Prolonged motility of fowl spermatozoa in vitro due to a low molecular weight factor(s) released from cultured embryonic cells

Fowl spermatozoa were incubated at 41°C in a supernatant removed from a 4-day culture medium of embryonic chick skeletal muscle cells. Their motility, as assessed at room temperature (20–25°C), was maintained for 48 h. Fertilizing ability was also retained for at least 24 h. In contrast, spermatozoa incubated in the fresh culture medium lost their motility and fertilizing ability rapidly. A filtrate of the 4-day culture medium, obtained by passing the fluid through an Amicon PM-10 ultrafiltration membrane, prolonged the motility of spermatozoa. These results suggested that a low molecular weight factor(s) (mol. wt. < 10 000) supplied by the cultured cells effectively prolonged the motility of spermatozoa.     

Maintenance of motility of fowl spermatozoa in vitro is prolonged by a low molecular weight factor derived from cultured chick embryo cells.

Gel filtration of a conditioned medium composed of the supernatant fluid removed from a 5-day culture of skeletal muscle cells from 9-day-old chick embryos with Bio-Gel P-2 revealed one peak of motility-prolonging activity (about 0.3 kDa), which was not present in fresh medium. Spermatozoa incubated in this fraction of the conditioned medium maintained their motility for at least 36 h at 37 degrees C. Both the formation of lipid peroxide and the leakage of lactic dehydrogenase of spermatozoa incubated in the conditioned medium fraction were lower than those incubated in the corresponding fresh medium. Initial rate of oxygen consumption of the spermatozoa incubated in the conditioned medium fraction increased compared with that of the fresh medium fraction. These results suggest that a low molecular weight factor(s) supplied by cultured cells effectively prolongs the motility of fowl spermatozoa, and that the effect could result from inhibition of the structural damage to the sperm membrane.     

Characterization of secretory proteins from cultured cauda epididymal cells that significantly sustain bovine sperm motility in vitro

Epididymis provides a safe environment in which stored-spermatozoa could survive for days before ejaculation. In vitro studies suggested that epididymal proteins seem to be implicated in sperm survival during coincubation with cultured epididymal cells. This study was basically designed to confirm if secretory proteins from bovine epididymal cell cultures provide sperm protection against rapid loss of sperm motility in vitro. Bovine spermatozoa were incubated in conditioned media (CM), which were prepared from cultured cauda epididymal cell (CEC). Motion parameters were recorded using a computer-assisted sperm analyzer. Sperm-free protein extracts from CM were fractionated by ultrafiltration through a 10-kDa cut off membrane. A significantly positive effect on sperm motility was observed when spermatozoa were incubated in CM (54 +/- 4%) and CM > 10 kDa (57 +/- 4%) compared to CM < 10-kDa fraction (30 +/- 3%) or fresh media (34 +/- 3%), after a 6-hr incubation period. This beneficial effect on sperm motility was abolished when the CM > 10-kDa fraction was heat-treated at 100 degrees C for 10 min. The CM > 10 kDa fraction provides factors that remained active even though spermatozoa were washed twice after a 2-hr preincubation period. To identify potential beneficial factors, bovine spermatozoa were incubated with radiolabeled proteins obtained using (35)S-methionine in culture medium. SDS-PAGE analysis of proteins extracted from CM-preincubated spermatozoa revealed the presence of a 42-kDa protein strongly associated to the sperm surface. This 42-kDa spot was trypsin-digested and identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) as a protein homologue to a 35-kDa bovine estrogen-sulfotransferase. This protein can play a role in epididymal biology and sperm function. Taken together, these results suggest that specific epididymal proteins can be implicated in the sperm protection in vitro, and can be characterized in our cell culture system.     

