New data on aberrant spermatozoa in the ejaculate of Sus domesticus

This paper describes 16 new types of aberrant spermatozoa observed by scanning electron microscopy in the ejaculate of two healthy, sexually mature Landrace boars. The new anomalies observed were 1) spermatozoa with folded tail and abnormal head; 2) tailless spermatozoa with an abnormal connecting piece; 3) immature spermatozoa with two tails of the same length, fused and coiled; 4) spermatozoa with two tails of the same length, fused and coiled, and a small, rounded head; 5) spermatozoa with two fused tails and a wide head; 6) spermatozoa with three tails of the same length, fused and coiled; 7) immature spermatozoa with two heads and two fused tails; 8) spermatozoa with two heads, one at each tip of the tail; 9) spermatozoa with a short, folded tail and a triangular head; 10) spermatozoa with a short tail lacking the intermediate piece; 11) spermatozoa with a short tail, without the main piece and with a long intermediate piece; 12) spermatozoa with a short tail, without the main piece and with a rough head; 13) spermatozoa with small, rounded head; 14) spermatozoa with small, aberrant heads; 15) spermatozoa with small, bacillary heads; and 16) immature spermatozoa with tapering heads.     

Flow microfluorometric analysis of living spermatozoa stained with Hoechst 33342

Bovine spermatozoa were stained with Hoechst 33342. The fluorescence distribution of stained spermatozoa was complex. Non-motile spermatozoa displayed a higher fluorescence than did motile spermatozoa. The fluorescence profile of the motile spermatozoa was bimodal. Sort and reanalysis, and orientation experiments suggested that there are two distinct populations of motile spermatozoa.     

Ultrastructural abnormalities of boar spermatozoa

Described here are the main ultrastructural malformations observed in spermatozoa of ejaculates collected from healthy, adult Landrace boars following 2 days of sexual abstinence. Previously semen had been collected 3 times per week. Sperm concentration in the cell-rich fraction of ejaculates was approximately 700,000 sperm/mm(3). The aberrant gamete forms did not exceed 2% of the total number of spermatozoa. Ultrastructural anomalies of spermatozoa were classified into 2 groups: head malformations and tail malformations. These consisted of: 1) spermatozoa with expanded and vacuolated acrosomes, 2) spermatozoa with myelin figures within the perinuclear space, 3) macrocephalic spermatozoa with 2 nuclei and a deformed acrosomal vesicle, 4) spermatozoa with an expanded acrosomal apex, 5) spermatozoa with nuclear vacuoles, 6) macrocephalic spermatozoa with a roundish head, 7) spermatozoa with swollen mitochondria, 8) spermatozoa with additional mitochondria over the mitochondrial sheath, 9) spermatozoa without the central microtubular pair, 10) spermatozoa without some peripheral doublets, 11) spermatozoa with 1 or 2 coiled tails, 12) spermatozoa with a folded tail and a disorganized connecting piece, 13) spermatozoa with a vesiculated tail, and 14) spermatozoa with 2 tails fused by their respective mitochondrial sheaths.     

Proacrosin/acrosin quantification as an indicator of acrosomal integrity in fresh and frozen dog spermatozoa

The scope of the present study was to evaluate the presence and activation of proacrosin/acrosin as a tool to determine the acrosomal status of fresh and frozen/thawed dog spermatozoa. Monoclonal antibody C5F11, directed against human acrosin, cross-reacted with dog spermatozoa and labeled the acrosome of both fresh and frozen/thawed dog spermatozoa. Frozen/thawed spermatozoa had a lesser proportion of labeled spermatozoa than fresh spermatozoa (P<0.05). When live spermatozoa were labeled with soybean trypsin inhibitor conjugated with Alexa 488 (SBTI-Alexa 488), the proportion of acrosome-labeled fresh spermatozoa was less than frozen/thawed spermatozoa (P<0.05). By using Western blots and enzymatic activity, frozen/thawed spermatozoa had a greater proportion of active acrosin than fresh spermatozoa. In addition, beta 1,4-galactosyl-transferase (GalT), a plasma membrane bound protein, remained attached to frozen/thawed spermatozoa. Proacrosin is activated during freezing/thawing of dog spermatozoa, and that proacrosin/acrosin may be a good indicator of acrosomal integrity of frozen/thawed spermatozoa.     

