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.     

Prolonged survival of mouse epididymal spermatozoa stored at room temperature

Summary: The viability and fertility of isolated mouse epididymal spermatozoa kept for up to 7 days at various temperatures (4°C, 22°C, and 37°C) were determined. Spermatozoa kept for 3 days at 22°C were still active, while those kept at 37°C or 4°C exhibited great reduction in motility within 2 days after isolation. In vitro fertilizing abilities of spermatozoa left for 0, 1, 2, and 3 days at 22°C were 69.2, 32.5, 9.5, and 4.9%, respectively, when the cleavage rate to two‐cell stage was examined. Transfer of two‐cell embryos produced in vitro with spermatozoa left for 1, 2, and 3 days at 22°C resulted in production of fetuses with efficiencies of respectively 30.2, 11.5, and 16.7%, which were lower (63.3%) than that of embryos derived from in vitro fertilization with fresh spermatozoa. These findings indicate that spermatozoa kept for up to 3 days at 22°C can fertilize oocytes, although at relatively low efficiency. genesis 31:147–155, 2001. © 2001 Wiley‐Liss, Inc.     

Biochemical changes in bull spermatozoa during capacitation in vitro

To evaluate the metabolic changes of bull spermatozoa (SPZ) during capacitation in vitro, SPZ were incubated for 0, 5 or 10 hours in the presence (co-culture) and absence (control) of monolayers of bovine oviduct epithelial cells, which promote capacitation-like changes in vitro. There was little change in the oxygen uptake of the SPZ after 5 hours, but after 10 hours there was a decrease, particularly in the co-cultured sample. After 5 hours there was little change in the cyclic adenosine monophosphate (cAMP) concentration of the co-culture or control SPZ, but by 10 hours the levels of cAMP decreased in both the co-cultured and control SPZ (P=0.06). The concentration of adenosine triphosphate (ATP) was somewhat decreased after 5 hours in both the co-cultured and control SPZ and the percentage of decline was much higher after 10 hours. Overall, there was no significant change in oxygen uptake or cAMP and ATP levels specifically associated with capacitation of bull SPZ.     

Assessment of fresh and stored duck spermatozoa quality via in vitro sperm-egg interaction assay

An in vitro sperm-egg interaction assay was used to measue the quality of duck spermatozoa in fresh and stored semen. The inner perivitelline layer (IPVL), which had been separated from laid duck eggs, was incubated with spermatozoa in vitro. The number of points of sperm hydrolysis in the IPVL in vitro was logarithmically correlated with the fertility of the eggs laid by inseminated females, for both fresh semen (r = 0.85, P < 0.001) and stored semen at 5 degrees C for 24 h (r = 0.84, P < 0.001). After semen storage, the ability of spermatozoa to hydrolyze the IPVL decreased by 67.4% compared with the values for fresh semen, whereas egg fertility and sperm motility decreased by 47.8% and 15.2%, respectively. These results suggest that the in vitro sperm-egg interaction assay accurately reflects the fertilizing ability of fresh and stored duck spermatozoa and detects spermatozoal damage due to semen storage more sensitively than motility or fertility tests.     

In vitro survival of bovine spermatozoa stored at room temperature under epididymal conditions