Bovine oviduct-specific glycoprotein: A potent factor for maintenance of viability and motility of bovine spermatozoa in vitro

In the cow, a specific glycoprotein-bovine oviduct-specific glycoprotein (BOGP)-is secreted by the epithelial cells of the oviduct at the follicular stage of the estrous cycle. In this study, we examined the effects of purified BOGP on the viability and motility of bovine spermatozoa in culture in vitro. Frozen-thawed bovine spermatozoa were incubated in modified Tyrode's solution (TALP) that contained purified BOGP (TALP-BOGP). In TALP-BOGP, both the viability and motility of bovine spermatozoa were more effectively maintained than in the control medium without any added protein. The increases in both the viability and motility of spermatozoa were dose-dependent. Spermatozoa were also incubated in TALP medium supplemented with bovine serum albumin, egg albumin, lactalbumin, or gastric mucin, and their viability and motility in these media were compared with that in TALP-BOGP. Both the viability and motility of spermatozoa were more effectively maintained in TALP-BOGP throughout a 12-hr incubation than in other media tested. An immunolabeling study demonstrated that a monoclonal antibody specific for BOGP reacted with the posterior region of the head, the middle portion, and the tail of spermatozoa that had been incubated with TALP-BOGP, suggesting that BOGP becomes specifically associated with particular regions of the spermatozoon. These results suggest that BOGP is a potent factor for maintenance of the viability and motility of sperm. On the basis of the present results, we also propose that BOGP may play an important role in sperm functions during the reproductive process. © 1995 wiley-Liss, Inc.     

Stimulation of sperm motility and oxygen consumption of fowl spermatozoa by a low molecular weight fraction of seminal plasma

Washed fowl spermatozoa were incubated in a phosphate buffer containing various concentrations of fowl seminal plasma at 41 degrees C, normal body temperature, and the motility and oxygen consumption of spermatozoa were determined. Immediately after the incubation, spermatozoa showed good motility in the various diluents. However, with concentrations of seminal plasma at or below 20%, spermatozoa quickly became immotile. In contrast, at concentrations higher than 40% seminal plasma, spermatozoa were motile even after 15 min. As the concentration of seminal plasma was increased, oxygen consumption of spermatozoa also increased. A filtrate of the seminal plasma, obtained by passing the fluid through an Amicon YM-2 ultra-filtration membrane (Mr less than 1000), also stimulated the motility and oxygen consumption of spermatozoa. These results suggest that some low molecular weight factor(s) in fowl seminal plasma stimulated motility and oxygen consumption of fowl spermatozoa at 41 degrees C. A physiological role of this factor(s) may be to assist passage of spermatozoa through the vagina after natural mating.     

Mechanism for procaine-mediated hyperactivated motility in guinea pig spermatozoa

Hyperactivated motility was studied in guinea pig spermatozoa. In the presence of the local anesthetic procaine, a high number of sperm cells (64%) showed hyperactivation when incubated in minimal culture medium with pyruvate, lactate, and glucose. Hyperactivated motility was dependent on glucose in the medium. Sperm ATP concentration was increased twofold in hyperactivated sperm when compared to procaine-treated nonhyperactivated cells. cAMP levels were also higher in hyperactivated cells than in control spermatozoa. Thus, in living spermatozoa high levels of ATP appear to be needed to generate hyperactivation. cAMP is present at a high concentration in hyperactivated spermatozoa, therefore a role of cAMP in hyperactivation cannot be excluded. Depletion of external Ca²⁺ did not inhibit procaine-induced hyperactivated motility. Hence, procaine canceled the requirement of external Ca²⁺ for sperm to express hyperactivated motility. © 1994 Wiley-Liss, Inc.     

In-vitro development of the fertilizing ability of hamster epididymal spermatozoa after co-culture with epithelium from the proximal cauda epididymidis

The epididymides of adult male hamsters were surgically ligated at the junction of the distal corpus and proximal cauda regions. After 3 days, spermatozoa recovered from the distal corpus displayed greater progressive motility and head to head agglutination in capacitating medium than did those from intact controls, but had low fertilizing ability (3% fertilization rate) in vitro or in vivo. When these spermatozoa were incubated for 6 h with epithelial cells from the proximal cauda epididymidis, previously cultured for 3 days, they maintained motility and exhibited a significant increase in fertilizing ability (30% and 29% in vitro and in vivo respectively). The fertilizing ability of distal corpus spermatozoa incubated with 3-day-old cultures without androgens, or 8-12-day-old epithelial cells with fibroblast overgrowth, or without epithelial cells, remained low (5%). Increase in sperm fertilizing ability was associated with increased sperm binding to the zona pellucida in vitro. These results demonstrate that, under suitable culture conditions, the final stages in the development of hamster sperm fertilizing ability can be achieved in vitro. Factors secreted by cultured epithelium from the proximal cauda epididymidis are implicated in this maturation process.     