Motion of individual human spermatozoa, both normal and lacking the outer dynein arms, during a continuous temperature rise

The effect of increasing temperature from 22-25 degrees C to 37 degrees C on various motion characteristics of individual normal human spermatozoa and spermatozoa lacking the outer dynein arms (LODA) was studied by using a new automatic microscopic tracking method. It was found that: 1) The curvilinear velocity (Vc, measured between 1-3 sec) of both normal and LODA spermatozoa, fluctuated more or less intensely between spermatozoa; this fluctuation was not thermodependent. 2) The average Vc in the two groups of spermatozoa increased with the rise in temperature at a similar rate (1 micron/sec/degrees C), but LODA spermatozoa had an initial Vc lower than that of normal spermatozoa (12.5 +/- 5.3 microns/sec and 34.2 +/- 8.2 microns/sec, respectively). 3) The profile of the Vc increase associated with the temperature rise was different for the two groups of spermatozoa: for LODA spermatozoa it was linear between 25-37 degrees C, whereas for normal spermatozoa a plateau was reached at about 31 degrees C. 4) Various patterns of trajectory were found for both normal and LODA spermatozoa; these patterns were unrelated to temperature. However, LODA spermatozoa had more linear trajectories than normal spermatozoa. 5) Plots derived from reaction rate theory showed that the activation enthalpy, delta H was a function of the increase of Vc for both normal and LODA spermatozoa, but that delta H was higher for LODA spermatozoa.     

Movement of turritella spermatozoa: direction of propagation and chirality of flagellar bends.

The marine snail, Turritella communis, produces two types of spermatozoa, named apyrene and eupyrene. Eupyrene spermatozoa are usually paired, but unpaired ones are involved in fertilization. Movements of these spermatozoa were analyzed using a video camera with a high-speed shutter. The eupyrene spermatozoa usually swim with the head foremost but are able to swim flagellum foremost. A reversal of the direction of their swimming was found to be the result of a change in the direction of flagellar bend propagation, which changed with calcium concentration. Reversal of the direction of bend propagation was accompanied by a reversal of direction of the rotational movement of the spermatozoa around their long axis, suggesting that the bending waves keep the sense of their three-dimensional form. The swimming speed of apyrene spermatozoa in natural seawater was about one-eighth of that of the eupyrene ones and remained almost constant in highly viscous medium.The swimming speed of conjugated eupyrene spermatozoa was the same as that of unpaired spermatozoa over a wide viscosity range (<3,000 cP). No advantage of swimming by two spermatozoa could be detected in Turritella spermatozoa.     

Quantitative comparison of the passage of homologous and heterologous spermatozoa through the uterotubal junction of the golden hamster

A quantitative method was used to determine whether the spermatozoa of foreign species could pass through the uterotubal junction (UTJ) of the hamster as efficiently as homologous (hamster) spermatozoa. Estrous female hamsters were artificially inseminated with epididymal spermatozoa of homologous and heterologous (foreign) species. The number and distribution of spermatozoa in the oviduct were determined several hours after insemination (shortly before ovulation). The passage of immotile (dead) hamster spermatozoa through the UTJ was also examined. It was found that the spermatozoa of all foreign species tested (rat, mouse, guinea pig, and rabbit), as well as immotile hamster spermatozoa, could pass through the UTJ but did so in much smaller numbers compared to live hamster spermatozoa. This was not specifically due to poor survival of foreign spermatozoa in the hamster uterus, as the viability of all inseminated spermatozoa (including hamster spermatozoa) was considerably reduced by 1 h after insemination. While a large number of live hamster spermatozoa were distributed throughout the caudal isthmus at the time of examination, none or only a very few foreign spermatozoa had advanced this far. The few foreign and immotile spermatozoa that reached the caudal isthmus were confined to the first ascending loop of this segment. Some possible causes for the small number and retarded advance of foreign spermatozoa in the hamster oviduct were discussed.     

