Effect of ferrous sulphate and ascorbic acid on motility, viability and lipid peroxidation of crossbred cattle bull spermatozoa

Numerous factors influence male fertility, one of these being the oxidative stress, which has elicited enormous interest recently. In sperm, induction of oxidation decreases motility and viability but increases lipid peroxidation (LPO). The optimum dose of ferrous ascorbate (FeAA: FeSO4 + ascorbic acid) for inducing oxidative stress by affecting motility, viability and LPO has been ascertained in local crossbred cattle bull spermatozoa. The fractions of spermatozoa suspended in 2.9% sodium citrate were subjected to three doses of FeAA (100 : 500, 150 : 750, 200 : 1000; μmol/l FeSO4 : μmol/l ascorbic acid). These fractions were assessed for various parameters. Increase in the incubation period and promoter concentration induced a decrease in motility and viability, but an increase in LPO. Among three doses of FeAA, 150 : 750 μmol/l ascorbic acid is suggested to be the optimum/best dose as it induces the oxidative stress/LPO to a significant extent and also maintains better motility and viability as compared with the other two doses, and such conditions may enhance the fertilising potential of bull spermatozoa.     

Preventive effect of Desferal on sperm motility and morphology

Transition metal ions, mainly iron, are involved in the generation of highly reactive hydroxyl radicals, which are the most powerful inducers of oxidative damage to all biomolecules. The lipids in sperm membranes are highly susceptible to oxidation. Sperm lipid peroxidation (LPO) leads to decrease of motility and reduction of likelihood for sperm‐oocyte fusion. The excess radical production may affect also the spermatozoa morphology. The aim of the present study was to investigate the effect of Desferal on the LPO, motility, and morphology of boar sperm subjected to oxidative stress. After collection, the ejaculates were equally separated and diluted in a commercial semen extender (experiment 1) or in physiological saline (experiment 2). The ejaculates of the 2 experiments were divided into aliquots, which were incubated with one of the following agents: FeSO₄ (0.1mM), H₂O₂ (0.5mM), or FeSO₄ + H₂O₂ (Fenton system), in the presence or absence of Desferal. The application of Desferal in the incubation medium had a protective effect against FeSO₄ + H₂O₂‐induced sperm damage, namely, decrease of LPO; decrease the quantity of immotile spermatozoa and decrease the number of morphological abnormalities, regardless of the used medium. In experiment 2, the presence of FeSO₄ in the incubation medium induced LPO in the same range as the combination FeSO₄ + H₂O₂, in which the effect was reduced by Desferal. Thus, the supplement of Desferal to media used for sperm storage and processing could be a useful tool for diminishing oxidative injury and improving the quality of the semen.     

Role of glutathione peroxidase in protecting mammalian spermatozoa from loss of motility caused by spontaneous lipid peroxidation

Mouse and human spermatozoa, but not rabbit spermatozoa, have long been known to be sensitive to loss of motility induced by exogenous H₂O₂. Recent work has shown that loss of sperm motility in these species correlates with the extent of spontaneous lipid peroxidation. In this study, the effect of H₂O₂ on this reaction in sperm of the three species was investi gated. The rate of spontaneous lipid peroxidation in mouse and human sperm is markedly enhanced in the presence of 1-5 mM H₂O₂, while the rate in rabbit sperm is unaffected by H₂O₂. The enhancement of lipid peroxidation, the rate of reaction of H₂O₂ with the cells, the activity of sperm glutathione peroxidase, and the endogenous glutathione content are highest in mouse sperm, intermediate in human sperm, and very low in rabbit sperm. Inac tivation of glutathione peroxidase occurs in the presence of H₂O₂ due to complete conver sion of endogenous glutathione to GSSG: No GSH is available as electron donor substrate to the peroxidase. Inactivation of glutathione peroxidase by the inhibitor mercaptosucci nate has the same effect on rate of lipid peroxidation and loss of motility in mouse and human sperm as does H₂O₂. This implies that H₂O₂ by itself at 1-5 mM is not intrinsically toxic to the cells. With merceptosuccinate, the endogenous glutathione is present as GSH in mouse and human sperm, indicating that the redox state of intracellular glutathione by itself plays little role in protecting the cell against spontaneous lipid peroxidation. Mouse and human sperm also have high rates of superoxide production. We conclude that the key intermediate in spontaneous lipid peroxidation is lipid hydroperoxide generated by a chain reaction initiated by and utilizing superoxide. Removal of this hydroperoxide by gluta thione peroxidase protects these sperm against peroxidation; inactivation of the peroxidase allows lipid hydroperoxide to increase and so increases the peroxidation rate. Rabbit sperm have low rates of superoxide reaction due to high activity of their superoxide dismutase; lack of endogenous glutathione and low peroxidase activity does not affect their rate or lipid peroxidation. As a result, these sperm are not affected by either H₂O₂ or mercapto-succinate. These results lead us to postulate a mechanism for spontaneous lipid peroxida tion in mammalian sperm which involves reaction of lipid hydroperoxide and O₂ as the rate-determining step.     

