Fertilizing capacity of bovine sperm may be maintained by binding of oviductal epithelial cells

The ability of the bovine oviduct to maintain the motility and fertilizing capacity of bovine sperm was investigated by incubating frozen-thawed sperm with endosalpingeal epithelial cells cultured on either tissue culture plastic (nonpolarizing) or Matrigel-coated Millicell (polarizing) substrata. Sperm were also incubated in medium alone or with cultured bovine tracheal epithelial cells. Motility was determined at 6-h intervals over a 48-h period. The fertilizing capacity of sperm was evaluated after 0, 24, and 30 h of incubation by adding oocytes to the culture and determining the incidences of fertilization and polyspermy. Motility was maintained for 48 h in sperm that bound to endosalpingeal epithelial cells, but to a greater extent with polarized cells (38.4% motile) than with nonpolarized cells (0.8%). Fertilizing capacity was maintained for 30 h in sperm incubated with endosalpingeal epithelial cells on Matrigel/Millicell, but not in sperm incubated in medium alone or with tracheal cells. Only sperm incubated with oviductal cells developed hyperactivated motility. Scanning electron micrographs revealed that sperm were bound by the rostral portion of the intact acrosome to the apical surface of polarized endosalpingeal cells. These results suggest that the oviduct may not only store sperm but may also maintain sperm viability and fertilizing capacity during the preovulatory period.     

Behavior of bull spermatozoa in bovine uterine tube epithelial cell co-culture: an in vitro model for studying the cell interactions of reproduction

Freshly ejaculated bull semen was centrifuged and spermatozoa were resuspended in modified sperm TALP. Bovine uterine tube epithelial cell monolayers (BUTC) were obtained from cows in the periovulatory phase of estrus. In Experiment 1, sperm aliquots were assigned to culture wells containing either BUTC, BUTC-conditioned TALP, or control TALP. Sperm heads attached to the monolayers within 1 h of co-culture. Attached spermatozoa showed vigorous tail motion. At 5, 8 and 11 h of incubation at 39 degrees C, the percentage of unattached sperm cells with intact acrosome membranes and percentage of motility of these cells was measured. Sperm-BUTC co-cultures were also fixed in situ for electron microscopy. Unattached spermatozoa in co-culture had more (P<0.05) acrosomal membrane loss, showed hyperactive motion and had an overall decrease in motility as compared to sperm cells in control or conditioned medium. Evaluation by electron microscopy showed BUTC attached spermatozoa to behave in the co-culture system similar to reports for spermatozoa found in uterine tubes in vivo. Microvilli of the BUTC appeared to actively entrap the spermatozoa. Mucus-type granules could be seen on acrosomal regions and vesiculation of acrosomal membranes was seen in some cells. In Experiment 2, 43% of the 12 x 10(6) sperm cells added to 2-cm(2) BUTC bound within 4 h of co-culture. By 7 h of co-culture 19% of the previously bound sperm cells had been released from the BUTC. Released cells had limited motility and were mostly dead (73%). Sperm cells remaining on the monolayer at 7 h showed vigorous tail motion and were gradually released from the BUTC over 48 h. Spermatozoa in co-culture interacted with the BUTC in a manner much like that seen in vivo, and sperm capacitation changes were stimulated by this interaction.     

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.     

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.     