Failure of the expression of phospholipid hydroperoxide glutathione peroxidase in the spermatozoa of human infertile males

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) was intensely expressed in mitochondria in the midpiece of human spermatozoa by immunostaining with anti-PHGPx monoclonal antibodies. The PHGPx not only reduced phospholipid hydroperoxide but also scavenged hydrogen peroxide in human spermatozoa. We found a dramatic decrease in the level of expression of PHGPx in the spermatozoa of some infertile males by immunoblotting with anti-PHGPx monoclonal antibodies. These individuals accounted for about 10% of the group of 73 infertile males that we examined. All seven patients with PHGPx-defective spermatozoa were classified as suffering from oligoasthenozoospermia, a defect in which both the number and the motility of spermatozoa are significantly below normal. Males with PHGPx-defective spermatozoa accounted for 26% of the 27 infertile males with oligoasthenozoospermia. No defects in expression of PHGPx in spermatozoa were observed in 31 fertile volunteers. After a 3-h incubation, the relative number of motile spermatozoa with low-level expression of PHGPx was significantly lower than that of spermatozoa with normal expression of PHGPx. The PHGPx-defective spermatozoa failed to incorporate rhodamine 123, revealing a loss of mitochondrial membrane potential. Ultrastructual analysis of mitochondria by electron microscopy demonstrated that the morphology of mitochondria in PHGPx-defective spermatozoa was abnormal. The results suggest that failure of the expression of mitochondrial PHGPx in spermatozoa might be one of the causes of oligoasthenozoospermia in infertile men.     

Motility and penetration competence of frozen-thawed miniature pig spermatozoa are substantially altered by exposure to seminal plasma before freezing

The objective was to determine if exposure of spermatozoa to seminal plasma before freezing decreases its freezability, assessed by percentage motile cells (using computer-assisted semen analysis) and in vitro penetration ability (using in vitro fertilization and chlortetracycline fluorescence assessment). Ejaculated spermatozoa from miniature pigs were washed by centrifugation within 20 min after collection, then incubated in seminal plasma or modified Hulsenberg VIII diluents (mHM). When the spermatozoa were cryopreserved, spermatozoa incubated in seminal plasma before freezing had significantly lower post-thaw motility than spermatozoa incubated in mHM. The incubation of spermatozoa in seminal plasma also significantly prevented frozen-thawed spermatozoa from penetrating the oocytes. The second experiment, using unfrozen spermatozoa, was to determine if the incubation of spermatozoa with seminal plasma reduced penetration ability before freezing, resulting in a significantly lower penetration rate after freezing (compared with spermatozoa incubated without seminal plasma). The penetration competence of unfrozen spermatozoa was significantly decreased by incubation in seminal plasma, but no difference in motility was observed between spermatozoa exposed to seminal plasma versus mHM. We concluded that ejaculated seminal plasma contained some factor(s) that modified the sperm before freezing and reduced the freezability and post-thaw penetration competence of spermatozoa.     

Viability of ram testicular spermatozoa following cryopreservation in rete testis fluid

Ram testicular spermatozoa, collected continuously from the cannulated testis, were frozen in rete testis fluid in straws using the cryoprotective agents egg yolk and glycerol. The effect of cryopreservation on the viability of the spermatozoa was assessed by studying their metabolism, morphology, ultrastructure, and radioiodination patterns. Freeze-thawing significantly depressed the respiration rate and glycolytic activity of testicular spermatozoa. Morphologically, there was little evidence of cryodamage in frozen-thawed testicular spermatozoa. Except for some slightly corrugated acrosomes and a more loosely attached plasma membrane over the sperm head, frozen-thawed testicular spermatozoa were indistinguishable from nonfrozen control spermatozoa. Surface radioiodination of frozen-thawed testicular spermatozoa was highly selective and resulted in a labeling pattern similar to that of the nonfrozen controls. In contrast, the radiolabeling pattern of frozen-thawed electroejaculated spermatozoa was characterized by high background radioactivity and low selectivity. These results confirm previous suggestions that testicular spermatozoa have a greater low-temperature tolerance than do ejaculated spermatozoa and indicate that cryopreservation of immature testicular spermatozoa in rete testis fluid with added egg yolk and glycerol may be a useful approach to extend the availability of these cells.     