Iron-induced luminescence as a method for assessing lipid peroxidation of frozen-thawed goat spermatozoa

Freezing/thawing procedures induce enhanced reactive oxygen species (ROS) formation in mammalian sperm and these ROS may be a cause for the decrease in sperm function following cryopreservation. In the present study, we used a chemiluminescence method to detect ROS-induced damage in goat spermatozoa. Iron-induced luminescence of fresh and frozen/thawed sperm cells was assessed using a luminometer. It was shown that the freezing/thawing procedure had a significant effect on some luminescence parameters. Semen freezing significantly increased the values of integral, peak max, T.half (rise) and T.max (peak) parameters. A significant correlation was observed between the percentage of motile spermatozoa and integral, peak max and T.half (rise) parameters. In conclusion, the results of the present study indicate that measurement of induced luminescence can be an alternative, sensitive and relatively simple method for assessing the effect of cryopreservation on oxidative damage to spermatozoa.     

Influence of image sampling frequency on the perceived movement characteristics of progressively motile human spermatozoa

Tracks of 30 progressively motile washed human spermatozoa were plotted manually from 200-Hz frame rate video recordings. Tracks at 100, 66.7, 50, 40, 33.3., 25, 20, 10, and 5 Hz were then constructed using every 2nd, 3rd, 4th, 5th, 6th, 8th, 10th, 20th, or 40th point. The 200-Hz tracks were analyzed by traditional manual methods, and all ten sets of tracks analyzed using a computer-assisted method (“Videomot,” developed originally to analyze 30-Hz tracks) to eliminate observer bias. Progression velocity (VSL) remained constant under all analysis conditions. Average path velocity (VAP) also remained essentially constant, although Videomot was less reliable at high frame rates due to problems in determining the average path. Curvilinear velocity (VCL) was very frame rate dependent (the 25-Hz mean value was only 56.5% of that at 200 Hz), and Videomot was more accurate than manual analysis at 200 Hz. Values of the amplitude of lateral head displacement (ALH) were acceptable at most frame rates. At < 66.7 Hz the inclusion of spurious curvilinear track deviations caused lower mean ALH values, and at 5 Hz ALH could not be measured since the track was essentially the average path. Beat/cross frequency (BCF) was also frame rate dependent; at high rates there was the same problem as with ALH measurements, while at ? 25 Hz the maximum BCF was restricted by the frame rate. We conclude that human sperm movement characteristics can be measured at frame rates ca. 30 Hz but only if the constraints affecting VCL and BCF values are understood and accepted. Finally, < 10 Hz can only give values for VSL and, perhaps, VAP.     

Initiation,prolongation, and reactivation of the motility of salmonid spermatozoa

The motility of salmonid spermatozoa initiated by dilution of the milt with ovarian fluid or isotonic saline is brief duration; it was believed that it can be activated only once in the life of the spermatozoon. Dilution of the milt with an equal volume of isotonic saline (0.12 M-NaCl) containing 5 mM-3-isobutyl-1-methylxanthine (MIX) prolonged and intensified sperm motiliy. When motility had stopped after initial mobilization with saline or ovarian fluid, it could be reactivated by addition of MIX; reactivated spermatozoa fertilized eggs. Dilution with saline containing K⁺ (24 mEq/liter) did not initiate sperm motility even in the presence of MIX. The spermatozoa were mobilized by subsequent with 0.12 M-NaCl. The concentration of adenosine triphosphate (ATP) in sperm suspensions dropped on dilution with saline and rose as motility ceased, but declined without subsequent recovery following dilution with MIX-saline. The concentration of cyclic adenosine monophosphate (cAMP) rose and fell sharply on initiation of motility and rose again after motility had declined. While salmonid spermatozoa can be mobilized by dilition with saline alone, the effectiveness of MIX in reactivating “spent” spermatozoa supports the assumption that cAMP plays a role in the initiation of sperm motility.     

