Fluorescence technologies for evaluating male gamete (dys)function.

For many years, andrologists have sought ways of assessing sperm fertility, especially of new sires entering the breeding chain. As knowledge of the complex processes that enable sperm to fertilize eggs has increased, it has become clearer that quantitative estimation of the fertilizing potential of a sire or an ejaculate is actually unlikely ever to be fully realized. Here, we propose that a better approach is to identify substandard males and semen samples. During the past decades, the use of fluorescence technologies in biomedical science has burgeoned, with the development of very powerful instrumentation such as confocal microscopy and flow cytometers of ever-increasing capabilities together with a vast range of fluorochromes and fluorochrome conjugates. This technology has been applied to andrology but thus far in only a relatively simple way. In this review, we offer strategies for assessing a large range of sperm functions thought to be related to fertilizing ability over a temporal window rather than at a single time point. From such an assessment profile, sperm samples that over-respond or do not respond sufficiently could be identified, termed dysfunctional and rejected. We outline the rationales behind such tests, present information on new potentially useful fluorochromes and current flow cytometer models that would be suitable for the multicolour multifunctional tests we propose, and we offer suggestions as to how andrologists might design such multicolour tests for themselves.

Response to capacitating stimuli indicates extender-related differences in boar sperm function.

Spermatozoa, especially those of the porcine species, are highly susceptible to in vitro chilling and ageing. Extenders are continuously developed to protect boar spermatozoa from chilling injury. New semen extenders and other modified preservation strategies require sensitive testing for essential sperm functions. The key process on the pathway of fertilization is capacitation. The aim of the present study was to examine whether the specific response to capacitating stimuli is sensitive enough to indicate different preservation capacities of extenders during hypothermic storage of boar spermatozoa. Semen was diluted in Beltsville Thawing Solution (BTS) and Androstar Plus and kept for 3 h at 22°C or stored at 17°C, 10°C, and 5°C. Semen was analyzed at 24 and 96 h of storage. Motility and membrane integrity remained at high levels, except for lower values when stored in BTS at 5°C. Washed subsamples were incubated in capacitating medium (Tyrode) and control medium and were assessed for intracellular calcium concentration and integrity of plasma membranes using a flow cytometer. On the basis of the loss of low-calcium live cells in a kinetic approach, the specific response to capacitation stimuli was determined. There was a higher loss of response in semen stored hypothermically in the standard extender BTS compared to Androstar Plus. Assessment of the extent of phospholipid disorder under capacitating and control conditions by use of merocyanine staining did not reveal any significant extender-related differences. A field insemination trial with 778 sows was performed to relate in vitro results to fertility. Fertility parameters did not differ in semen stored up to 48 h at 10°C in Androstar Plus compared to controls stored at 17°C in BTS. In conclusion, assessment of specific reactivity to capacitating stimuli appears to be a sensitive tool for detection of extender-dependent alterations in functionality of chilled boar spermatozoa.

The specific response to capacitating stimuli is a sensitive indicator of chilling injury in hypothermically stored boar spermatozoa.

Boar spermatozoa are sensitive to storage temperatures below 15 °C. Chilling injury causes loss of motility and membrane integrity in a minority of cells, whereas the main population displays sublethal changes compromising fertility. In this study, changes of the response to capacitation conditions in hypothermically stored boar spermatozoa have been examined using a kinetic approach with well-defined test and control media. Ejaculates of seven boars were diluted in Beltsville Thawing Solution kept for 3 h at 22 °C or cooled to 17, 10 and 5 °C and stored for 24 and 96 h. At each time point, the standard sperm parameters motility and membrane integrity were evaluated. Subsequently, washed subsamples were incubated in capacitating and control medium before flow cytometric analysis of intracellular calcium content using the Fluo-3 probe and changes in phospholipid disorder using merocyanine. Kinetic changes of response parameters were monitored in viable (plasma membrane intact) cells. Chilling led to a loss of standard sperm quality traits in a minor subpopulation of cells, whereas storage length had no effect on these parameters. However, responses to incubation as determined by the loss of live cells with low intracellular calcium content showed marked changes in relation to storage conditions. The specific responsiveness to capacitation conditions decreased in close relation to storage temperature and length. In contrast, the merocyanine probe revealed to be limited to detect effects of hypothermic storage. Using Fourier transform infrared spectroscopy, no influence of chilling on membrane phase behaviour was found that might implicate decreased sperm function. In conclusion, assessment of response to capacitating media by monitoring intracellular calcium levels provides a sensitive measure for chilling injury in extended boar semen, and therefore, deserves implementation in hypothermic storage tests.