Effect of bovine sperm separation by either swim-up or Percoll method on success of in vitro fertilization and early embryonic development

The objectives of these experiments were to characterize separation of frozen-thawed bovine spermatozoa on a Percoll gradient and then to compare sperm separation by either a swim-up or Percoll gradient procedure for the ability of spermatozoa to fertilize oocytes in vitro. The Percoll gradient was a 45 and 90% discontinuous gradient. Initial experiments found that centrifugation of semen on the Percoll gradient for 15 min at 700 g was sufficient to obtain optimal recovery of motile spermatozoa. Most of the nonmotile spermatozoa were recovered at the interface of the 45 and 90% Percoll layers, while the motile spermatozoa were primarily in the sperm pellet at the bottom of the gradient. When frozen-thawed semen from each of 7 bulls was separated by swimup, a mean +/- SEM of 9% +/- 1 of the motile spermatozoa were recovered after the procedure. In contrast, more spermatozoa were recovered after Percoll gradient separation (P < 0.05), with 40% +/- 4 of the motile spermatozoa recovered. The effect of separation procedure on in vitro fertilization found swim-up separated spermatozoa penetrated a mean +/- SEM of 74% +/- 5 of the oocytes, while fewer oocytes were penetrated by Percoll separated spermatozoa at 52% +/- 8 (P < 0.05). There was no effect of the separation procedure on the rates of polyspermy as measured by sperm/penetrated ova, with a mean +/- SEM of 1.25 +/-.09 for swim-up separated spermatozoa and 1.14 +/-.07 for Percoll separated spermatozoa (P>0.05). A carry over of Percoll into the fertilization medium with the Percoll separated spermatozoa was found not the cause for the decreased penetration of oocytes by these spermatozoa. In 2 of 3 bulls tested, the decreased penetration of oocytes by Percoll separated spermatozoa could be overcome by increasing the sperm concentration during fertilization from 1 x 10(6) to 5 x 10(6)/ml. When development of embryos fertilized by either swim-up or Percoll separated spermatozoa was compared for the semen from 2 bulls, a difference in cleavage rate was found in favor of swim-up separated spermatozoa (P < 0.05), but there was no effect of separation procedure on development (Day 7) to the morula + blastocyst or blastocyst stage (P>0.05). The disadvantages of the Percoll procedure could easily be overcome and the procedure was faster and yielded a six-fold greater recovery of motile spermatozoa than the swim-up method.     

Validation of a rapid, large-scale assay to quantify ATP concentration in spermatozoa

Quantification of ATP content in spermatozoa is a useful assay for evaluating sperm function; however, most detection methodology relies on assessing single samples. We have developed and validated a highly repeatable assay that permits simultaneous measurement of up to 78 samples. A key feature of this assay includes combination of a phosphatase inhibition and ATP extraction step that permits maximal detection of ATP and sample storage at -20 degrees C prior to assay. The assay was validated for spermatozoa from three different species, including turkey, rooster and boar. The sensitivity of the assay differed between avian and mammalian spermatozoa, with 2.5 x 10(6) spermatozoa being the lowest number of turkey and rooster spermatozoa that could be assayed compared to 2.5 x 10(5) boar spermatozoa. Concentrations of ATP in fresh turkey semen ranged from 2.14 to 15.6 nmol/10(9) spermatozoa; similarly, freshly collected rooster semen contained from 2.16 to 21.4 nmol ATP/10(9) spermatozoa. Evaluation of turkey semen that had been stored at 4 degrees C for 24 h revealed a decline in ATP concentrations (2.35 +/- 0.34 nmol ATP/10(9) spermatozoa). Likewise, cryopreserved rooster spermatozoa contained lower concentrations of ATP (0.05 +/- 0.01 nmol ATP/10(9) spermatozoa) than non-stored spermatozoa. Boar spermatozoa contained similar concentrations of ATP, whether fresh (74.2 +/- 8.1 pmol ATP/10(6) spermatozoa), stored for 1 day (77.0 +/- 8.1 pmol ATP/10(6) spermatozoa) or 5 days (81.96 +/- 8.1 pmol ATP/10(6) spermatozoa). For all three species, assay variation was low (inter-assay, 0.66-1.9% CV; intra-assay, 1.3% CV).     

Assessment of Pisum sativum agglutinin in identifying acrosomal damage in stallion spermatozoa.