The motility of rabbit spermatozoa recovered from the female reproductive tract

The motility of rabbit spermatozoa recovered from the vagina, endocervix, uterus, and four regions of the oviduct was assessed visually by phase-contrast microscopy at intervals from one minute to 16 hours after a single mating. The percentage of motile cells in each sample was dependent on the temperature of recovery, ie, 23° vs 37°C, but was not influenced by the temperature of observation. Spermatozoa in the lower isthmus of the oviduct were the most temperature sensitive population to recovery at 23°C. When all manipulations and observations were performed at 37°C, the percentage of spermatozoa with progressive motility varied according to the region sampled and interval after mating. Populations from the vagina, uterus and upper regions of the oviduct usually had a high proportion of progressively motile cells with vigorous flagellar activity. Fewer spermatozoa showed progressive movement on recovery from the endocervix and lower 2 cm of the tubal isthmus and their flagellar activity was generally depressed. The decrease in flagellar beat frequency noted in the latter regions may be a major factor limiting sperm ascent in the female tract. A unique pattern of “activated” motility was seen exclusively in populations taken from the oviducts at 6 to 16 hours after mating. This motility pattern, consisting of alternating episodes of linear progressive and vigorous nonprogressive movement, may be analogous to the activated motility described for capacitated rodent spermatozoa.     

Sperm motility and cryopreservation of spermatozoa in freshwater gobies

Testicular sperm motility and methods for the cryopreservation of spermatozoa in the freshwater goby Rhinogobius sp. CB (Cross Band type) were examined. Spermatozoa were almost immotile upon dilution with 300 mOsm kg⁻¹ of NaCl, KCl and mannitol solutions but began to swim in solutions with concentrations <200 mOsm kg⁻¹. The highest percentage and longest duration of motility was obtained in the 0 and 100–200 mOsm kg⁻¹ solutions, respectively. The highest post-thaw motility, c. 50% of motility before cryopreservation, was obtained when spermatozoa were diluted with an extender of 10% methanol and 90% artificial seminal plasma, cooled at −10·0 ± 1·1° C min⁻¹ (mean ± s . e .) to −50° C and plunged into liquid nitrogen. Spermatozoa were cryopreserved in a 50 μl acrylic haematocrit tube to store the small amount of milt. As the cryopreservation method described above was applicable to the endangered Rhinogobius sp. BI (Bonin Island type), it is probable that this method can be used for other species of freshwater gobies.     

Lipid peroxidation in plumbagin administered rats

Plumbagin was administered to rats at a concentration of 1,2,4,8 and 16 mg per kg body weight. After 24 h lipid peroxide levels were found to decrease in subcellular fractions of liver. Plumbagin inhibited ascorbate and nicotinafde adenine dinucleotide phosphate (reduced) dependent lipid peroxidation but was without any effect on cumene hydroperoxide dependent lipid peroxidation. Injection of 16 mg of plumbagin per kg body weight was found to decrease liver total reduced glutathione and also fcrosomal glucose-6-phosphatase. The results are discussed with reference to the anti- and prooxidant properties of plumbagin.     

Computerized analysis of motility, motility patterns and motility parameters of spermatozoa of carp following short-term storage of semen

A computer-aided semen analysis system was used to assess the % motile cells following storage of carp semen in 11 different buffers at 2, 5 or 22° C. BWW and TLP were the most suitable storage buffers because carp semen stored at 5° C in these buffers following activation showed no significant decrease in % motile spermatozoa up to 24 h. But, in most of the other buffers (Fish Ringer, Cytomix, Cortland, FRT, Mannitol, FPS, NAS and TSM) the motility potential was lost by 2 h. Storage was best at pH 6–9 and at 5° C. Carp spermatozoa exhibit three distinct motility patterns, namely 'linear', 'circular' and 'haphazard', the proportion of spermatozoa with a particular motility pattern depending on storage buffer and time. All spermatozoa with a linear trajectory had high VSL, STR and LIN; those moving in circles had low VSL, STR, LIN and BCF and those with a haphazard trajectory were distinct in that they had the highest ALH and their VSL, STR, LIN and BCF were higher than the circular moving spermatozoa and lower than the spermatozoa exhibiting linear trajectory. The study also demonstrates a pronounced time-dependent decrease in VCL, VAP, VSL and ALH of carp spermatozoa following activation with water or low osmolality solutions. This study provides for the first time data related to seven motility parameters of carp spermatozoa and demonstrates how these parameter values could be used to evaluate quality of carp milt following storage in different buffers. It confirms that carp spermatozoa exhibit linear or circular trajectories and provides evidence for a third type of trajectory described as haphazard. All three motility patterns could be discriminated objectively on the seven motility parameters.     