In this study, environmental conditions mimicking those prevailing in the epididymis were used for storing ejaculated bull spermatozoa in vitro during 4 days at ambient temperature. These conditions were low pH, high osmolarity, high sperm concentration and low oxygen tension. Hepes-TALP was used as basic storage medium. Fresh spermatozoa were stored at a concentration of 10 x 10(6)spermatozoa/ml in Hepes-TALP of different pH (pH 4, 5, 6, 7 or 8), and osmolarity (100, 300, 400, 500, 600 or 800 mOsm/kg), and under different atmospheric conditions (nitrogen gassed or aerobic). Spermatozoa were also stored undiluted or at different concentrations: 10x 10(6), 100 x 10(6), 500 x 10(6) or 1 x 10(9)spermatozoa/ml. Sperm parameters such as membrane integrity, motility, mitochondrial membrane potential or DNA fragmentation were used to assess semen quality after storage. Adjustment of the pH of Hepes-TALP to pH 6 yielded significantly better results than storage at all other pH values. Isotonic Hepes-TALP (300 mOsm/kg) had a less detrimental effect on spermatozoa than hypo- and hyperosmotic versions. No differences in sperm parameters were observed when spermatozoa were incubated under aerobic or under nitrogen gassed storage conditions. Optimal sperm concentration in vitro is 10 x 10(6)spermatozoa/ml. This is in contrast with the in vivo situation, where spermatozoa are stored at high concentration. However, better results at high sperm concentrations were obtained when spermatozoa were diluted for less than 5 min in Triladyl-egg yolk-glycerol diluent immediately after ejaculation.     

Capacitation-like changes occur in mouse spermatozoa cooled to low temperatures

Previously we showed that > 70% of mouse spermatozoa cooled slowly from 37°C to 4°C and warmed have undergone capacitation-like changes as examined by a chlortetracycline staining assay. These membrane changes are reflected in the ability of cooled spermatozoa to achieve fertilization rates in vitro similar to those of uncooled controls when added to oocytes immediately upon warming. The aim of this study was to determine the nature of these membrane changes. We found they were not dependent upon the rate of cooling to 4°C and similar changes were observed when spermatozoa were cooled to higher temperatures (10° and 20°C), but it took longer for 50% of the spermatozoa to undergo such changes (3, 18, and 27 min for spermatozoa held at 4°, 10°, and 20°C, respectively). Mixing cooled spermatozoa with oocytes immediately upon warming produced fertilization rates similar to fresh spermatozoa capacitated in vitro for 90 min before the oocytes were added. The rate of sperm penetration as determined by the fluorescent DNA stain Hoescht 33258 was also similar. However, the penetration time for cooled spermatozoa was significantly shortened when they were preincubated for 90 min before being added to oocytes. We conclude that membrane changes resembling capacitation (1) occur during cooling to temperatures above freezing, (2) are independent of cooling rate, (3) proceed faster at lower temperatures, and (4) obviate the need for prior capacitation in vitro before mixing with oocytes. Mol. Reprod. Dev. 46:318–324, 1997. © 1997 Wiley-Liss, Inc.     

Dibutyryl cyclic AMP decreases capacitation time in vitro in mouse spermatozoa

Epididymal mouse sperm suspensions were preincubated for various times in medium containing glucose and/or dibutyryl cyclic AMP and then assessed for fertilizing ability in vitro, loss of the acrosome and motility changes. Capacitation time was significantly reduced by exposure to glucose and 0.1 mM-dbcAMP for 30 min as evidenced by early and synchronous fertilization of eggs, compared with glucose alone. Although this was accompanied by a precocious development of whiplash motility, the rate of acrosome loss in isolated sperm suspensions was not accelerated by the presence of exogenous cyclic nucleotide. Exposure of spermatozoa to 1 mM-dbcAMP in the presence of glucose resulted in very poor fertilization, but the effect could be prevented by withholding glucose until eggs were introduced; this may be due to free butyrate in the system since the inclusion of 1 mM-butyrate in glucose-containing medium had a similar inhibitory effect. Although cyclic nucleotide supported the acrosome reaction but not motility changes, no fertilization was obtained unless zonae were removed, when a low level of fertilization (30%) was observed. Both whiplash motility and acrosome loss are thus obligatory for sperm penetration of the zona and glycolytic metabolism supports both changes, perhaps by promoting endogenous generation of cyclic AMP to act as an intermediary in these two distinct phenomena.     