Comparison of the longevity of motility of stallion spermatozoa incubated at 38 degrees C in different capacitating media and containers

This study was designed to compare the effects of different media and containers on longevity of motility of spermatozoa during in vitro incubation at 38 degrees C in either air or 5% CO2 atmosphere. Three ejaculates were collected from each of 4 stallions. The media tested were skim milk-glucose, modified Krebs/Ringer and Hank's salts solution for incubation in an air atmosphere, and modified Krebs/Ringer and Brackett and Oliphant (BO) defined medium for incubation in a 5% CO2 atmosphere. All samples were incubated in 5-mL borosilicate glass tubes filled with 3 mL of extended spermatozoa, 5-mL borosilicate tubes filled with 6 mL (topped) of extended spermatozoa, 35-mm Petri dishes filled with 3 mL of extended spermatozoa, and 35-mm Petri dishes with 200-microL microdroplets of extended spermatozoa under sterile mineral oil. For all treatments, individual samples were removed at 2, 4, 6 and 12 h of incubation to determine the percentage of motile cells. Overall, spermatozoa incubated in Petri dishes in both 3-mL and microdroplet treatments had significantly higher motility than those incubated in glass tubes (P<0.01). At 6 and 12 h of incubation in Petri dishes, progressive motility was significantly higher for spermatozoa extended in the Hank's salts solution than in the other media. Both the medium and container used significantly affected the longevity of motility of spermatozoa incubated at 38 degrees C.     

ATP Content and Sperm Motility of Extended Bovine Semen under Different Storage Conditions

The effects of incubation temperature (+20°C vs +35°C) and media type on the ATP content and motility of spermatozoa were determined in fresh bovine semen in order to develop a method for assaying post-thaw quality. Semen was obtained from 3 bulls at 2 occasions. The spermatozoa were washed using a Ficoll-containing medium before being resuspended in each of 4 different media (I. 0.9 % NaCl; II. Trisbuffer solution; III. seminal plasma; IV. seminal plasma + Tris-buffer solution) and incubated for 6 h. The least-squares means for ATP content were higher (p ≤ 0.05) at +20°C than +35°C for all media except no. I. By contrast, the least-squares means for sperm motility were higher (p ≤ 0.05) at +35°C than at +20°C in media II and III. A decrease over time in ATP content and motility at both temperatures was also observed. The single most important factor responsible for changes in ATP content and sperm motility was the temperature and the medium, respectively.     

Expansion of human embryonic stem cells in defined serum-free medium devoid of animal-derived products

Human embryonic stem cells (hESCs) can serve as an unlimited cell source for cellular transplantation and tissue engineering due to their prolonged proliferation capacity and their unique ability to differentiate into derivatives of all three-germ layers. In order to reliably and safely produce hESCs, use of reagents that are defined, qualified, and preferably derived from a non-animal source is desirable. Traditionally, mouse embryonic fibroblasts (MEFs) have been used as feeder cells to culture undifferentiated hESCs. We recently reported a scalable feeder-free culture system using medium conditioned by MEFs. The base and conditioned medium (CM) still contain unknown bovine and murine-derived components, respectively. In this study, we report the development of a hESC culture system that utilizes a commercially available serum-free medium (SFM) containing human sourced and recombinant proteins supplemented with recombinant growth factor(s) and does not require conditioning with feeder cells. In this system, which employs human laminin coated surface and high concentration of hbFGF, the hESCs maintained undifferentiated hESC morphology and had a twofold increase in expansion compared to hESCs grown in MEF-CM. The hESCs also expressed surface markers SSEA-4 and Tra-1-60 and maintained expression of hTERT, Oct4, and Cripto genes similar to cells cultured in MEF-CM. In addition, hESCs maintained in this culture system were able to differentiate in vitro and in vivo into cells of all three-germ layers. The cells maintained a normal karyotype after prolonged culture in SFM. In summary, this study demonstrates that the hESCs cultured in defined non-conditioned serum-free medium (NC-SFM) supplemented with growth factor(s) retain the characteristics and replicative potential of hESCs. The use of defined culture system with NC-SFM on human laminin simplifies scale-up and allows for reproducible generation of hESCs under defined and controlled conditions that would serve as a starting material for production of hESC derived cells for therapeutic use.     