Effect of oviductal epithelial cells on fertilization of pig oocytes in vitro

The incidence of polyspermy is reduced by co-culture of pig oocytes with oviductal cells. It is not known whether the effect is due to soluble factors secreted into the medium. Oviductal epithelial cell monolayers and cell-conditioned media were prepared and their effects on fertilization of pig oocytes were examined. In vitro matured pig oocytes were inseminated with ejaculated boar spermatozoa at a concentration of 1 x 10(5) or 1 x 10(6) cells/ml and co-cultured in one of 5 culture systems: an oviductal epithelial cell monolayer, a fibroblast monolayer, an oviductal epithelial cell-conditioned medium, or a fibroblast-conditioned medium, and medium alone (modified-TCM199). In all 5 systems, the majority (range 85 to 100%) of the oocytes were penetrated by sperm. When oocytes were inseminated with spermatozoa at a concentration of 1 x 10(5) cells/ml, the percentages of monospermic oocytes were significantly higher in the oocytes co-cultured with oviductal epithelial cells and fibroblasts than that of the oocytes cultured without these cells. In contrast, when oocytes were inseminated with spermatozoa at a concentration of 1 x 10(6) cells/ml, the percentages of monospermic oocytes were significantly higher in the oocytes co-cultured with epithelial cells than those cultured with the fibroblasts and in the control medium. The suppressive effect on polyspermy was observed in the oviductal epithelial cells-conditioned medium when oocytes were inseminated with spermatozoa at both concentrations of 1 x 10(5) and 1 x 10(6) cells/ml. The effect was absent in the fibroblasts-conditioned medium. Moreover, the effect of the epithelial cells was maintained during the culture period, whereas the proportion of monospermic oocytes co-cultured with fibroblasts showed a gradual decrease, reaching 0% after 16 h. These results suggest that a soluble factor(s) derived from the oviductal epithelial cells decreased the number of spermatozoa penetrating the oocytes without suppressing the high rate of fertilization.     

肝素处理山羊精子体外获能的研究

系统研究了作用浓度、时间和温度以及输卵管上皮细胞和卵丘细胞对肝素处理山羊精子体外获能后的精子活力、质膜完整性、顶体完整率、获能比例及受精和卵裂的影响,为改善山羊精子体外获能效果和研究获能机理提供了必要的数据。主要实验结果如下：1、在获能液中添加5、10、25、50和100μg/mL肝素处理45min时,添加50和100μg/mL肝素精子获能比率最高（分别为55%和56%）,但添加100μg/mL肝素处理后顶体完整率明显（P<0.05）低于对照组。说明山羊精子获能的最佳肝素浓度为50μg/mL。2、肝素作用时间（0, 10, 20, 30, 45, 60 和120 min）的延长,获能精子比例逐渐提高。其中,肝素处理45～120 min各组的获能精子比例差异不显著（P>0.05）,处理120 min组的精子活力和质膜完整率显著低于其它各组。说明50μg/mL肝素处理精子获能的最佳时间是45～60 min。3、在42℃和38.5℃下处理时,获能精子比例显著高于15℃和37℃,但42℃处理后精子活力和顶体完整率显著低于其它温度。因此,385℃为山羊精子获能的最佳温度。4、与输卵管上皮细胞共培养获能精子比例显著高于对照组和卵丘细胞组,但精子活力、质膜完整率和顶体完整率差异不显著。输卵管上皮组的受精率（91.3％）和卵裂率（72.2％）显著高于对照组（81.2％,65.0％）。说明与输卵管上皮细胞共培养能显著提高肝素处理山羊精子体外获能的效果。     

Direct contact between boar spermatozoa and porcine oviductal epithelial cell (OEC) cultures is needed for optimal sperm survival in vitro

Oviductal epithelial cell (OEC) co-culture prolongs sperm viability and motility in vitro in a number of species including humans and horses. This study has sought to determine the effects of homologous OEC co-culture on boar sperm function. To determine whether the effects on spermatozoa were specifically caused by co-culture with or by OEC secretions, or by both factors together, a number of co-culture and cell-conditioned medium (CM) experiments were conducted. Firstly, Percoll-washed spermatozoa were co-cultured with OECs and pig kidney epithelial (LLC-PK1) cells, and in medium without cells. Secondly, Percoll-washed spermatozoa were incubated with CM derived from both OECs and LLC-PK1 cells and in unconditioned medium. A number of sperm function parameters were assessed after 5, 30, 60, 90, 120, and 180 min, and 24h of co-culturing or incubation with CM. Of all the sperm function parameters investigated, the percentage (%) viability data yielded the most interesting results. OECs (mean+/-S.E.M.; 31.2+/-1.10) were better than LLC-PK1 cells (24.3+/-0.93) at prolonging the viability of unbound spermatozoa after 24h of co-culturing (P<0.05). Also after 24h, the viability of spermatozoa bound to the OECs (77.6+/-1.83) was significantly higher than in the case of the LLC-PK1 cells (53.5+/-1.43; P<0.001). Other sperm function parameters, e.g., capacitation and motility, were also influenced by OEC co-culturing and incubation with CM, although to a lesser degree. In conclusion, porcine homologous OEC co-culture and CM incubation specifically affect sperm function. However, we propose that it is OEC co-culturing, rather than OEC-CM, that has the greater influence.     