Sperm head abnormalities are associated with excessive omega‐6 fatty acids in two finch species feeding on sunflower seeds

In a rapidly changing world, it is important to understand how urban environments impact wildlife. For example, supplementary feeding of birds, though well‐intended, might have unexpected negative effects on the health of individual animals. Sunflower seeds are commonly provided in garden bird feeders, but they contain high levels of linoleic acid (LA), an omega‐6 polyunsaturated fatty acid (PUFA). Omega‐6 PUFAs are associated with increased oxidative stress, which can damage cell membranes, and in particular sperm cells. We assessed the level of LA in the blood of two seed‐eating finch species, greenfinches Chloris chloris and hawfinches Coccothraustes coccothraustes, caught in and in environments with direct access to sunflower seed feeders (Norway), and compared these with the level of LA in a smaller number of individuals sampled in in a rural area with low incidence of sunflower seed feeders (Czech Republic). Furthermore, we investigated the relationship between the proportion of LA in the blood (as well as the proportion of 10 other fatty acids) and sperm quality (the frequency of sperm head abnormalities and sperm swimming speed). We found that both finch species, but particularly greenfinches caught near feeders, exhibited levels of LA that were considerably higher than those previously reported for other wild birds. We also found that the proportion of LA was positively correlated with the frequency of abnormal sperm heads (sperm missing the acrosome), while there was no significant effect of fatty acid composition on sperm swimming speed. Our results indicate that the sperm quality of finches may be negatively affected by a high intake of sunflower seeds, adding to a growing body of research showing that supplementary feeding may have detrimental side effects for urban animals. This is particularly relevant for the greenfinch, which is currently affected by disease and population declines.     

The onset and maintenance of hyperactivated motility of spermatozoa from the mouse

Estimates were made of the proportion of freely motile mouse spermatozoa displaying hyperactivated motility by an objective photographic method employing stroboscopic illumination under dark-field conditions and examining displacements of the sperm head and bend angles of the sperm tail. In media known to support in vitro fertilisation hyperactivation gradually appeared reaching about 40% by 6 hr incubation, and it was not promoted by 2 mM caffeine or 0.1 mM Bt₂ cAMP or washing the cells free of epididymal fluid. Raising the osmolarity of the medium to 400 mOSM with electrolytes, but not nonelectrolytes, did promote hyperactivation (60% by 2 hr) suggesting that the ionic strength of the medium was important. Hyperactivation in high ionic strength media could be prevented by removing or chelating Ca²⁺, or replacing Ca²⁺ with Ba²⁺ or Mg²⁺, when nonhyperactivated motility was maintained, but Sr²⁺, like Ca²⁺, permitted hyperactivated motility. Hyperactivation in low ionic strength medium could be promoted by the ionophore A23187, suggesting that Ca²⁺ movement into the cells is important. Of a range of glycolytic substrates tested supporting nonhyperactivated motility in the presence of lactate, only glucose supported hyperactivation. Addition to glucose— or Ca²⁺ — free, high ionic strength media after 2 hr increased hyperactivation immediately (glucose) or after a lag of 2 hr (Ca²⁺) suggesting that glucose acts on a Ca²⁺ — primed system. Removal from high ionic strength medium, chelation of Ca²⁺ or inhibition of glucose metabolism did not prevent hyperactivation continuing once it had been initiated, indicating different requirements for initiation and maintenance of this form of motility.     