Cyclic nucleotide metabolism in mouse epididymal spermatozoa during capacitation in vitro

The role of cyclic nucleotides in sperm capacitation is equivocal. Using conditions known to support mouse sperm capacitation after 120 min incubation in vitro, the cAMP and cGMP contents of epididymal spermatozoa were measured and the cGMP/cAMP ratio determined. The initial high cAMP content detected upon release of spermatozoa decreased within 30 min to a lower plateau, which was then maintained throughout incubation. With the cGMP content remaining approximately constant, the cGMP/cAMP ratio increased over 120 min. In the presence of 2 mM caffeine, an increased cAMP content was noted at 0 and 30 min before a fall to the plateau level. To investigate cyclic nucleotide metabolism, adenylate cyclase and phosphodiesterase activities were compared in two sperm populations, one essentially uncapacitated and the other incubated for 120 min. Adenylate cyclase activity, higher in the presence of 2 mM Mn²⁺ compared to Mg²⁺, showed increased activity at 120 min compared to 30 min incubation, while phosphodiesterase activity decreased during this period. The ability of spermatozoa to form adenosine and inosine from cAMP indicated endogenous 5′-nucleotidase and deaminase, as well as phosphodiesterase, activities. Although the endogenous cAMP content appeared to remain constant during the time that acrosome loss, hyperactivated motility and fertilizing ability can be demonstrated, activities of the enzymes responsible for cAMP metabolism indicate an increased potential for cAMP availability and turnover. The increased cGMP/cAMP ratio may also play a role during capacitation.     

Freezing of stored, chilled dog spermatozoa

The aims of this study were to find out if dog spermatozoa can be stored chilled for 1 or 2 days prior to freezing without a deterioration in post-thaw vitality and longevity, and to compare two extenders; the Uppsala Equex-2 (UE-2) and a TRIS egg yolk extender (EYT). Pooled dog semen was frozen immediately after collection, or was extended and stored at 4 degrees C for 1 or 2 days before freezing. Sperm motility and acrosome integrity were evaluated before freezing and for 6h post thaw at 38 degrees C, while sperm plasma membrane integrity was evaluated post thaw. There were no effects of pre-freeze storage time or extender on post-thaw motility or plasma membrane integrity, but a significant effect of extender (P < 0.0153) on post-thaw acrosomal integrity was found, UE-2 being better than EYT. There was a significant (P < 0.0001) negative effect of post-thaw storage time on acrosome integrity, but this was not influenced by pre-freeze storage time or extender. In conclusion, we found that dog spermatozoa can be frozen after 1 or 2 days of cold storage without significant deterioration in post-thaw motility, acrosome integrity or sperm plasma membrane integrity compared to when frozen immediately after collection. The UE-2 extender was superior to the EYT extender for freezing of cold stored dog spermatozoa.     

Effect of sperm coating on the survival and penetrating ability of in vitro stored bovine spermatozoa

The aim of this study was to examine the effect of sperm coating on the survival and penetrating ability of in vitro stored diluted spermatozoa. Bovine semen was collected by means of an artificial vagina connected with a tube containing 5 ml of the commercial Triladyl diluent supplemented with 20% egg yolk and 6.7% glycerol (EYTG). Both EYTG and seminal plasma were removed by centrifugation and the spermatozoa were stored under different in vitro storage conditions. In the first and second experiment, "control" and "coated" spermatozoa were stored in Hepes-TALP (pH 6 and 7) at room temperature. After 4 days of storage, the progressive motility, membrane integrity, mitochondrial membrane potential or DNA integrity of the spermatozoa were evaluated before and after Percoll centrifugation. The in vitro penetration rate of the spermatozoa was examined only after Percoll centrifugation. A significantly (P<0.05) positive influence of sperm coating was observed on the tested sperm characteristics and penetration rate of spermatozoa when they were stored in Hepes-TALP at pH 7, but not at pH 6. In the last experiment, the influence of the storage medium Hepes-TALP (pH 7) or EYTG was investigated on motility, membrane integrity, mitochondrial membrane potential and in vitro penetration potential of "coated" spermatozoa stored at room temperature or at 4 degrees C during 4, 5 and 6 days. After 6 days of storage, a significantly (P<0.05) higher percentage of motile and membrane intact spermatozoa with high mitochondrial membrane potential was obtained in EYTG at both temperatures leading to a significantly higher in vitro penetration rate. These results indicate that sperm coating could preserve sperm characteristics and penetrating capacity of fresh bovine spermatozoa stored in egg yolk containing diluent for up to 6 days.     

