperm cryosurvival was judged in vitro by microscopic assessments of individual sperm motility (SM) and of plasma membrane and acrosome (NAR) integrities. Statistically significant variations were found (P <.05) between stags for most of the seminal parameters evaluated. The thawing procedure did not have an effect on the seminal characteristics evaluated after this process, except for SM (P <.05), with the best overall recovery rates after freezing and thawing found with the use of protocol I. Our results also show a differential resistance to return to isosmotic conditions of spermatozoa thawed using the different thawing protocols. In the second experimental series (insemination artificial trial), with spermatozoa from 3 stags, results of fertility were statistically higher (69.7% vs 42.4%, P =.014) when spermatozoa were thawed at 37 degrees C for 20 seconds than were warmed at 60 degrees C for 8 seconds. Therefore, thawing protocol I, which provides slow thawing rates, was the most beneficial for epididymal spermatozoa thawing of the cervid subspecies analyzed in this study. In summary, high in vitro survival and in vivo fertility of frozen-thawed deer epididymal spermatozoa were dependent on warming rates, but each stag exhibited its own sensitivity to cryopreservation.     

Motility of Seminal Plasma-Free Spermatozoa in the Presence of Several Physiological Compounds

The purpose of this pilot study was to get information whether the motility and velocity of washed human spermatozoa can be affected by different compounds usually found in seminal plasma. The following purified substances wee added to washed spermatozoa in physiological concentrations: bradykinin, angiotensin I, II, III, spermine, spermidine, acetylcarnitine, LH and FSH. Sperm motility and velocity were measured by the method of multiple exposure photography after 30 minutes of incubation at 22 degrees C and 37 degrees C including appropriate controls. Bradykinin improved sperm velocity at 22 degrees C. Angiotensin I and II, acetylcarnitine and LH stimulated sperm velocity at 37 degrees C. The latter two substances increased also sperm motility at 37 degrees C. Angiotensin III, spermine, spermidine and FSH showed no effect on sperm motility neither at 22 degrees C nor at 37 degrees C. These observations indicate that distinct physiological compounds found in seminal plasma stimulate directly sperm motility and/or velocity in vitro and support the assumption that the sperm motility stimulating principle of human semen is complex and of multifactorial origin.     

Human sperm mitochondrial function related to motility: a flow and image cytometric assessment

Current evaluation of male fertility, routinely estimated by sperm count, motility, and morphology, provides only crude information about the fertility state of individuals. Both flow and image cytometry were applied to mitochondrial activity and sperm motility respectively. Sperm samples from fertile donors were concomitantly measured for Rhodamine 123 (Rh123) uptake (an estimation of mitochondrial activity), percentage of dead cells, and motility characteristics, such as percentage of motility, curvilinear velocity, and amplitude of lateral head displacement. These measurements were done under experimental conditions known to modulate sperm motility (temperature and time course survival in a capacitating medium). Bimodal distributions were found for Rh123 uptake. Flow cytometry-derived parameters were essentially time-dependent whereas motility characteristics were primarily temperature-dependent. Correlations were found between various flow cytometry-derived parameters and motility characteristics. Most of the correlations were obtained after a 24 h incubation in a capacitating medium. The most significant correlation in every experimental condition concerned the percentage of motile spermatozoa and the Rh123 uptakes. The drop in motility observed after a 24 h incubation was paralleled by a markedly lower drop in mitochondrial activity. The data suggest that these two complementary techniques represent an improvement in basic and/or clinical assessment of the functional spermatozoa status.     

Cryopreservation of ram semen facilitates sperm DNA damage: relationship between sperm andrological parameters and the sperm chromatin structure assay