The use of fluorescein-conjugated Pisum sativum agglutinin (FITC-PSA) was evaluated for its ability to distinguish acrosome-intact from acrosome-damaged stallion spermatozoa. Incubation of fresh (acrosome-intact) and frozen-thawed (acrosome-damaged) spermatozoa with FITC-PSA resulted in acrosome-intact spermatozoa that exhibited no fluorescence, while acrosome-damaged spermatozoa exhibited fluorescent staining over the rostral portion of the head and equatorial segment. Experiments using mixtures of various ratios of acrosome-intact and acrosome-damaged spermatozoa determined the precision (intrasample coefficient of variation), and linearity (increased percentage of spermatozoa with PSA binding, with increased percentage of frozen-thawed spermatozoa in a sample) of FITC-PSA binding. The binding of FITC-PSA increased in samples as the portion of frozen-thawed (acrosome-damaged) to fresh (acrosome-intact) spermatozoa increased. A positive correlation existed (r = 0.98, P less than 0.05) between the percentage of FITC-PSA bound sperm and the proportion of damaged spermatozoa added to a sample. Location of PSA lectin binding on acrosome-damaged spermatozoa, determined by electron microscopy using gold-conjugated PSA, was to components of the outer acrosomal membrane and acrosomal matrix. These results demonstrate that FITC-PSA binding may be useful in determining acrosomal integrity of fresh and frozen-thawed stallion spermatozoa.     

Morphological changes of mouse spermatozoa during in vitro storage

In addition to normal spermatozoa, spermatozoa with bent and coiled tails, as well fragments of spermatozoa, such as single heads and tails and complexes of heads and tails, were found in a suspension of epididymal spermatozoa obtained from mice kept in the state of sexual deprivation for a long time. The dynamics of these elements was traced in the suspensions maintained at 10 and 35°C. At 37°C, the numbers of normal spermatozoa and spermatozoa with bent tails significantly decreased, while that of spermatozoa with coiled tails remained unchanged. At 10°C, the number of only spermatozoa with bent tails decreased. Based on the published data and our results, we propose that spermatozoa unclaimed for fertilization not only disintegrate into characteristic fragments, but also undergo a sequence of forms characteristic for the haploid phase of multicellular organism life cycle.     

Studies on the influence of aneuploidy of spermatozoa obtained from patients with oligozoospermia on their structure

The presence of aneuploidy in spermatozoa influences their biological characteristics, especially their ability to fertilise the ovum. The aim of the present study was to investigate if aneuploidy is accompanied by any changes in the morphology of spermatozoa in oligozoospermic patients. For this purpose, the percentage of aneuploid cells in sperm and the correlation between the specific morphological forms of spermatozoa and aneuploidy were evaluated. The study proved a negative correlation between DNA content of aneuploid and normal spermatozoa. A weak positive correlation was demonstrated between the presence of aneuploid spermatozoa and DNA content of spermatozoa with large heads. No such correlations could be detected for DNA content of the remaining morphological forms of spermatozoa. Thus, men with a lowered number of spermatozoa and/or with abnormal spermatozoal morphology should have their spermatozoal DNA content tested in order to evaluate the degree of aneuploidy, especially in cases where in vitro fertilisation is intended.     

Effects of cryopreservation on Ca2+ signals induced by membrane depolarization, caffeine, thapsigargin and progesterone in boar spermatozoa

Although the fertilizing ability of spermatozoa is greatly reduced after freezing, complete understanding of alterations induced by cryopreservation has not been elucidated. The present study evaluates the effects of cryopreservation on the Ca2+ handling of boar spermatozoa using several sperm activators. Intracellular Ca2+ signals from single spermatozoa were measured using confocal Ca2+ imaging of unfrozen samples and of other spermatozoa after having been frozen. Elevation of the external K+ concentration elicited a three times larger Ca2+ increase in fresh spermatozoa than in cryopreserved spermatozoa. Caffeine elicited Ca2+ transients with some oscillations in the fresh spermatozoa, but not in the thawed spermatozoa. Depletion of the Ca2+ store with thapsigargin induced a rapid rise in Ca2+ in the control but generated a smaller increase of Ca2+ after thawing. Exposure to progesterone induced a biphasic rise of the Ca2+ level in the fresh spermatozoa only. Sperm viability was reduced by cryopreservation. Resting Ca2+ levels in fresh and cryopreserved spermatozoa were similar. Longer incubation (2.5 h) of thawed spermatozoa partly recovered the Ca2+ response to the interventions. These results suggest that cryopreservation reduces the responsiveness of spermatozoa to depolarization, modulators of the internal Ca2+ store and progesterone in terms of the Ca2+ signal, thus providing a possible mechanism for reduced fertility observed in cryopreserved boar spermatozoa.     