Sperm midpiece length predicts sperm swimming velocity in house mice

Evolutionary biologists have argued that there should be a positive relationship between sperm size and sperm velocity, and that these traits influence a male''s sperm competitiveness. However, comparative analyses investigating the evolutionary associations between sperm competition risk and sperm morphology have reported inconsistent patterns of association, and in vitro sperm competition experiments have further confused the issue; in some species, males with longer sperm achieve more competitive fertilization, while in other species males with shorter sperm have greater sperm competitiveness. Few investigations have attempted to address this problem. Here, we investigated the relationship between sperm morphology and sperm velocity in house mice (Mus domesticus). We conducted in vitro sperm velocity assays on males from established selection lines, and found that sperm midpiece size was the only phenotypic predictor of sperm swimming velocity.     

Binding of the lipid peroxidation product 4-hydroxynonenal to human polymorphonuclear leukocytes

4-Hydroxynonenal (HNE) is produced during peroxidation of polyunsaturated fatty acids. It exerts a chemokinetic effect on human polymorphonuclear leukocytes (PMN). Investigations of this mechanism were performed. The results indicate that [³H]-HNE binding to PMN results both in non-specific bonds to the numerous SH groups of the cells and in binding to a saturable, reversible and specific HNE site. Scatchard analysis revealed that this is a single site with an apparent affinity constant of 319 nM and a density of 1·57 pmol (10⁶)^?1 cells. This specific binding site may be involved in the chemokinetic effect of HNE.     

Lipid peroxidation and growth inhibition of human microvascular endothelial cells

Peroxidation products of polyunsaturated fatty acids may cause growth inhibition of cells in culture. This study was carried out to elucidate to what extent peroxidation products may be found in growth media, with and without cells and albumin, using thiobarbituric acid-reactive substances (TBARS) and protein carbonyl groups as measures of peroxidation. The growth of human microvascular endothelial cells was studied as influenced by docosahexaenoic (C22:6, n - 3), arachidonic acid (C20:4. n - 6), and serum albumin. Cell growth was strongly inhibited by the fatty acids, and the inhibition was related to the concentration of TBARS in the medium. Defatted albumin (0.5 g/100 ml) nullified the increase of TBARS in the medium and released the growth inhibition by the fatty acids. With polyunsaturated fatty acids (PUFA) there was a time- and concentration-dependent increase in media TBARS, observed both with and without cells, but the TBARS increase was somewhat greater in the presence of cells. Surprisingly, TBARS in cell-free media also increased somewhat upon increasing the albumin concentration from 0.5 to 5 g/100 ml, and the TBARS increase differed among various preparations of albumin. Unexpectedly, the albumin that had not been defatted gave the lowest TBARS values. The amount of protein carbonyl groups did not differ among various albumin preparations. It is concluded that PUFA may autooxidize in media used for cell cultures, and thereby cause an unspecific growth inhibition, which can be prevented by a low albumin concentration. However, even defatted albumin preparations may contain lipid peroxidation products, the causes and implications of which remain to be elucidated.     

Morphological abnormalities in the spermatozoa of fertile and infertile men

The morphological analysis of the spermatozoa from fertile and infertile men was performed using light and electron microscopy to clarify the relationship between sperm morphology and fertility. Semen samples obtained from 22 partners of pregnant women were prepared according to the protocol standardized in an international collaborative study. Semen samples from 17 patients with asthenozoospermia or varicocele were collected in a hospital. Abnormalities in the spermatozoa were classified into three types for the tails, two for the midpieces, and six for the heads according to the criteria adapted from WHO guidelines (World Health Organization, 1999: WHO laboratory manual for the examination of human semen and semen-cervical mucus interaction (4th edition)). Approximately 14% of the spermatozoa from the fertile men had abnormal tails at the light microscopic level while approximately 44% had abnormal heads. Most types of abnormalities found in the spermatozoa from the asthenozoospermic and varicocele patients were encountered in those from the fertile men, although the semen from the fertile men contained a higher percentage of normal spermatozoa than that from the patients. These results were also confirmed at the ultrastructural level. Most abnormal cell types are encountered in semen from fertile men, although the incidence of abnormalities is low.     

