Effect of epididymis handling conditions on the quality of ram spermatozoa recovered post-mortem

Post-mortem spermatozoa recovery is an important technique for obtaining germplasm reserves from genetically valuable animals or endangered species. However, there are many factors that influence the outcome of this technique. We have studied the effect of the interval between animal's death and sperm recovery (0, 24 or 48 h) on the quality and freezability of ram spermatozoa from cauda epididymidis. Storage temperature of epididymis (room temperature or 5 degrees C) was also analysed. Spermatozoa were diluted with Tes-Tris-Fructose solution supplemented with egg yolk (10%) and glycerol (4%), and frozen using a programmable biofreezer (-20 degrees C/min). Pre-freeze and post-thaw sperm samples showed viable spermatozoa up to 48 h after the animal's death, although their quality declined significantly as post-mortem storage time increased. Epididymis sperm stored at 5 degrees C showed better motility and a lower percentage of abnormal forms than epididymis stored at room temperature after 24 and 48 h. The fertilizing ability of cauda epididymis ram spermatozoa obtained at 0 and 24h after the animal's death is similar to that of ejaculated spermatozoa. Therefore, a good protocol for post-mortem semen collection in rams when epididymal spermatozoa cannot be collected immediately, is to preserve the epididymis at 5 degrees C and process the samples in the first 24h after the animal's death.     

Influence of staining and sampling procedures on goat sperm morphometry using the Sperm Class Analyzer

Computer-assisted sperm morphometry analysis (ASMA) has improved the assessment of sperm morphology, but the results depend on the use of adequate sampling and staining procedures of spermatozoa from individual species. In this study, the Sperm Class Analyzer ASMA system was used for the morphometric analysis of goat sperm heads. Semen samples, obtained from four bucks, were used to evaluate the influence of three staining procedures (Diff-Quik, Hemacolor and Harris' Haematoxylin) on the accuracy of image processing and sperm morphometry, the effect of the number of cells analysed and the repeatability of the method. These experiments were performed to obtain objective, accurate and reliable sperm morphometric measurements of goat spermatozoa. Diff-Quik and Harris' Haematoxylin were significantly (p<0.05) more accurate than Hemacolor. However, Diff-Quik obtained the highest proportion of correctly analysed sperm heads (86.06%) and the lowest coefficients of variation on the image processing and morphometric measurements. The staining methods affected significantly the sperm dimensions (p<0.001) with increased values from Diff-Quik than Hemacolor and Harris' Haematoxylin, respectively (Diff-Quik>Hemacolor>Harris' Haematoxylin). No differences in morphometric parameters were found when 100, 150, 175 or 200 spermatozoa were analysed. The repeatability of results obtained was very high since no differences were found when measuring the same sperm on multiple attempts. In conclusion, to obtain objective, accurate and repeatable sperm morphometric measurements by the Sperm Class Analyzer system in goats, the analysis of 100 spermatozoa from slides which have been previously stained with Diff-Quik is recommended.     

A pilot study on post-thawing quality of Iberian red deer spermatozoa (epididymal and electroejaculated) depending on glycerol concentration and extender osmolality

The optimization of cryopreservation extenders is a fundamental issue for adequately performing germplasm banking on wild species. We have tested two glycerol concentrations (4 and 8%), and three extender osmolalities (320, 380 and 430 mOsm/kg; before adding cryoprotectants), for cryopreservation of epididymal and ejaculated sperm samples from Iberian red deer. All the extenders were based on Tes-Tris and fructose (for osmolality adjustment), and complemented with 20% egg yolk. Epididymal and ejaculated sperm samples were obtained from the cauda epididymis (post-mortem) and using electroejaculation, respectively. Samples were diluted 1:1 with each extender and equilibrated for 2 h at 5 degrees C. Then, they were diluted down to 100x10(6) sperm/mL and frozen at -20 degrees C/min. Post-thawed samples were assessed for motility (CASA), HOS test, proportion of swollen (osmotically challenged) cells in the untreated sample, viability and acrosomal status. For epididymal samples, 8% glycerol rendered a slightly higher proportion of intact acrosomes on viable spermatozoa than 4%; regarding extender osmolality, 380 and 430 mOsm/kg rendered higher motility results, and the 430 mOsm/kg yielded the lowest proportion of swollen spermatozoa. For ejaculated samples, 4% glycerol yielded more viable spermatozoa than 8%; for extender osmolality, 320 mOsm/kg rendered the highest percentages of progressively motile and viable spermatozoa, although 380 mOsm/kg extender was not significantly different. These results show that sample source influences extender suitability, and that extenders should be isoosmotic or rather slightly hyperosmotic. Future studies should test multiple glycerol concentrations and extender osmolalities in order to adjust them to these kinds of sample.     