Micronucleus formation in 2-cell embryos after in vitro X-irradiation of mouse spermatozoa

Mouse spermatozoa were exposed in vitro to various X-ray doses and added to a medium containing superovulated oocytes. The percentage of fertilized oocytes was determined 24 h after sperm addition and 2-cell embryos were investigated for micronucleus formation. The fertilization rate was drastically decreased after exposure to 470 cGy whereas smaller doses had no effect. The dose-response relationship for micronucleus formation per embryo was linear-quadratic and the number of embryos with micronuclei increased linearly with dose. The distribution of micronuclei among 2-cell embryos showed a significant overdispersion relative to the Poisson distribution after sperm irradiation for doses above 188 cGy.     

Cryopreservation and ensuing in vitro fertilization ability of boar spermatozoa from epididymides stored at 4 degrees C

The influence of prolonged storage of boar epididymides on post-thaw sperm motility, and in vitro fertilization was evaluated. Twenty pairs of epididymides were obtained from Large White boars, and spermatozoa from one of each of the pairs were immediately collected and frozen (control group). The remaining epididymides were cooled to 4 degrees C and stored for 1, 2 or 3 d, after which spermatozoa were collected and frozen (experimental groups Day 1, 2 and 3, respectively). Sperm motility was maintained throughout the dilution procedure and then dropped (P < 0.01) after freezing and thawing. During storage the motility of nonfrozen spermatozoa decreased significantly (P < 0.01), reaching a value equal to that of frozen-thawed spermatozoa on Day 3. In vitro fertilization experiments revealed significantly (P < 0.05) lower penetration rates using Day 1, 2 and 3 stored spermatozoa (12, 13 and 2%, respectively) than that of the control group (40%). Oocyte penetration ability seemed to be reflected by acrosome integrity. However, the motility of spermatozoa with the ability to penetrate oocytes in Day 1 and Day 2 groups did not differ from that of the controls. The motility of spermatozoa lacking penetration ability, on the other hand, gradually decreased as the storage period was prolonged. This suggests that the sperm motility and penetration ability are affected by different mechanisms during the cold storage of epididymides. Finally, control and experimental groups exhibited high incidences of monospermic penetration (64 to 90%) and of male pronuclear formation (67 to 71%). These data suggest that cryopreservation of spermatozoa from boar epididymides stored at 4 degrees C for 1 to 2 d can be used for conserving male germ cells when epididymal spermatozoa can not be collected immediately and cryopreserved.     

Effect of different fractions of seminal plasma on the fertilizing ability of fowl spermatozoa stored in vitro.

This paper describes the effects of whole seminal plasma and of dialysed seminal plasma on the fertilizing ability of fowl spermatozoa stored for 24 h at 4 degrees C. The fertilizing ability of fowl semen diluted 1:1 with Beltsville Poultry Semen Extender and stored for 24 h at 4 degrees C was enhanced after replacement of the homologous seminal plasma by the diluent (89 versus 77% fertilization rate). Better results were obtained with seminal plasma dialysed against water before sperm storage to discard the less than 1 kDa or the less than 50 kDa fractions. It was concluded that low molecular weight seminal plasma fractions could damage the fertilizing ability of spermatozoa during storage at 4 degrees C, whereas high molecular weight fractions appeared to enhance fertilizing ability.     