Sperm capacitation in vitro in the eld's deer

Sperm capacitation was examined in the endangered Eld's deer (Cervus eldi thamin). Sperm motility and viability (percentage of sperm cells with intact membranes) were assessed in vitro over time after attempting to induce capacitation in TALP alone and TALP supplemented with calcium (10 mM CaCl2), dibutyryl cAMP (1 mM dbcAMP), or fetal calf serum (20% FCS). Sperm aliquots were evaluated at 0, 3, 6, 9, and 12 h for motility, viability, and ability to acrosome react after exposure to calcium ionophore (A23187, CI; 10 microM) or lysophosphatidylcholine (LC; 100 microg/mL). Fresh sperm aliquots in TALP + 10 mM CaCl2 exposed to CI had fewer (P < 0.05) intact acrosomes than the TALP control (TALP alone) or dbcAMP and FCS treatments after 9 h. Mean (+/- SEM) percentage of intact acrosomes of spermatozoa incubated in medium with increased CaCl2 declined (P < 0.05) from 80.2 +/- 2.6% (0 h) to 49.7 +/- 7.3% after prolonged incubation (9 h). The proportion of capacitated fresh spermatozoa was not influenced by LC treatment. Capacitation was not induced (P > 0.05) by any of the presumptive sperm capacitators after freeze-thawing. Likewise, neither CI nor LC induced the acrosome reaction (AR) in these spermatozoa, suggesting that the freeze-thawing process may have caused membrane damage. Results revealed that the supplementation of medium with CaCl2 evokes capacitation in some spermatozoa. However, Eld's deer spermatozoa appear remarkably resistant to conventional stimulators of capacitation and the AR.     

Intracellular signaling cascades induced by relaxin in the stimulation of capacitation and acrosome reaction in fresh and frozen-thawed bovine spermatozoa

Relaxin is one of the 6-kDa peptide hormones, which acts as a pleiotropic endocrine and paracrine factor. Our previous studies revealed that sperm capacitating medium containing relaxin induced capacitation and acrosome reaction (AR) in fresh and frozen-thawed porcine or bovine spermatozoa. However, the intracellular signaling cascades involved with capacitation or AR induced by relaxin was unknown. Therefore, the present study was designed to investigate the intracellular signaling cascades involved with capacitation and AR induced by relaxin in fresh and frozen-thawed bovine spermatozoa. Spermatozoa were incubated in sperm Tyrode's albumin lactate pyruvate (Sp-TALP) medium supplemented with (40 ng ml(-1)) or without relaxin, and subjected to evaluation of chlortetracycline staining pattern, cholesterol efflux, Ca(2+)-influx, intracellular cyclic adenosine monophosphate (cAMP) and protein tyrosine phosphorylation. Capacitation and AR were increased (P<0.05) in both fresh and frozen-thawed spermatozoa incubated with relaxin. Cholesterol effluxes were greater in the fresh (P<0.01) and frozen-thawed (P<0.05) spermatozoa incubated with relaxin than the spermatozoa incubated without relaxin. Ca(2+)-influxes were also significantly stimulated by relaxin in the fresh (P<0.01) and frozen-thawed (P<0.05) spermatozoa. The Sp-TALP medium containing relaxin influenced the generation of intracellular cAMP in the fresh (P<0.01) and frozen-thawed (P<0.05) spermatozoa, and exhibited higher exposure of protein tyrosine phosphorylation in both sperm types than the medium devoid of relaxin. Therefore, the results postulate that relaxin exerts the intracellular signaling cascades involved with capacitation and AR through accelerating the cholesterol efflux, Ca(2+)-influx, intracellular cAMP and protein tyrosine phosphorylation in fresh and frozen-thawed bovine spermatozoa.     