Single-layer centrifugation separates spermatozoa from diploid cells in epididymal samples from gray wolves, Canis lupus (L.)

Sperm samples may be used for assisted reproductive technologies (e.g., farmed or endangered species) or as a source of haploid DNA or sperm-specific RNA. When ejaculated spermatozoa are not available or are very difficult to obtain, as is the case for most wild endangered species, the epididymides of dead animals (e.g., animals that have been found dead, shot by hunters or poachers, or that that require euthanasia in zoological collections) can be used as a source of sperm. Such epididymal sperm samples are usually contaminated with cellular debris, erythrocytes, leukocytes, and sometimes also bacteria. These contaminants may be sources of reactive oxygen species that damage spermatozoa during freezing or contribute undesired genetic material from diploid cells. We used single-layer centrifugation through a colloid formulation, Androcoll-C, to successfully separate wolf epididymal spermatozoa from contaminating cells and cellular debris in epididymal samples harvested from carcasses. Such a procedure may potentially be applied to epididymal sperm samples from other species.     

Live mice from cryopreserved embryos derived in vitro with cryopreserved ejaculated spermatozoa

This study was undertaken to try to reduce the number of animals required to maintain mouse strains by banking of embryos or spermatozoa. The principal objective was to cryopreserve ejaculated mouse spermatozoa, using a method recently developed for epididymal spermatozoa. Within 30 min after mating, ejaculated spermatozoa were flushed from the uterus of mated females; shortly afterwards, epididymal spermatozoa were also collected from the same males that had mated with the females. The average values for spermatozoal motility and viability of ejaculated specimens of nine males were 43 and 46%, respectively, and for epididymal specimens, the corresponding values were 60 and 52%. In experiment 1, ejaculated or epididymal spermatozoa were incubated with oocytes for 0.5 to 4 h. As evidenced by development into two-cell embryos within 24 h, kinetics of fertilization of the two spermatozoa types were similar. In experiment 2, ejaculated and epididymal spermatozoa of three males were separately cryopreserved in medium containing raffinose, glycerol, and egg yolk. Samples were cooled and seeded at -4 degrees C, cooled to -70 degrees C at 20 degrees C/min, and then were placed into liquid nitrogen for storage. When cryopreserved epididymal or ejaculated spermatozoa were thawed at > 1,000 degrees C/min and used for in vitro fertilization, > 60% of oocytes cleaved, and approximately 95% of cleaved embryos developed into morulae or blastocysts. When embryos produced with cryopreserved spermatozoa were transferred into recipients, 18 and 22 live pups were obtained from 62 and 54 embryos resulting from ejaculated or epididymal spermatozoa, respectively. This study documented the feasibility of cryopreserving ejaculated spermatozoa as an effective alternative to preserving germ plasm from genetically valuable mice.     

Effect of sperm concentration during preincubation in a defined medium on fertilization in vitro of pig follicular oocytes

Pig follicular oocytes cultured in a defined medium for 28-29 h were inseminated in vitro by epididymal or ejaculated boar spermatozoa that were preincubated in a modified KRB solution at various sperm concentrations for 4 h at 37 degrees C. Sperm concentration at insemination was 2 X 10(6) cells/ml. When epididymal spermatozoa were preincubated at concentrations of 4-16 X 10(8) cells/ml, 71-75% of oocytes were penetrated. In contrast, preincubation at a low concentration (0.8 X 10(8) cells/ml) resulted in a low penetration rate (11%). Epididymal spermatozoa preincubated at a concentration of 4 X 10(8) cells/ml could also penetrate denuded oocytes. None of the oocytes were penetrated by epididymal spermatozoa that were exposed to seminal plasma before preincubation or by ejaculated spermatozoa. After preincubation, whiplash motility was observed in the epididymal spermatozoa, but not in the ejaculated spermatozoa.     