Analysis of motility parameters from paddlefish and shovelnose sturgeon spermatozoa

Ninety to 100% of paddlefish Polyodon spathula were motile just after transfer into distilled water, with a velocity of 175 μm s^-1, a flagellar beat frequency of 50 Hz and motility lasting 4–6 min. Similarly, 80–95% of shovelnose sturgeon Scaphirhynchus platorynchus spermatozoa were motile immediately when diluted in distilled water, with a velocity of 200 μm s^-1, a flagellar beat frequency of 48 Hz and a period of motility of 2–3 min. In both species, after sperm dilution in a swimming solution composed of 20 mM Tris–HCl (pH 8·2) and 20 mM NaCl, a majority of the samples showed 100% motility of spermatozoa with flagella beat frequency of 50 Hz within the 5 s following activation and a higher velocity than in distilled water. In such a swimming medium, the time of motility was prolonged up to 9 min for paddlefish and 5 min for sturgeon and a lower proportion of sperm cells had damage such as blebs of the flagellar membrane or curling of the flagellar tip, compared with those in distilled water. The shape of the flagellar waves changed during the motility phase, mostly through a restriction at the part of the flagellum most proximal to the head. A rotational movement of whole cells was observed for spermatozoa of both species. There were significant differences in velocity of spermatozoa between swimming media and distilled water and between paddlefish and shovelnose sturgeon.     

Analysis of lipid peroxidation mechanisms in human spermatozoa

The mechanisms by which ferrous ion promoters induce malondialdehyde generation by human spermatozoa have been investigated in order to provide a rational basis for the quantification and interpretation of lipid peroxidation assays. Incubation of human spermatozoa with a ferrous ion promoter in the presence of thiobarbituric acid (TBA) led to the generation of the bone fide malondialdehyde-TBA adduct. The importance of iron in the stimulation of lipid peroxidation was emphasized by the ability of Desferal* and EDTA to suppress malondialdehyde generation. Paradoxically, when the concentration of EDTA relative to iron was equimolar or greater, the suppression of malondialdehyde formation was accompanied by the generation of hydroxyl radicals. These results suggested that the addition of promoter did not effect the first-chain initiation of lipid peroxidation but favored an alternative mechanism involving the catalytic decomposition of pre-existing lipid peroxides. This conclusion was reinforced by the inability of reagents that would limit the formation (superoxide dismutase and/or catalase) or availability (mannitol, formate) of hydroxyl radicals, to influence malondialdehyde generation. While hydroxyl radicals were not directly involved in Fe²⁺-promoted malondialdehyde generation, the existence of significant correlations between reactive oxygen species production and the outcome of the TBA assay, suggested that Fenton chemistry might be important in the initiation of peroxidative damage. It is proposed that the impeded propagation of peroxidation initiated by Fenton or Haber Weiss reactions would lead to the accumulation of lipid peroxides in the spermatozoa and it is these peroxides that are induced to decompose during the Fe²⁺-promoted TBA assay, stimulating a lipoperoxidative chain reaction and malondialdehyde formation. © 1993 Wiley-Liss, Inc.     

Lipid peroxide formation in relation to membrane stability of fresh and frozen thawed stallion spermatozoa

In this study we used a new method to detect reactive oxygen species (ROS) induced damage at the level of the sperm plasma membrane in fresh and frozen-thawed stallion sperm. Lipid peroxidation (LPO) in sperm cells was assessed by a fluorescent assay involving the labeling of stallion sperm with the LPO reporter probe C11-BODIPY(581/591). The peroxidation dependent spectral emission shift of this membrane probe could be localized using inverted spectral confocal microscopy and quantified on living and deteriorated sperm cells using flow cytometry. Mass spectrometric analysis of the main endogenous lipid class, phosphatidylcholine (PC), was carried out to determine the formation of hydroxy- and hydroperoxyphosphatidylcholine in fresh sperm cells. Peroxidation as reported by the fluorescent probe corresponded with the presence of hydroxy- and hydroperoxyphosphatidylcholine in the sperm membranes, which are early stage products of LPO. This allowed us to correlate endogenous LPO with localization of this process in the living sperm cells. In absence of peroxidation inducers, only relatively little peroxidation was noted in fresh sperm cells whereas some mid-piece specific probe oxidation was noted for frozen-thawed sperm cells. After induction of peroxidation in fresh and frozen-thawed sperm cells with the 0.1 mM of lipid soluble ROS tert-butylhydrogen peroxide (t-BUT) intense probe oxidation was produced in the mid-piece, whereas the probe remained intact in the sperm head, demonstrating antioxidant activity in the head of fresh sperm cells. At higher levels of t-BUT, probe peroxidation was also noted for the sperm head followed by a loss of membranes there. Frozen-thawed sperm were more vulnerable to t-BUT than fresh sperm. The potential importance of the new assays for sperm assessments is discussed.