Peritoneal fluid from women with endometriosis impairs human spermatozoa functionality

The success of mammalian fertilization depends largely on spermatozoa physiological events. However, the manner in which endometriosis influence morpho-functional spermatozoa biomarkers is poorly defined. Here, we studied in vitro the effect of peritoneal fluid (PF) from women with endometriosis (PFE) and non-endometriosis (PFNE) on spermatozoa parameters. This research was prospective and double-blind. A total of 45 PF samples were collected from women with (n = 25) and without endometriosis (n = 20). Semen samples were obtained from normozoospermic donors (n = 15) and cultured with 20 % (v/v) of PF or commercial culture medium (controls) during 0, 24, and 48 h. The outcome measures were spermatozoa/viability, motility, tyrosine phosphorylation (TP) and spontaneous acrosomal reaction. In addition, plasma membrane sugars were characterized by lectins [Aleuria aurantia agglutinin (AAA), Concanavalin A (ConA), Peanut agglutinin (PNA), and Wheat germ agglutinin (WGA)]. After a 24 -h culture, results reported a significant decrease in motility in cells cultured with PFE compared to the control, together with differences in the AAA and WGA-binding sites. Moreover, spermatozoa in contact with PFNE presented a significantly lower level of acrosome spontaneous reaction. At 48 h, no differences were observed in the biomarkers studied between the PFNE and the control, excluding ConA-binding sites. On the other hand, cells cultured with PFE exhibited significantly less motility, TP, and differences in the relocation of spermatozoa surface sugars. Viability was not affected in any culture condition. Overall, the effects of PFE could negatively affect spermatozoa quality, contributing to explain and diagnose the infertility associated to male partners of women with endometriosis.     

Effects of ouabain on the response to osmotic changes in dog and boar spermatozoa

Although there is much information on the response of spermatozoa from different species to osmotic changes, little has been reported about the mechanism/s by which spermatozoa react to similar changes in the osmotic pressure of the medium. In this study we examine the effect of inhibition of Na (+)K (+), ouabain-sensitive ATP-ase on the response of canine and porcine spermatozoa when they are incubated in hypoosmotic and hyperosmotic media. The presence of ouabain slightly decreased the percentages of total and progressive motility, and increased the percentages of altered acrosomes (from 13.0 +/- 0.3% to 17.2 +/- 0.4% in the presence of 10(-4) M ouabain) and, specially, swollen tails (from 0.6 +/- 0.1% to 5.9 +/- 0.2% in the presence of 10(-4) M of ouabain) in fresh dog semen, although it did not affect these parameters in boar semen samples. Moreover, ouabain increased the percentage of both altered acrosomes and swollen tails in canine spermatozoa incubated in 100 mOsm and in 900 mOsm media at concentrations higher than 10(-5) M and 10(-7) M, respectively. The percentage of viability of canine spermatozoa was not modified by ouabain after incubation in 100, 300 or 900 mOsm media. Furthermore, ouabain did not significantly affect boar spermatozoa incubated in 100, 300 or 900 mOsm media. Although ouabain induced a significant decrease in L-lactate production in canine spermatozoa in an isoosmotic medium (from 4.7 +/- 0.4 micromol mg protein x 60 min to 2.6 +/- 0.3 micromol mg protein x 60 min in the presence of 10(-4) M ouabain), there was no significant effect on L-lactate production in boar spermatozoa. These results indicate that while dog spermatozoa acted against changes in the osmotic pressure by a mechanism(s) related to Na (+)K (+), ouabain-sensitive ATP-ase, boar spermatozoa reacted to some mechanism(s) not related to ionic pumps.     