We hypothesized that cryopreservation and incubation in conditions that mimic the female genital tract following insemination increases the susceptibility of ram sperm DNA to denaturation. Ram sperm samples (n = 12) underwent the sperm chromatin structure assay (SCSA) and semen quality tests, including motility parameters, viability, and chlortetracycline fluorescence (CTC) patterns. We also assessed correlations between SCSA variables and semen quality parameters. Analyses were performed for both fresh and cryopreserved samples at 0, 3, and 20 hours of incubation in synthetic oviductal fluid (SOF; 39 degrees C, 5% CO(2)). The SCSA variables, mean alpha t (X alpha(t)) and standard deviation of alpha t (SD alpha(t)), were higher because of cryopreservation (P <.05, P <.001, respectively) after 20 hours in SOF. For both fresh and frozen spermatozoa, SCSA values (X alpha(t), SD alpha(t), and the percentage of cells outside the main population of alpha(t) [%COMP alpha(t)]) increased during incubation in SOF. Motility was negatively correlated with both SD alpha(t) and %COMP alpha(t), ranging from -0.39 (P <.01) to -0.59 (P <.001) for both fresh and cryopreserved semen; viability also was negatively correlated with X alpha(t), SD alpha(t), or %COMP alpha(t) (-0.36; P <.05, -.40 and -.46; P <.01, respectively) in fresh semen. The %COMP alpha(t) was positively correlated to the percentage of CTC pattern AR (P <.001) and negatively correlated to the percentages of patterns F and B (-0.33 to -0.60, P <.05 to P <.001). Variation among ejaculates within ram was observed (P <.01). Cryopreservation clearly facilitates DNA damage in physiological conditions. The low to moderate correlations between SCSA variables and classical semen quality parameters indicate that the SCSA provides additional information to standard tests for evaluating ram sperm quality.     

Incubation of frozen-thawed llama sperm with seminal plasma

Seminal plasma is intimately connected to sperm physiology and particularly in South American Camelids, has demonstrated to be involved in multiple physiological reproductive events. Different percentages of seminal plasma (0%, 10% and 50%) were added to thawed llama semen samples with the objective of evaluating the interaction with cryopreserved sperm over time (0, 1.5 and 3 hr at 37°C). A total of 20 ejaculates from five adult llama males (n = 5; r = 4) were evaluated. A significant decrease in sperm motility, membrane function and live sperm was observed in all thawed samples (0%, 10% and 50%) at 0 hr when compared to raw semen. Neither morphology nor chromatin condensation was altered in all thawed samples (p > .05), but a significant increase in the percentage of spermatozoa with fragmented DNA was observed after thawing all samples versus raw semen. When evaluating thawed samples over time, a significant decrease of motility and membrane function was observed, while the percentages of total live sperm were preserved over the 3 hr of incubation in all final concentrations evaluated. To conclude, the addition of 10% or 50% of seminal plasma was incapable of preserving motility or membrane function of frozen-thawed llama sperm during 3 hr of incubation.     

Dynamics of sperm DNA damage in fresh versus frozen-thawed and gradient processed ejaculates in human donors

The rate of increase of sperm DNA fragmentation (rsDF) in fresh and frozen-thawed and processed sperm samples after a density gradient for sperm selection was analysed after 0, 0.5, 1.5, 4.5, 6, 24, 48 and 72 h of incubation at 37 °C, in five donors with proven fertility. The results showed that: (i) sperm DNA fragmentation (sDF) at baseline in fresh samples (14.3 ± 3.3) was lower than that obtained after freeze-thawing and selection (19.4 ± 4.1), significant differences; (ii) After 6 h of incubation the mean sDF in fresh samples (24.2 ± 10.2) was significantly lower than that in frozen-thawed samples (45.3 ± 7.1); (iii) Subsequently, the rsDF in fresh semen samples was 1.6% per h after 6 h of incubation, while after thawing and selection the rsDF was 4.3% per h; The tendency to increase in sDF showed high R(2) values (R(2) = 0.90) for exponential functions in case of fresh samples, whereas R(2) values for linear functions were higher after sperm selection (R(2) = 0.97). These results indicate that differences in sperm DNA fragmentation dynamics before and after storage are an important issue that must be considered for storage of sperm to be used for artificial reproduction techniques.     

Comparative study of the kinetics of the acrosome reaction and survival of human spermatozoa in various media

The in-vitro kinetics of the acrosome reaction and the survival of human spermatozoa were studied under different capacitating conditions. Human preovulatory follicular fluid (FF), isotonic BWW (N-BWW) and hypertonic BWW (H-BWW) were tested. Motile sperm selected by migration in these media were examined after 1, 3, 5 and 22 h of incubation under 5% CO2. The kinetics of the reaction in the population of live, morphologically normal sperm was dependent on both the culture medium and time of incubation. In the first hour, the mean percentage of acrosome-reacted sperm in H-BWW and FF was significantly greater than in N-BWW. The proportion of reacted cells increased significantly after 3 h in N-BWW (P = 0.001), after 5 h in FF (P = 0.03) and after 22 h in H-BWW (P = 0.01). A significant decrease in sperm viability was registered at 3 and 22 h of incubation (P less than 0.002) in all media. These results demonstrate that both H-BWW and FF stimulate the acrosome reaction while survival is optimal in the latter.