Vitrification of human ICSI/IVF spermatozoa without cryoprotectants: new capillary technology.

The aim of this study was to develop and to test the standardized aseptic technology of permeable cryoprotectant-free vitrification of human spermatozoa in capillaries (for intracytoplasmic sperm injection [ICSI] or in vitro fertilization [IVF]). To test the effect of vitrification on basic sperm parameters, each of 68 swim-up-prepared ejaculates from oligo-astheno-terato-zoospermic patients were aliquoted and distributed into 3 groups: 1) nontreated control, 2) 10 µL of spermatozoa cryopreserved by slow conventional freezing with glycerol-contented medium, and 3) 10 µL of spermatozoa vitrified in 50-µL plastic capillaries in culture medium with 0.25 M sucrose. Spermatozoa motility (1, 24, and 48 hours after warming), plasma membrane integrity, acrosomal integrity, and spontaneous capacitation-like changes were determined after warming. Aseptic cryoprotectant-free vitrification showed a significantly stronger cryoprotective effect compared with conventional freezing. One hour after warming, motility, plasma membrane integrity, and acrosomal integrity were significantly higher than is observed for conventionally frozen spermatozoa (28% vs 18%, 56% vs 22%, and 55% vs 21%, respectively; P < .05), although lower than in fresh spermatozoa (35%, 96%, and 84%, respectively; P < .05). Capacitation-like changes did not differ significantly between vitrified and conventionally frozen samples (8% vs 9%, respectively; P > .1) (2% in fresh spermatozoa). The newly developed technology of aseptic vitrification of human spermatozoa in capillaries can effectively preserve these cells from cryo-injures. Spermatozoa, vitrified by this technology, are free from seminal plasma owing to swim-up preceding vitrification and are free from permeable cryoprotectants. They are ready for further use immediately after warming without any additional treatment. Therefore, the reported technology has a great potential for use in ICSI/IVF.

Cryoprotectant-free vitrification of human spermatozoa in large (up to 0.5 mL) volume: a novel technology.

BACKGROUND: The aim of this study was to develop and to test the aseptic technology of cryoprotectant-free vitrification of human spermatozoa in large volume (for intrauterine insemination). Spermatozoa, vitrified by this technology, are free of permeant cryoprotectants and are ready for further use immediately after warming without any additional treatment (centrifugation or separation in the gradient for removal of cryoprotectant). METHODS: Each of 52 swim up-prepared ejaculates were divided into three aliquots and distributed into three treatment groups: Group 1: non-treated control; Group 2: spermatozoa cryopreserved by slow conventional freezing with glycerol-containing medium, and Group 3: spermatozoa vitrified in 0.5 mL insemination "French" straws in culture medium with 0.25 M sucrose. Sperm motility 1, 24 and 48 hours after warming, plasma membrane integrity and capacitation-like changes (spontaneous "cryo-capacitation" and acrosome reaction) were assessed after freezing-thawing. RESULTS: In contrast to conventional freezing, spermatozoa vitrified with aseptic cryoprotectant-free technology displayed superior functional characteristics. The motility rate, integrity rates of cytoplasmic, and acrosomal membranes were significantly higher after vitrification than after conventional freezing (76% vs 52%, 54% vs 28% and 44% vs 30%, respectively) (p < 0.05). However, there were no differences between vitrification and conventional freezing in the presence of glycerol in terms of percentage of spermatozoa expressing CTC-capacitation pattern (11% vs 10%, respectively) (p > 0.1). CONCLUSIONS: A basic protection from cryo-injury can be achieved for human spermatozoa using the novel technology of aseptic cryoprotectant-free vitrification in large volumes.

Assessment of storage effects in liquid preserved boar semen.

Fertility of extended boar semen declines within the first 72 h of storage in vitro. Standard semen assessment, such as motility and membrane integrity, allows detection of lethal damage of spermatozoa. However, conventional sperm assessment often lacks standardization and does not allow identification of sub-lethal changes of sperm quality during the initial 72 h of storage. In the present brief review, recent strategies for quality assessment of liquid preserved boar semen are discussed and basic implications for experiments designed to detect storage effects are given.

Mathematical analysis of mis-estimation of cell subsets in flow cytometry: viability staining revisited.