Birth of live Spanish ibex (Capra pyrenaica hispanica) derived from artificial insemination with epididymal spermatozoa retrieved after death

As a consequence of increasing limitations to maintaining genetic variability in endangered wildlife species, methods of assisted reproduction widely used in domestic animals are being applied to nondomestic species. However, practical efforts have met limited success to date. The Spanish ibex (Capra pyrenaica hispanica) is a wild caprine originating exclusively in the mountains of Spain. This study was designed to evaluate the fertilizing capability of cryopreserved Spanish ibex epididymal spermatozoa recovered postmortem. For this purpose, we have previously evaluated the effect of time elapsed between death and sperm recovery on spermatic parameters, and the fertilization ability of frozen-thawed spermatozoa using heterologous in vivo fertilization by intrauterine insemination in domestic goat (Capra hircus). The time of death significantly affected most sperm quality parameters (motility, viability and intact acrosomes). The fertility obtained by heterologous artificial insemination was 18.7%, and only goats inseminated with spermatozoa recovered within 8h after death became pregnant. Our findings showed that heterologous in vivo fertilization is a useful method to evaluate the fertilizing capacity of sperm samples in rare or wild species. Sperm samples, with verified fertilization ability in the previous trial, were used to inseminate a total of six ibex females. Inseminations resulted in one pregnancy. The study demonstrated for the first time the feasibility of applying artificial insemination in Spanish ibex.     

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.     

Epithelial and stromal uterine cells cultured in vitro protect bovine sperm from hydrogen peroxide

It is known that large amounts of leukocytes colonize the uterus, and that these leukocytes can produce considerable quantities of hydrogen peroxide (H2O2) and other reactive oxygen species that are toxic to sperm. It has been shown recently that oviductal fluid has a catalase that helps to maintain sperm motility. Therefore, the current experiment was performed to determine if a similar mechanism of protection exists against peroxides within uterine cells. Sperm motility and velocity were recorded after a 6h incubation in 1) conditioned media in the presence of endometrial cells, 2) conditioned media without endometrial cells, 3) control media (48h without cells) over endometrial cells, or 4) control media alone. All these treatments were performed in the presence or absence of added catalase. Conditioned media, endometrial cells and catalase had a significant positive effect on the maintenance of sperm motility and velocity. Addition of anti-catalase antibodies did not neutralize the beneficial effect of the conditioned media. However, the concentrations of aromatic amino acids, known substrates for sperm amino acid oxidase, were significantly lower in uterine conditioned media as compared to control medium. This reduction of aromatic amino acids was in correlation with reduced H2O2 production by sperm as estimated by chemiluminescence. These results suggest that epithelial and stromal uterine cells do not maintain sperm motility by secreting catalase in the conditioned media, but rather by reducing the levels of aromatic amino acids and thus of peroxides generated in the presence of spermatozoa.     

Influence of storage time on functional capacity of flow cytometrically sex-sorted boar spermatozoa