The application of in vitro sperm competition test to evaluate the impact of ZP-derived peptides on fertilization capacity of cat sperm

The present study aimed to establish a sensitive in vitro assay to assess the binding capacity of cat spermatozoa. Cat oocytes and epididymal sperm cells were isolated from gonads and cultured for in vitro fertilization. Before fertilization, the sperm cells were incubated either in 10 microM green dye Fluo-3-AM or 10 microM orange dye CellTracker Orange CMTMR (Molecular Probes), respectively. After removing the dyes by washing, sperm cells stained with each dye were added to medium drops containing oocytes in various proportions and cultured for 16 h at 37 degrees C, 5% CO(2). The oocytes were examined using fluorescence microscopy. Sperm bound to oocytes, and stained with different colors, were counted. When fresh epididymal sperm were mixed in at a specific proportion, the number of sperm bound to the zona pellucida (ZP) of oocytes reflected the proportion of differently colored sperm in the medium. This indicated that neither dye influenced the binding capacity of cat sperm. Mixing fresh and cryopreserved sperm, however, resulted in a higher number of fresh sperm bound to the oocyte surface in comparison to frozen-thawed sperm. Also, the pre-incubation of cat sperm cells with ZP derived peptide reduced the sperm binding capacity by 40%. In conclusion, the presented sperm competition assay allows assessment of fertilizing capacity of cat spermatozoa in vitro when a mixture of two different populations is used. The applied supravital fluorescence dyes do not affect motility and binding capacity of sperm cells and were clearly distinguishable under fluorescence microscopy. We demonstrate that the assay can be used to study the impact of sperm treatment, such as cryopreservation or pre-incubation in bioactive peptides, on fertilizing capacity.     

Rabbit sperm function after co-culture with uterine epithelial cells

The effects of spermatozoa:uterine epithelial cell interactions in vitro on various sperm functions were studied using monolayers of uterine epithelial cells, endometrial stromal cells and fetal fibroblasts. Epithelial and stromal cells were isolated from uteri of rabbits in estrus, while fibroblasts were derived from 12-d-old rabbit fetuses. Twenty-nine to 31 h after culture initiation, washed, ejaculated rabbit spermatozoa were incubated with epithelial cells, uterine stromal or fetal fibroblastic cells, medium or conditioned medium. Sperm viability and loss of acrosome were measured after 10 to 20 h of incubation. Progressive sperm motility and fertilizing ability, which was assessed by an in vivo fertilization assay, were determined after 12 h co-culture. Sperm viability decreased throughout the culture period and was not affected by treatment. Sperm co-cultured with epithelial cells or incubated in medium had fewer acrosomes after 20 h than after 10 h. Fewer sperm co-cultured with stromal or fibroblastic cells lost their acrosomes. Progressive motility was positively affected by sperm-epithelium interaction as 39% of the co-cultured sperm were motile compared with 18% of the sperm incubated in media. In vivo fertilization experiments suggested that sperm incubated with epithelial cells or in medium had similar fertilizing ability. The co-culture of sperm with uterine cells provides an in vitro model to evaluate the effect of gamete-genital tract interaction on sperm function.     

Influence of seminal plasma on fresh and post-thaw parameters of stallion epididymal spermatozoa