Many research projects in cell biology now use flow cytometry for analysis or for isolation of specific cell types. In such studies, cell viability is obviously a crucial issue. However, many studies appear to rely upon light-scattering characteristics to identify and gate out non-viable cells, despite the fact that reliable identification of such cells can only be achieved through staining with impermeable fluorescent nuclear dyes such as propidium iodide or 7-amino actinomycin. In this paper we apply mathematical analysis to the theoretical problem of quantifying cell sub-populations labeled with two or more fluorescent markers, comparing situations in which dead cells have been identified with those in which cell viability has not been assessed. We demonstrate that in all cases in which dead cells are present within the population, percentages of live sub-populations in different subsets are mis-estimated. In cases where the pattern of marker expression differs greatly between live and dead cells, or where the proportion of dead cells is high, this mis-estimation will be aggravated; the subsets pattern will therefore be biased in a population selected only on the basis of light-scatter behavior. The importance of accurately detecting and gating out dead cells is illustrated by an experimental example accompanying the mathematical analysis. To conclude, identification of dead cells by means of viability stains should be an absolute routine in practical flow cytometry, so as to avoid mis-estimation in sorting or analysis.

Cytometric solutions in veterinary andrology: Developments, advantages, and limitations.

Cytometric methodologies are becoming increasingly important in veterinary andrology as means of assessing sperm function. However, as yet, flow cytometric techniques in veterinary andrology have not kept up in sophistication with those in other areas of biology and medicine. In this brief review, we consider the present state of cytometry in andrological procedures for evaluating the fertility of domestic animal sires. We outline the aspects of sperm physiology, paying particular attention to the changes that take place during the process known as capacitation, which prepares the sperm for interaction with the egg. We then examine briefly but critically the cytometric techniques that are currently in commercial use or are being established in research laboratories for testing sperm characteristics. Current limitations and potential developments in semen assessment are discussed. Recent research knowledge offers possibilities for applying more subtle flow cytometric approaches to distinguish different levels of fertilizing potential in semen samples. For example, linking field fertility data to multiparametric kinetic studies of sperm capacitational changes rather than "single parameter-single time point" estimations may reveal that slower rather than rapid changes indicate high fertility. Moreover, the development of multicolor flow cytometric procedures as a means of evaluating multiple functional parameters in individual cells would reduce the uncertainties always inherent in predicting fertility from in vitro sperm evaluation tests.

Identifying non-sperm particles during flow cytometric physiological assessment: a simple approach.

Flow cytometry is now being used more frequently to determine sperm functional characteristics during semen assessment for artificial insemination. With this methodology, viable and potentially functional cells are detected as unstained events differentiated from non-sperm events through their light-scattering characteristics. However, it can be shown mathematically that identification of sperm on the basis of light scatter leads to significant overestimation of unstained viable cells and underestimation of responding cells in tests of sperm function (subpopulations expressing different fluorescence patterns). We have developed a simple and cost-efficient flow cytometric approach for identifying non-sperm particles that can be carried out in parallel with functional assessments. Our method is based on the sperm's osmotic intolerance. Diluted in water, lethal osmotic shock causes major damage to the cell membranes, and all sperm will stain with propidium iodide (PI). Particulate material which is not PI-positive can then be quantitatively evaluated by FACS analysis and the results substituted in mathematical equations to provide true values for sperm counts and subpopulations. In practical tests, the percentage of non-sperm particles determined by this technique was closely comparable to the figure obtained either by SYBR14/PI staining or by PI/CFDA staining. As well as being valuable with respect to tests of sperm function, the procedure is also suitable for obtaining accurate sperm counts during routine semen evaluation.

Systematic misestimation of cell subpopulations by flow cytometry: a mathematical analysis.

Various sources of variability in flow cytometric determination of cell concentration have previously been investigated with respect to andrologic applications. Although common aspects related to the variation between samples, variation between operators, and accuracy have been extensively studied, specific sources of false-count estimation have found less attention. In particular, a major and well-recognized source of misestimation of cell counts (i.e., contamination of the sample by non-sperm particles) has not to date been characterized in detail. We show here by means of original mathematical research that not only the cell counts but also the percentages of cells expressing different fluorescence patterns are affected by the presence of alien particles often neglected in studies involving flow cytometric characterization. We demonstrate that there is a systematic overestimation in the proportion of unstained (viable) cells detected by flow cytometry in cases where the non-sperm particles are not excluded from analysis by additional identification other than light-scatter characteristics. Moreover, we provide an exact mathematical estimate for the magnitude of this overestimation, and we discuss the consequences for diagnostic applications and studies on sperm physiology, specifically for studies on sperm capacitation and evaluation of cryopreserved semen. Finally, equations are derived for the correction of the flow cytometric values for use in practical applications.