Sex-sorting of boar spermatozoa is an emerging biotechnology, still considered suboptimal owing to the slowness of the process, which requires long sorting periods to obtain an adequate number of spermatozoa to perform a non-surgical insemination. This period involves storage of sorted cells that could impair their functional capacity. Here, we have studied how the storage of sex-sorted boar spermatozoa affects their functional capacity. Sorted spermatozoa were assessed at various times (0, 2, 5h or 10h) during storage after sorting and compared with diluted and unsorted spermatozoa for sperm motility patterns, plasma membrane and acrosomal integrity and their ability to penetrate homologous IVM oocytes. Sex-sorted sperm motility and membrane integrity only decreased significantly (p<0.05) by the end of the storage period (10h) compared to unsorted spermatozoa. Sperm velocity, ALH and Dance increased significantly (p<0.05), immediately post-sorting, returning to unsorted sperm values during storage. Acrosome integrity was not seriously affected by the sorting process, but decreased (p<0.05) during storage after sorting. Sorted spermatozoa stored 2h after sorting did not differ from unsorted in penetration rates and numbers of spermatozoa per oocyte, reaching the highest (p<0.05) penetration rates and sperm numbers per oocyte, when co-cultured for 6 or more hours. Non-storage or storage for 5h or 10h negatively (p<0.05) affected sperm penetration ability. In conclusion, although flow cytometrically sex-sorted spermatozoa are able to maintain motility, viability and acrosomal integrity at optimal levels until 10h of storage after sorting, fertilizing ability is maintained only over shorter storage times (<5h).     

Quality and fertilizing ability of electroejaculated cat spermatozoa frozen with or without Equex STM Paste

An optimal protocol for cat semen cryopreservation has not yet been defined. Addition of Equex STM Paste has been tested for epididymal cat spermatozoa but not for ejaculated cat spermatozoa. Furthermore, the effect of Equex STM Paste on fertilizing ability of cryopreserved semen has never been evaluated in that species. Therefore, the aims of the current study were to investigate if addition of Equex STM Paste to a freezing extender for electroejaculated cat (Felis catus) semen would improve postthaw sperm quality and if sperm fertilizing ability after cryopreservation with or without Equex STM Paste was preserved. Semen was collected by electroejaculation and frozen in a Tris-glucose-citrate egg yolk extender supplemented with (0.5% vol/vol) or without Equex STM Paste. In Experiment 1, sperm motility, membrane integrity, and acrosomal status were determined immediately after collection and at 0, 3, and 6 h postthaw. In Experiment 2, frozen semen from the two groups was used for in vitro fertilization (IVF) of in vitro-matured cat oocytes. Cleavage rate was recorded 30 h after IVF, and embryo development was evaluated on Days 6 and 7 of culture. In Experiment 1, the rate of motile spermatozoa after freezing-thawing was higher when Equex STM Paste was added to the freezing extender, but progressive motility score was not influenced (P > 0.05). Sperm membrane integrity was positively affected (P < 0.05) by the addition of the detergent. Intact acrosomes after thawing were similar (P > 0.05) between groups. Even if the decreasing rates of motility and membrane integrity were more rapid in presence of Equex than those in controls, total motility and sperm viability were similar at 3 and 6 h after thawing (P > 0.05). In Experiment 2, there was no difference in fertilizing ability and embryo development between the two groups (P > 0.05). The results of this study demonstrate that the addition of Equex STM Paste in the freezing extender avoids the loss of motile spermatozoa and maintains fertilizing ability of frozen-thawed spermatozoa.     

A technique for the evaluation of sperm penetrating ability and quality of bovine semen processed in an extender made with Brackett-Oliphant medium and egg yolk

Egg yolk-sodium citrate (EYC) semen extender was compared with an extender made of Brackett-Oliphant medium and egg yolk (BOEY). Ejaculates were divided into equal portions, processed and frozen. Semen was thawed and evaluated for quality. Additional semen was thawed, stained with Hoechst 33342 and the spermatozoa capacitated, after which they were co-incubated with zona-free hamster oocytes to determine their penetrating ability. Sperm penetration of non-compressed, unfixed oocytes was evaluated using an optical sectioning technique on a standard research microscope. Sperm penetration was considered successful if a fluorescing sperm head was observed within the living oocyte in a hanging drop of fertilization medium. There were small differences in percentage of secondary abnormalities and percentage of progressive motility immediately after thawing between spermatozoa extended in EYC or BOEY diluent. There were no differences due to by extender composition in percentage of spermatozoa with intact acrosomes or percent of progressively motile after a 3 h incubation at 37 degrees C, nor the percentage of spermatozoa with head abnormalities. While there were significant correlations between all seminal quality characteristics, no quality measurements were correlated to percentage of oocyte penetration. The new penetration evaluation method allowed for examination of the fertilized oocytes using fluorescent microscopy initially and again after re-incubation for further development.     