Comparison between different markers for sperm quality in the cat: Diff-Quik as a simple optical technique to assess changes in the DNA of feline epididymal sperm

The majority of wild felids, as well as some domestic cats, have low sperm concentration in their ejaculates, and a high proportion of abnormal spermatozoa. We have employed several possible semen quality markers to further characterize cat epididymal sperm. Methods included possible apoptotic reporters, such as the annexin V assay to monitor exposure of phosphatidylserine (PS) on the outer leaflet of the plasma membrane, as well as cell integrity; and the TUNEL assay to quantify DNA breaks. Sperm surface ubiquitination, another putative marker of sperm quality, was also monitored. The annexin V assay revealed a high percentage of sperm with PS exposure, and the TUNEL assay pointed to high levels (13+/-12%) of sperm with DNA breaks. Correlations were found between apoptotic markers (but not ubiquitination) and semen parameters. In parallel to this analysis, cat sperm morphology was evaluated using the Diff-Quik optical stain, which has been used in human reproduction laboratories. Several types of abnormalities could be characterized with this method. Remarkably, head staining abnormalities detected using the Diff-Quik staining method were strongly correlated with, and could accurately predict, sperm DNA defects detected in the same sample using the TUNEL assay. We therefore suggest that sperm morphology analysis using Diff-Quik could be used in field conditions to assess sperm status, due to the simplicity of the procedure and the equipment involved.     

Preservation of ejaculated and epididymal stallion spermatozoa by cooling and freezing

The suitability of ejaculated and epididymal stallion spermatozoa for cooled storage (5 degrees C) and cryopreservation was examined in 5 ejaculates from each of 6 stallions and in spermatozoa recovered from the cauda epididymidis after castration of these stallions. The percentage of progressively motile spermatozoa, examined by subjective estimation (cooled samples) or by computerized analysis (frozen-thawed samples), was used as parameter. In ejaculated semen samples containing 5 and 25% seminal plasma in a skim milk glucose extender, the lower amount of seminal plasma supported spermatozoal motility significantly better throughout storage at 5 degrees C. Addition of 5 or 25% seminal plasma to perfused epididymal spermatozoa (0% seminal plasma) resulted in a significant stimulation of spermatozoal motility by 25% seminal plasma at 0 h (P<0.05) and to a lesser extent at 24 and 48 h. Post-thaw motility of ejaculated as well as epididymal spermatozoa was not influenced by slow cooling to 15 degrees or 5 degrees C with or without glycerol prior to rapid freezing in liquid nitrogen vapor. During cooled storage, seminal plasma had a stimulatory effect on epididymal spermatozoa and depressed motility in ejaculated spermatozoa. Results on cryopreservation indicate that freezability of equine spermatozoa is already determined when spermatozoa leave the tail of the epididymis.     

Sperm penetration in vitro of pig follicular oocytes matured in culture.

Boar ejaculated and epididymal spermatozoa were preincubated in modified KRB or the isolated oviduct and uterine horn of an oestrous sow for 4.5-5 h at 37 degrees C before introduction into medium containing ovarian oocytes previously cultured for 24 h. At examination 17-20 h after insemination 60.6% of the total oocytes had reached at least the 2nd metaphase. The proportions of oocytes penetrated (i.e. enlarged sperm head or male pronucleus and corresponding sperm tail) were 0, 10.0 and 16.7% with ejaculated spermatozoa, and 3.3, 19.6 and 26.4% with epididymal spermatozoa preincubated in modified KRB, oviduct and uterus, respectively. Although the proportion of oocytes with morphologically normal male and female pronuclei was low (10/36 = 27.8%), the results suggest that boar spermatozoa can be capacitated in the isolated genital tract of an oestrous sow and that capacitation of epididymal is better than that of ejaculated spermatozoa.     