Cryobiological determinants of frozen semen quality, with special reference to stallion.

Success in cryopreserving stallion semen has been very variable. Several different freezing regimes have been published. However, because extenders and procedures used in each regime have differed, direct comparison of these techniques has been very difficult, and controlled studies comparing different techniques have not been reported. A number of different factors affect sperm cryosurvival. In this article we review briefly current cryopreservation procedures for stallion semen, and then in more detail cryobiological determinants of sperm function, and mechanisms of cryoinjury and cryoprotectant action. Specific attention is given to data relating to stallion sperm. The complexity of sperm cell biology is believed to be an important factor when developing improvements in stallion semen cryopreservation. It may be assumed that impairment of cell function resulting from cold and osmotic shock is a main source of stallion sperm sensitivity to conventional freezing procedures. Further physiological studies on stallion sperm are required to understand the mechanisms by which cryopreservation alters sperm function and influences selection of sperm with higher fertilizing potential. Such studies should focus especially on the processes involved in sperm volume regulation, sperm-oviduct interaction, capacitation and cellular signalling, and on the alterations in these processes caused by cryopreservation.

Can external quality control improve pig AI efficiency?

External quality control programmes carried out by central laboratories have been long established in human andrology with the aim of enhancing the accuracy and reproducibility of semen assessment. Compared to human, demands on boar semen assessment in AI stations are more complex, with the need both to identify boars with poor ejaculate quality and to monitor individual boar differences for semen storage. Additionally, appropriate assessment serves as a control instrument to ensure the security and efficiency of semen processing. Despite current limitations regarding the ability of sperm assays to estimate the potential fertility of males, it is evident that boar fertility is related to certain conventional semen tests, e.g. sperm morphology. In central studies carried out on stored semen from 11 AI stations, flow cytometric assessment of plasma and acrosome membrane integrity proved to be more sensitive in detecting sperm damage associated with ageing and temperature stress as compared to light microscopy. Membrane integrity of stored semen differed between AI stations indicating significant influences of semen processing on sperm quality. Thus external control of semen quality in reference laboratories may be useful to monitor the efficiency of internal semen quality control in individual AI stations, to identify males with lower semen quality and/or poor response to semen storage, and to verify the precision of sperm counting. The possibility that central laboratories with sufficient resources may be able to identify functionally different responding sperm subpopulations for better estimation of fertility is discussed. Ideally, external quality control schemes for AI stations would comprise application of validated tests with high relevance for fertility (including bacterial status), analysis of semen processing on the AI station, and training courses for laboratory personnel.

Determinants of sperm quality and fertility in domestic species.

Fertilization success cannot be attributed solely to the absolute number of vital, motile, morphologically normal spermatozoa inseminated into the female but more especially to their functional competence. A range of in vitro tests has therefore been developed to monitor crucial aspects of sperm function: their ability to adapt to changing osmotic conditions, to bind to the oviductal epithelium, and to undergo capacitation in an appropriate and timely manner. The tests employ flow cytometry in conjunction with fluorescent techniques, electronic cell counting, and computer-assisted image area analysis. The highly quantitative analysis provided by electronic sizing and flow cytometry enables assessment of representative cell numbers in a very short time with high reproducibility. More importantly, it allows the detection of physiological heterogeneity within an ejaculate in terms of the development of cell subpopulations and enables the kinetic analysis of changes in living cell suspensions. The tests offer a promising strategy for evaluating fertility in domestic animals. The capability for volume regulation ensures that sperm recover from the tonic shocks experienced at ejaculation and during cryopreservation. Assessment of capacitation in vitro provides valuable information on both the sperm's ability to respond to fertilizing conditions and the sequence and rates of ongoing capacitation/destabilization processes. The monitoring of response to capacitating conditions in kinetic terms allows the sensitive and adequate detection of sperm populations expressing fertilization attributes and their ability to respond to external stimuli in a timely manner. However, subfertility is likely to be associated with a suboptimal response (i.e. too high or too low) rather than a minimal response.

Signalling pathways involved in the control of sperm cell volume.