Supplementation of 17β-estradiol and progesterone in the co-culture medium of bovine oviductal epithelial cells and ovine spermatozoa reduces the sperm kinematics and capacitation

This study investigated the effect that bovine oviductal epithelial cell (BOEC) and ovine spermatozoa co-culture exposed to different hormonal environments had on ram sperm function over the course of a 24-h incubation period. Ram cooled-stored spermatozoa were selected by swim-up and then co-cultured separately for 24?h at 38.5?°C under 5% CO₂ with either: (1) Fert-TALP medium (positive control [POSControl]), (2) Fert-TALP medium supplemented with 17β-estradiol (E2) and progesterone (P4) at concentrations similar to follicular phase (Follicular NEGControl), (3) Fert-TALP medium supplemented with E2 and P4 concentrations similar to luteal phase (Luteal NEGControl), (4) BOEC cultured in the same medium as that of the Follicular NEGControl group (Follicular BOEC group), or (5) BOEC cultured in the same medium as that of the Luteal NEGControl group (Luteal BOEC group). The sperm kinematics, capacitation status, and plasma membrane (PM) integrity were evaluated at different intervals. Sperm PM integrity was not affected (P ? 0.05) by BOEC co-culture, regardless of the phase of the estrous cycle. After 4?h of incubation, the Luteal BOEC group presented lower (P?<? 0.05) progressive motility and total motility than the Luteal NEGControl group while the Follicular BOEC group showed lower (P?<? 0.05) velocimetric parameters and progressive motility than the Follicular NEGControl group. Throughout the incubation period, both BOEC co-culture groups showed a decrease (P?<? 0.05) in their capacitation rate in comparison to the POSControl group. Conversely, the Luteal BOEC group presented a higher (P?<? 0.05) non-capacitated rate than both the POSControl and Luteal NEGControl groups. In conclusion, BOEC co-culture with ovine spermatozoa at either the follicular or luteal phase decreases sperm kinematics and delays sperm capacitation.     

The role of osmotic resistance on equine spermatozoal function

Cryopreservation requires exposure of sperm to extreme variations in temperature and osmolality. The goal of this experiment was to determine the osmotic tolerance levels of equine sperm by analyzing motility, viability, mitochondrial membrane potential (MMP), and mean cell volume (MCV). Spermatozoa were incubated at 22 degrees C for 10 min in isosmolal TALP (300 mOsm/kg), or a range of anisosmolal TALP solutions (75-900 mOsm/kg), for initial analysis, and then returned to isosmolal conditions for 10 min for further analysis. Total sperm motility was lower (P < 0.05) in anisosmolal conditions compared to sperm motility in control medium. When cells were returned to isosmolal conditions, only sperm previously incubated in 450 mOsm/kg TALP were able to recover to control levels of motility. Sperm viability and MMP were lower (P < 0.05) when exposed to hypotonic solutions in comparison to control solutions. Sperm suspensions that were returned to isosmolal conditions from 75, 150, and 900 mOsm/kg had lower (P < 0.05) percentages of viable sperm than control suspensions (300 mOsm/kg). MMP was lower (P < 0.05) in cells previously incubated in 75 and 900 mOsm/kg when returned to isosmolal, as compared to control cells. MCV differed (P < 0.05) from control cell volume in all anisosmolal solutions. Cells in all treatments were able to recover initial volume when returned to isosmolal medium. Although most spermatozoa are able to recover initial volume after osmotic stress, irreversible damage to cell membranes may render some sperm incapable of fertilizing an oocyte following cryopreservation.     