Effect of seminal plasma on the motility of epididymal and ejaculated spermatozoa of the ram and bull during the cryopreservation process

Experiments were conducted to investigate the effect of seminal plasma on sperm motility during the cryopreservation process. Ejaculated and epididymal spermatozoa from the ram and the bull were washed by centrifugation and resuspended in either seminal plasma or a modified Tyrode's medium (TALP) prior to dilution in medium suitable for cryopreservation. Resuspension of washed ejaculated ram spermatozoa in seminal plasma resulted in higher percentages of motile spermatozoa than resuspension in TALP after the spermatozoa were cooled to 5 degrees C (52 vs 35%), and after thawing (14 vs 9%), respectively. Resuspension of epididymal ram spermatozoa in seminal plasma had no beneficial effect in maintaining sperm motility after cooling (78 vs 73%); however, seminal plasma was beneficial to epididymal ram spermatozoa after thawing (34 vs 3%), respectively. Resuspension of washed ejaculated bull spermatozoa in either seminal plasma or TALP had no effect on the percentage of motile spermatozoa after cooling to 5 degrees C (73 vs 75%) or after thawing (60 vs 60%), respectively. In addition, seminal plasma had no beneficial effect on the percentage of motile epididymal bull spermatozoa when compared with that of TALP-treated spermatozoa after cooling (75 vs 72%) or after thawing (66 vs 63%), respectively. Seminal plasma from different sires (ram and bull) affected epididymal sperm motility. The ability of sperm cells to withstand damage during cryopreservation, however, appears to reside in the sperm cells themselves, probably due to sperm cell composition.     

In vitro fertilization of porcine oocytes with fresh and frozen-thawed ejaculated or frozen-thawed epididymal semen obtained from identical boars

The objective of this study was to compare the in vitro fertilizing capacity of porcine spermatozoa from fresh and frozen-thawed semen and frozen-thawed epididymal spermatozoa obtained from identical boars. Prior to IVF, fresh spermatozoa were capacitated in TCM 199. Frozen semen samples were stored in 0.25-ml plastic straws using a lactose/glycerol/orvus-es-paste extender. Cumulus-oocyte-complexes (COC) obtained from superovulated prepuberal gilts were fertilized in vitro within 2 h after aspiration with one of the semen samples. After final dilution for IVF, frozen-thawed epididymal semen samples showed motility rates (72.2 +/- 5.6%) similar to those of spermatozoa in fresh semen (76.4 +/- 4.5%), while sperm motility decreased in frozen-thawed ejaculated semen (40.2 +/- 9.4%). Considerable individual differences in sperm motility between boars were observed for ejaculated semen but not for epididymal semen. Enhanced fertilizing capacity of frozen-thawed epididymal spermatozoa was confirmed by pronucleus formation and cleavage rates, with significantly more embryos developing to the 2- and 4-cell stages compared with the groups fertilized with fresh or with frozen-thawed ejaculated semen (59.7 vs 14.6 and 16%). In conclusion, consistent in vitro fertilization rates with minimal semen variability are obtained using frozen-thawed epididymal semen. Following a modified freezing protocol, epididymal spermatozoa can easily be frozen in small containers for IVF, with higher resultant motility and fertilization rates than with ejaculated semen.     

ATP-induced reactivation of ram testicular, cauda epididymal, and ejaculated spermatozoa extracted with Triton X-100