The ability to maintain cellular volume is an important general physiological function, which is achieved by specific molecular mechanisms. Hypotonically induced swelling results in the opening of K+ and Cl- ion channels, through which these ions exit with accompanying water loss. This process is known as regulatory volume decrease (RVD). The molecular mechanisms that control the opening of the ion channels in spermatozoa are as yet poorly understood. The present study investigated pathways of osmo-signalling using boar spermatozoa as a model. Spermatozoa were diluted into isotonic and hypotonic Hepes-buffered saline in the presence or absence of effector drugs, and at predetermined intervals volume measurements were performed electronically. Treatment with protein kinase C (PKC) inhibitors staurosporine, bismaleimide I and bismaleimide X led to dose-dependent increases of both isotonic and hypotonic volumes (P<0.05). However, as the isotonic volume was affected more than the hypotonic volume, the kinase inhibitors appeared to improve RVD, whereas activation of PKC with phorbol dibutyrate blocked RVD. The increase in isotonic cell volume induced by bismaleimide X was observed in chloride-containing medium but not in the medium in which chloride was replaced by sulphate, implying that PKC was involved in the control of chloride channel activity, e.g. by closing the channel after volume adjustment. The protein phosphatase PP1/PP2 inhibitors calyculin and okadaic acid increased the isotonic volume only slightly but they greatly increased the relative cell volume and blocked RVD. The activation of RVD processes was found to be cAMP-dependent; incubation with forskolin and papaverine improved volume regulation. Moreover, papaverine was able to overcome the negative effect of protein phosphatase inhibitors. The mechanism of sperm RVD appears to involve (a) alterations in protein phosphorylation/dephosphorylation balance brought about by PKC and PP1 and (b) a cAMP-dependent activating pathway.

Enhanced binding of sperm with superior volume regulation to oviductal epithelium.

The plasma membrane is a key organelle with respect to sperm fertilizing ability. A sensitive way of testing plasma membrane functionality is to examine the sperm ability to moderate its swelling in response to hypo-osmotic stress (volume regulatory ability) using an electronic cell counter to assess cell volume changes. In this study of frozen-thawed bull sperm, we examined the relationship among sperm-oviductal epithelium binding capacity, osmotically induced swelling response, volume regulatory ability, and standard spermatologic parameters. Sperm cell volume distributions were measured under iso-osmotic conditions and after hypo-osmotic stress. The relative volume shift was calculated by comparing modal values of the cell volume distributions during transition from iso-osmotic to hypo-osmotic conditions. Significant correlations were found between volumetric parameters and sperm-oviduct binding capacity. Both the relative volume shift and regulative volume decrease correlated positively and significantly with the sperm-oviduct binding capacity. No significant correlations were found between sperm volumetric parameters and any of the standard sperm parameters with the exception of forward motility of Percoll-washed sperm. However, the use of multiple regression models improved the prediction level for binding capacity when motility parameters were combined with membrane integrity and volumetric parameters (R2 = .84). Spermatozoa of bulls with high nonreturn rates responded to hypotonicity as "perfect osmometers." Subfertile bulls had lower binding indices and deficiencies in volume recovery after hypotonic challenge, indicating that intact volume regulatory ability is a necessary prerequisite for binding to oviductal epithelium and is related to fertility. Volumetric parameters therefore could be used as tools in semen evaluation programs.

Binding of boar spermatozoa to oviductal epithelium in vitro in relation to sperm morphology and storage time.

In vitro short-term storage of boar semen for up to 72 h before insemination negatively affects fertility, but this often remains undetected during semen quality assessment. One important sperm function is the ability to form the functional sperm reservoir in the oviduct. In the present study, we used the modified oviductal explant assay to study sperm binding to oviductal epithelium in vitro in diluted boar semen stored for 24 or 72 h. First, we determined the kinetics of in vitro sperm binding to oviductal epithelium in relation to co-incubation time of sperm and oviductal tissue pieces. Then, we studied how the binding of sperm to oviductal epithelium was affected by in vitro semen storage and by differences among individual boars. Sperm binding after different incubation times was significantly higher when semen was stored 24 h than after 72-h storage (P < 0.05), and peaked at 30-90 min of incubation. Sperm binding differed between boars (n = 44), and was negatively correlated to the percentage of sperm with cytoplasmic droplets (R = -0.51, P < 0.001). There were no significant changes in motility, acrosome integrity and propidium iodide stainability during the 72-h storage period. However, sperm-binding indices were significantly lower after 72 h in vitro storage than after 24-h storage in sperm from boars with normal semen quality (P < 0.05); in contrast, the binding capacity of sperm from boars with higher percentages of morphologically altered sperm remained at a low level. The sperm-binding capacity of sperm from four of the five boars with known subfertility was lower than the mean binding index minus one standard deviation of the boar population studied here. It is concluded that changes in the plasma membrane associated with in vitro ageing reduce the ability of stored boar sperm to bind to the oviductal epithelium. This study shows the potential of sperm-oviduct binding as a tool to assess both male fertility and changes in sperm function associated with in vitro ageing.