Encapsulation of porcine spermatozoa in poly-lysine microspheres

Three experiments were conducted to examine the effects of incubating porcine spermatozoa in concentrated samples, to determine the viability of sperm encapsulated in microspheres and to evaluate the potential of microencapsulating porcine spermatozoa for use in artificial insemination. In Experiment 1, sperm incubated at 4, 15, 20 or 37 degrees C and at concentrations of 7.5, 15, 30, 60 or 120 x 10(6) sperm/ml lost motility over a 16-h incubation period. Sperm motility was significantly lower at 4 degrees C than at 15, 20 or 37 degrees C and was significantly higher in more concentrated samples. In Experiment 2, sperm were encapsulated in poly-lysine microspheres at concentrations of 30, 60 or 120 x 10(6) sperm/ml and incubated in vitro at 4, 15 or 20 degrees C. Unencapsulated samples were incubated at similar concentrations and temperatures and served as controls. Motility and percentage of sperm with intact acrosomes were estimated at 2, 4, 8 and 16 h of incubation. The procedure of encapsulation did not affect sperm motility or acrosomal morphology; however, there was an accelerated loss of motility in encapsulated samples. There were no differences in acrosomal morphology between the two groups across time. In Experiment 3, sperm were encapsulated at a concentration of 120 x 10(6) sperm/ml and 20 ml of capsules were inseminated into estrous sows. Uterine contents were flushed at 3, 6 and 24 h after insemination and examined for capsules. Capsules containing motile sperm were recovered from sows at 3 and 6 h, but not at 24 h. These results demonstrate that porcine spermatozoa can be encapsulated in microspheres and that these capsules can be inseminated into estrous females, but the sperm undergo an accelerated loss of motility in vitro and in vivo.     

Direct binding of boar ejaculate and epididymal spermatozoa to porcine epididymal epithelial cells is also needed to maintain sperm survival in in vitro co-culture

The aim of the present study was to compare the influence of cultured epididymal epithelial cells (EEC) from corpus, caput or cauda, oviductal epithelial cells (OEC) and non-reproductive epithelial cells (LLC-PK1) on function and survival of epididymal and ejaculated spermatozoa, in the latter case to determine whether such influence differed between morphologically normal and abnormal spermatozoa. For this purpose, either spermatozoa were directly co-cultured with EEC from caput, corpus, or cauda, OEC and LLC-PK1 cells (experiment 1) or a membrane-diffusible insert was included in these co-cultures (experiment 2). EEC cultured from the three epididymal regions did not differently affect the sperm parameters. Morphologically normal spermatozoa presented a higher ability to bind EEC, OEC, and LLC-PK1 than abnormal spermatozoa with cytoplasmic droplets or with tail/head malformations. Epididymal spermatozoa were more able to bind EEC during the first 24 h of co-culture, while ejaculated spermatozoa presented a higher capacity to bind OEC between 30 min and 3 h of co-incubation. In all cases, the ability to bind to epithelial cells was higher when they were co-cultured with EEC and OEC than with LLC-PK1. After 2 h of co-culture, the viability of epididymal spermatozoa was better maintained when they bound EEC than when they bound OEC. Conversely, the viability of ejaculated spermatozoa was better maintained when bound OEC than when bound EEC after 24 and 48 h of co-culture. Our work, apart from corroborating the involvement of morphologically normal spermatozoa in the formation of sperm reservoir, highlights the importance of direct contact spermatozoa-EEC in maintaining the sperm survival in in vitro co-culture, and also suggests that a specific binding between EEC and epididymal spermatozoa exists.     

Calcium alters capacitation and progressive motility of uterine spermatozoa from +/+ and congenic tw32/+ mice.

The importance of calcium-dependent sperm processes for fertilization in vitro is well known, but their interaction with sperm transport in vivo is not yet clear. To determine whether exposure to calcium alters sperm physiology after incubation in the uterus, spermatozoa from +/+ mice were incubated in medium with 1.7 mM calcium prior to artificial insemination (AI). Spermatozoa from congenic tw32/+ mice were also tested because their flagella are hypersensitive to calcium. As a control, spermatozoa were incubated in calcium-deficient medium before AI. When recovered from the uterus 60 min post-AI, neither prior exposure to calcium nor genotype affected numbers of spermatozoa, or percentage of motile or acrosome-reacted spermatozoa. However, significantly more calcium-treated spermatozoa were capacitated and significantly fewer were progressively motile than spermatozoa preincubated without calcium. In addition, significantly fewer spermatozoa from tw32/+ mice than from +/+ mice were progressively motile. These results suggest that uterine sperm physiology is changed by prior exposure of sperm to calcium. Since the level of progressive motility of spermatozoa recovered from the uterus was correlated with their ability to reach the oviduct (as determined in a previous study), these data support the hypothesis that progressive motility of uterine spermatozoa is important for passage to the oviduct and fertility.     