Respiration and Motility in Starfish Spermatozoa at Various Times in the Breeding Season

In the spermatozoa of Asterias amurensis , patterns of changes in the respiratory rate and motility following dilution of dry sperm in sea water varied among batches and were classified into three types. The type I spermatozoa were immotile and exhibited quite low respiratory rate. Ethylenediamine tetraacetic acid made the type I spermatozoa motile and elevated their respiratory rate. The type II spermatozoa were also immotile and the respiratory rate remained quite low for about 5 min after the dilution. Thereafter, they spontaneously became motile and the respiratory rate increased. The type III spermatozoa became motile upon their dilution and exhibited high respiratory rate. Differences in the motility and respiratory rate of spermatozoa among batches probably result from degree of their maturation. In moving spermatozoa, the ADP and AMP levels increased at the expense of ATP. 2,4-Dinitrophenol elevated the respiratory rate only in immotile spermatozoa, which showed a high ATP level and quite low ADP level, but did not made them motile. Oligomycin inhibited the respiration of both motile and immotile spermatozoa. Probably, the respiratory rate is made low by a shortage of ADP in immotile spermatozoa and is enhanced by ADP production due to the initiation of their movement.     

The fate of the normal-anucleated spermatozoa in inseminated females of the silkworm Bombyx mori

Both typical (haploid) and atypical (anucleated) spermatozoa reach the receptaculum seminis of inseminated females of Bombyx mori intermingled. However, only typical spermatozoa both leave the receptaculum and fertilize the eggs. Atypical spermatozoa, which are in fact anucleated flagellar apparatuses, probably function in transporting typical fertilizing spermatozoa to the receptaculum seminis. In the male ejaculatory duct both kinds of spermatozoa are wrapped with extra-cellular sleeves that presumably protect them on their way to the receptaculum. Typical spermatozoa “hatch” from the sleeves before leaving the receptaculum to fertilize the eggs. The presence of a centriole in the extra-testicular spermatozoa of this species supports the generalization that insect spermatozoa do have a centriole at the base of the flagellum.     

Staining procedure to detect viability and the true acrosome reaction in spermatozoa of various species

A simple dual stain procedure (DS) for simultaneously determining sperm viability and acrosomal status is described. The DS includes the use of the vital stain trypan blue to detect live and dead spermatozoa and Giemsa to detect the presence or absence of an acrosome. For staining, spermatozoa are washed, incubated with trypan blue, washed, dried onto slides, and subjected to Giemsa. Dead spermatozoa stain blue in the postacrosomal region while live spermatozoa remain unstained. The acrosome stains light purple–dark pink while acrosome-free sperm remain unstained. This staining pattern enables differentiation of spermatozoa which have undergone a true acrosome reaction (TAR) from those which have undergone a false acrosome reaction (FAR). Incubation of bull, boar, ram, and stallion spermatozoa for 60 minutes at 37°C in the presence of calcium ionophore A23187 increased the proportion of spermatozoa undergoing a TAR in all species except the stallion. Incubation of bull spermatozoa for up to 24 hours at 37°C resulted in a decrease over time in the percentage of live acrosome-intact spermatozoa and a simultaneous increase in the percentage of spermatozoa categorized as having undergone a TAR and FAR. The DS could be a useful technique in evaluating sperm viability and acrosomal status in fertilization and clinical studies.     

Motility characteristics and membrane integrity of cryopreserved human spermatozoa.

The motility characteristics of washed spermatozoa from 50 normal ejaculates were measured by time-lapse photography, before and after cryopreservation. Plasma membrane integrity was assessed by the hypo-osmotic swelling test and with the supravital fluorescent dye bisbenzimide (H33258). There was a marked decline in the percentage of progressively motile spermatozoa after cryopreservation, the extent varying widely among donors. Results were, however, consistent between different ejaculates from the same individual. The ability of spermatozoa to survive cryopreservation could not be predicted from the properties of the semen beforehand. The mean velocity of the spermatozoa was significantly reduced after freezing, but the lateral head displacement was unaltered. There was a significant reduction in the proportion of spermatozoa with intact plasma membranes after cryopreservation and the results of the hypo-osmotic swelling test and H33258 tests correlated closely. There was no correlation between the declines in the percentage of motile spermatozoa, or intact spermatozoa and the sperm velocity. We conclude that membrane rupture is not the sole cause of loss of motile spermatozoa during freezing and that the decrease in the proportion of motile spermatozoa is caused, at least in part, by a separate process from that responsible for the decrease in the average swimming speed of spermatozoa.