Regulatory and necrotic volume increase in boar spermatozoa.

Spermatozoa of many species initially respond to hypotonicity as perfect osmometers. Thereafter they undergo a regulatory process resulting in a decrease in cell volume, similar to that reported for somatic cells. Regulatory volume increase (RVI), a complementary process which is assumed to occur following initial shrinkage of sperm volume after exposure to a hypertonic medium, has not yet been described in detail for spermatozoa. In this study, we investigated whether spermatozoa are able to regulate their volume after hypertonic stress and whether this ability is maintained in preserved sperm. Cell volume changes were recorded using electronic cell sizing. Sperm response to the ion channels blockers quinidine, tamoxifen, and dydeoxyforskolin, and to protein kinase/phosphatase inhibitors lavendustin, staurosporine, and vanadate was studied to investigate possible mechanisms of RVI. Annexin V staining was used in combination with propidium iodide to determine whether hypertonic stress may induce apoptosis. Overall protein tyrosine phosphorylation under hypertonic conditions was measured via flow cytometry using antiphosphotyrosine antibody. Spermatozoa exposed to hypertonic stress initially responded with an abundant subpopulation according to the perfect osmometer model and recovered their volume from this shrinkage after 20 min. RVI was inhibited by quinidine and tamoxifen, which indicates the involvement of the important cellular ions sodium and chloride in this process. Volume regulatory ability was essentially maintained during storage of liquid semen. However, the response of the sperm population was heterogeneous. A second population raised, containing spermatozoa with larger volumes, which demonstrated irregularities in the volume response with respect to osmotic challenge, ion channel blockers, and storage. Under hypertonic conditions, both protein kinase inhibitors (PKI) led to increased isotonic volumes and to elevated initial relative volumes and subsequent volume decrease. RVI was inhibited by the vanadate. Hypertonic stress did not result in an increase in early apoptotic cells, but produced a shift toward late necrotic cells. Substitution of sodium and chloride by choline and sulfate resulted in decreased isotonic volume of sperm treated with lavendustin. Tyrosine phosphorylation levels were reduced after 20 min under hypertonic conditions. It was concluded that RVI is regulated via a protein tyrosine kinase-dependent pathway, and that dephosphorylation occurs when volume regulation is required. The necrotic volume increase (NVI) is associated with the accumulation of sodium and chloride following uncontrolled opening of the channels. The ability to regulate volume after exposure to hypertonic conditions is important for sperm functionality and can have practical applications in spermatological diagnostics and cryopreservation.

Volume regulatory function and sperm membrane dynamics as parameters for evaluating cryoprotective efficiency of a freezing extender.

In the past years a series of functional assays has been developed to determine the structural, morphological and functional integrity of the plasma membrane and sperm acrosomal membrane. Cell volume regulation is an important physiological function crucial for the success of cryopreservation. In this study, the effects induced by freezing-thawing were judged by evaluating the functional characteristics of frozen-thawed semen samples submitted to secondary stress such as osmotic challenge or incubation under capacitating conditions, following cryopreservation. Prior to freezing, dog semen samples were diluted in the presence or absence of Equex STM Paste, which contains sodium dodecyl sulphate (SDS) as the active ingredient. Cell volume regulation and capacitation and calcium ionophore-induced membrane dynamics were assessed in freshly diluted and frozen-thawed semen samples by electronic volume measurement and flow cytometry. Cryopreservation led to a disturbance of the volume regulatory function and to a rapid decrease in the proportion of acrosome-reacted live spermaotozoa. Extender containing Equex STM Paste had a protective effect on isotonic cell volume, on regulatory function under hypertonic conditions, and on the proportion of live acrosome-reacted cells. The evaluation of the functional state of sperm submitted to secondary stress after freezing-thawing leads to a more subtle characterization of sperm function and helps improve the cryoprotective efficiency of the extender.