Fresh and post-thaw parameters (motility, morphology and viability) of stallion epididymal spermatozoa that have been and have not been exposed to seminal plasma were evaluated, and directly compared to fresh and post-thaw parameters of ejaculated spermatozoa. Six sperm categories of each stallion (n=4) were evaluated for motility, morphology and viability. These categories were fresh ejaculated spermatozoa (Fr-E), fresh epididymal spermatozoa that had been exposed to seminal plasma (Fr-SP+), fresh epididymal spermatozoa that had never been exposed to seminal plasma (Fr-SP-), frozen-thawed ejaculated spermatozoa (Cr-E), frozen-thawed epididymal spermatozoa that had been exposed to seminal plasma prior to freezing (Cr-SP+) and frozen-thawed epididymal spermatozoa that had never been exposed to seminal plasma (Cr-SP-). Results show that seminal plasma stimulates initial motility of fresh epididymal stallion spermatozoa while this difference in progressive motility is no longer present post-thaw; and that progressive motility of fresh or frozen-thawed ejaculated stallion spermatozoa is not always a good indicator for post-thaw progressive motility of epididymal spermatozoa. This study shows that seminal plasma has a positive influence on the incidence of overall sperm defects, midpiece reflexes and distal cytoplasmic droplets in frozen-thawed stallion epididymal spermatozoa while the occurance of midpiece reflexes is likely to be linked to distal cytoplasmic droplets. Furthermore, seminal plasma does not have an influence on viability of fresh and frozen-thawed morphologically normal epididymal spermatozoa. We recommend the retrograde flushing technique using seminal plasma as flushing medium to harvest and freeze stallion epididymal spermatozoa.     

Ion-mediated resistance to osmotic changes of ram spermatozoa: the role of amiloride and ouabain

The aim of this study was to evaluate whether the Na+/K+ and Na+/H+ exchange can maintain the function of fresh ram spermatozoa. We analyzed the quality parameters of spermatozoa from fresh ram ejaculates incubated in iso- (about 300 mOsm), hypo- (about 100 mOsm) and hyperosmotic (about 900 mOsm) media in the presence of ouabain a specific inhibitor of the Na+/K+ ATP-ase or amiloride, a specific inhibitor of the Na+/H+ antiporter. Ouabain increased the percentage of morphologically altered acrosomes in isoosmotic media (from about 10% to 15% in control to about 30% with 10(-4) M ouabain) and decreased the percentage of total motility (from about 80% in control to about 50% to 55% with 10(-4) M ouabain). Ouabain decreased the mean linearity component of motility and decreased the frequency of head displacement. The addition of ouabain increased the percentage of altered acrosomes in the hypo- and hyperosmotic media, although it did not modify viability in either media. Ouabain also increased the percentage of swollen tails in the hypoosmotic medium at higher concentrations of the inhibitor. Amiloride increased the percentage of altered acrosomes in all media although its effect was less intense than that of ouabain. In isoosmotic media, total motility was decreased from about 80% in control to about 65% with 10(-4) M amiloride. Motile spermatozoa incubated with amiloride showed a clear decrease of mean velocity and mean linearity and increased frequency of head displacement. In the hyperosmotic medium, adding amiloride decreased the percentage of viability and altered tails at concentrations as low as 10(-6) to 10(-5) M. Our results indicate that the active mechanisms which control Na+ transport play a significant role in the maintenance of function in ram spermatozoa subjected to different osmotic environments. These mechanisms may be important in maintaining ram sperm function both "in vivo" and "in vitro".     

Ability of thawed tiger (Panthera tigris) spermatozoa to fertilize conspecific eggs and bind and penetrate domestic cat eggs in vitro.

Electroejaculates from tigers were collected and half was used fresh to inseminate tiger eggs in vitro and domestic cat eggs stored in a hypertonic salt solution. The remainder was pelleted, frozen in a solution of 20% egg yolk, 11% lactose and 4% glycerol, thawed and cultured with tiger and domestic cat eggs. The motility index ((sperm % motility)+(status rating x 20))/2 for thawed spermatozoa was about 86% of that in fresh aliquots. Of the 49 tiger oocytes inseminated in vitro with fresh spermatozoa, 34 (69.4%) cleaved, compared with 33 of 47 oocytes (70.2%) cultured with thawed spermatozoa (P > 0.05). Embryos generated by either sperm treatment could develop in vitro to the 16-cell or morula stage. Fresh and thawed tiger spermatozoa were equally capable (P > 0.05) of binding and penetrating the outer and inner zona pellucida of domestic cat eggs. These results demonstrate the ability of frozen-thawed tiger spermatozoa to (i) penetrate homologous and heterologous eggs and (ii) result in conspecific, advanced development of preimplantation embryos in vitro.     

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.