Effect of liquid storage and cryopreservation of boar spermatozoa on acrosomal integrity and the penetration of zona-free hamster ova in vitro

The effect of liquid storage and cryopreservation of boar spermatozoa on sperm motility, acrosomal integrity, and the penetration of zona-free hamster (ZFH) ova was examined. The sperm penetration assay (SPA) provides valuable information on specific events of fertilization and is a potentially useful indicator of sperm fertility. Ejaculated semen from 4 boars was subjected to 3 treatments: fresh (FRE, no storage), liquid-stored (LIS, stored at 18°C for 3 days), and frozen (FRO, frozen by pellet method and stored at ?196°C for 3 days). A highly motile sperm population was isolated by the swim-up procedure (1 hr). FRE and LIS were incubated an additional 3 hr at 39°C in a Tris-buffered medium to elicit capacitation and the acrosome reaction. Sperm motility and acrosomal integrity were assessed before and after incubation. For the SPA, sperm and eggs were incubated at 39°C for 3 hr in Hams F-10 medium. Each egg was assessed for sperm penetration, sperm binding, and stage of development. Percentages of sperm motility and sperm with a normal apical ridge (NAR) prior to incubation were 78 and 78 (FRE), 75 and 69 (LIS), and 28 and 50 (FRO). After incubation, percentages of motility, NAR, and acrosome-reacted sperm were 34, 10, and 73 (FRE); 43, 24, and 51 (LIS); and 18, 13, and 59 (FRO). A somewhat higher (P < .05) percentage of ZFH ova was penetrated by FRE (45.8) than by LIS (42.0). Penetration of ZFH ova by FRO was markedly (P < .05) reduced (30.2). Sperm penetration was not significantly correlated with motility or acrosomal integrity before or after incubation, regardless of treatment. These data suggest that the SPA can be used in conjunction with conventional measures of semen analysis in assessing the potential fertilizing capacity of boar sperm and that liquid storage is superior to frozen storage with respect to preserving sperm fertility.     

In vitro capacitation of boar ejaculated spermatozoa: Effect of conditioned media prepared from preincubated sperm suspension

The fertilizing ability of boar ejaculated spermatozoa was examined in vitro after prcincubation at a concentration of 2.5 × 10⁸/ml for 4 hr in several conditioned media (CM). For preparation of CM, boar spermatozoa were incubated in a modified Krebs-Ringer bicarbonate solution (TYH) at concentrations of 20 to 40 × 10⁸/ml for several hours up to 4 hr; then their supernatant fluids were collected by centrifugation. When boar ejaculated spermatozoa were preincubated in TYH alone, 14.1% of oocytes were penetrated by them as we reported previously. On the other hand, preincubating them with CM, their fertilizing ability was elevated according as the incubation time of CM preparation was lengthened. The fertilization rate reached 75.0%, using 4 hr-incubated CM for the preincubation medium. The effect of CM was not deteriorated by heat treatments (56°C, 30 min, or 100°C, 5 min). The components of CM were separated at a molecular weight of 25,000 by ultrafiltration, and high fertilization rate (69.8%) was obtained when low molecular weight fraction was used for the preincubation medium. Sperm extracts prepared from directly frozen-thawed sperm suspension and 0.1–10 mM of taurine or hypotaurine had no effect on the fertilizing ability of boar spermatozoa. These results suggest that substances stimulating boar sperm capacitation were accumulated from viable spermatozoa into the medium during incubation and that the effective substances were heat-stable and of low molecular weight and were not taurine and hypotaurine.