Apoptotic-like changes in the spermatozoa of fresh and stored boar semen and the quality of embryos produced in vivo

The aim of this study was to determine the apoptotic-like changes in the spermatozoa of fresh and stored boar semen and to investigate the relationship between this phenomenon and the quality of embryos produced in vivo. The experiments were divided into two series. In the first series, ten ejaculates were collected from five boars, which were crossbreeds of the Polish Landrace and Large White breeds. The semen was stored as a liquid until Day A (the day on which sperm motility decreased to 30%). Three fluorescence methods were used to evaluate semen quality: an assay to assess the early changes in sperm membrane integrity using the fluorophore YO-PRO-1, an assay for phosphatidylserine (PS) translocation across the plasma membrane using fluorescein-labeled annexin-V and the mitochondrial-specific probe JC-1 (5,5',6,6'-tetrachloro-1,1',3,3' tetraethylbenzimidazolylcarbocyanine iodide) for measuring changes in mitochondrial membrane potential. Our results showed that liquid preservation of boar semen causes apoptotic-like changes in the sperm, and a significant increase in both: apoptotic sperm (YO-PRO-1(+)/PI(-)) and early apoptotic sperm (annexin-V(+)/PI(-)) were observed between Day 0 (fresh semen) and Day A only in semen from three of the five boars. In the second series of experiments, the semen from boar nos. 1, 2, and 3 was selected for insemination of superovulated gilts. The fertilizing capacity of fresh and stored semen with different levels of apoptotic spermatozoa was measured based on the morphology and the number of cells of embryos that were obtained after insemination with this semen. Our studies indicated no significant differences in the fertilization rate of gilts after insemination with fresh and stored semen with increased levels of apoptotic spermatozoa. After insemination with stored semen, a significantly greater number of degenerated embryos were observed, but the morphologically normal blastocysts obtained after insemination with either fresh or stored semen had a similar number of nuclei.     

Cryopreservation of mouse spermatozoa

Recently, it has become possible to freeze a large number of mouse spermatozoa immediately after collection from the epididymides of a small number of males. The cryopreservation of spermatozoa is simpler, less time-consuming, and less costly than that of embryos for maintaining various strains of mice with induced mutations. This chapter attempts to provide a realistic overview of the cryopreservation of mouse spermatozoa and to describe a detailed procedure for mouse sperm freezing. Received: 16 December 1999 / Accepted: 17 December 1999     

Functional changes and motility characteristics of Japanese Black bull spermatozoa separated by Percoll

Spermatozoa from two Japanese Black bulls (Bull-ATF and Bull-KTG) were separated by centrifugation at 700 x g for 15min in modified TALP with or without 45-90% Percoll. Control washed spermatozoa and those collected from the bottom of 45 and 90% Percoll fractions were examined for viability and membrane integrity (using Hoechst bis-benzimide 33258 or propidium iodide and 6-carboxyfluorescein diacetate (PI-CFDA)), acrosomal status (using fluorescence isothiocyanate (FITC) conjugated Pisum Sativum agglutinin (PSA) and Peanut agglutinin (PNA), Naphthol Yellow S and Erythrosin B (NE) or triple staining (TS)), capacitation status (using chlortetracycline (CTC)), motility characteristics (using a computer-assisted sperm motion analysis system (CASA)) and for in vitro fertility. Percoll-separated spermatozoa showed greater viability and membrane integrity than controls, as determined by supravital staining. Differences were observed in the results regarding viability and acrosomal status of spermatozoa among sperm staining methods. Bull-ATF, which showed significantly greater in vitro fertility than Bull-KTG (P<0.05), showed a significantly higher rate of CTC-B-pattern (capacitated) spermatozoa (P<0.01) than Bull-KTG. The motility characteristics of control washed spermatozoa and those separated by 45-90% Percoll were analyzed by CASA. More motile and progressively motile spermatozoa were observed in the fraction at the bottom of the 90% Percoll solution than in the 45% Percoll fraction or in controls (P<0.01). Moreover, the spermatozoa of Bull-KTG, which showed lower in vitro fertility than Bull-ATF, did not show significant differences in motility from those of Bull-ATF. These results provided basic information about Japanese Black bull spermatozoa, and suggested that spermatozoa with greater motility and viability can be obtained by Percoll separation than without separation. However, Percoll separation did not enhance their in vitro fertility.