It was possible to demembrante and reactivate not only freshly collected testicular, cauda epididymal, and ejaculated ram sperm but also sperm that had been stored for several days at 0 degrees C and for several months at -196 degrees C in rete testis fluid or egg yolk citrate media. Sperm were usually washed free of seminal plasma before demembranation, but this was not essential for reactivation. Bovine serum albumin (1.0%) in the wash medium increased the survival of sperm, but more than 0.25% in the extraction medium decreased reactivation. A macro-molecular component of cauda epididymal fluid also inhibited the reactivation of testicular sperm. Triton X-100 concentrations between 0.01% and 1.00% in the extraction medium were satisfactory for demembranating the sperm. Rapid cooling (i.e., cold shock) mimicked the effect of detergent in making the sperm responsive to added ATP and demonstrated that damage to ram sperm in cold shock does not involve the axoneme. Ejaculated and cauda sperm were reactivated immediately on addition of ATP and activity persisted for up to 10 min. Testicular sperm, on the other hand, required about 4 min to become fully reactivated. The optimal ATP concentration for activation of sperm was 0.1-1.0 mM. Magnesium ions (0.1-1.0 mM) were important for reactivation, and testicular sperm required a higher magnesium concentration than did cauda or ejaculated sperm. Manganese ions were almost as effective as magnesium for reactivating cauda epididymal and ejaculated sperm. Cobalt and cadmium ions were much less active for cauda and ejaculated sperm and none of these ions were effective for testicular sperm. Fluoride (25-50 mM) inhibited reactivation. The presence of 50 microM cAMP in the extraction medium or preincubation of testicular sperm with theophylline or caffeine increased low levels of activation, but this was not evident with ejaculated or cauda sperm. We conclude that the motor apparatus is already functionally assembled in spermatozoa on leaving the testis, but some fine adjustment must take place during maturation in the epididymis.     

Effects of exposure of epididymal boar spermatozoa to seminal plasma on the binding of zona pellucida proteins during in vitro capacitation

The purpose of the investigation was to determine whether seminal plasma plays a role in the increase during in vitro capacitation of the number of boar spermatozoa with enhanced binding of zona pellucida proteins. Ejaculated spermatozoa and spermatozoa collected from the caudae epididymides of boars were incubated at 39 degrees C in a Tyrode's IVF medium. During incubation, the zona binding ability of individual spermatozoa was assessed with fluorescein-conjugated solubilized zona pellucida proteins (FITC-sZP), using a flow cytometer. Propidium iodide (PI) was included to simultaneously monitor cell viability. During incubation of ejaculated spermatozoa, a percentage of the spermatozoa expressed enhanced binding of FITC-sZP. The percentage of viable spermatozoa with enhanced binding reached a maximum of 37% (S.D.=8, averaged over five boars) after 2-3 h. In epididymal sperm, a similar maximum was observed after incubation in vitro, but a longer time of incubation was needed (6 h). Also, the rate of cell death of epididymal sperm was much lower than that of ejaculated sperm. When epididymal spermatozoa was exposed to seminal plasma in vitro, the time needed to reach a maximal percentage of viable spermatozoa with enhanced FITC-sZP binding was similar to that in ejaculated semen. However, the rate of cell death was still much lower than in ejaculated sperm. We concluded that the binding sites on the sperm surface that are involved in the increased binding of zona proteins during incubation under IVF conditions were not derived from the seminal plasma. The cellular processes leading to the increased binding capacity were accelerated by exposure of the sperm to seminal plasma.     

Evaluation of rabbit sperm acrosomal integrity and fertilizing ability by use of vital stains.

Three staining procedures to detect sperm acrosome integrity were compared via electron microscopy. Stains were applied to epididymal, freshly ejaculated, in vivo capacitated, and sonicated sperm cells in addition to spermatozoa displaying sequentially removed plasma and outer and inner acrosomal membranes. Sequential membrane removal procedures resulted in removal of plasma membranes from 73% of all sperm cells, removal of plasma and outer acrosomal membranes from 74% of all sperm cells, and removal of plasma and outer and inner acrosomal membranes from 87% of all sperm cells as determined by electron microscopy. Live/dead staining results were not statistically different from subjective microscopic motility evaluations (P less than 0.005) for epididymal, sonicated, freshly ejaculated, and in vivo capacitated sperm samples. All three stains assessed were similarly capable of detecting the acrosome status of freshly ejaculated and of sonicated spermatozoa compared to data obtained by electron microscopy (P = 0.010). However, only the Bryan-Akruk stain afforded data that were closely correlated with data obtained via electron microscopy for all sperm types assessed; the latter included in vivo capacitated spermatozoa and sperm cells rendered free of plasma membranes. Results confirmed an earlier report by successfully effecting sequential removal of rabbit acrosomal membranes and documented use of the Bryan-Akruk acrosomal stain for evaluation of sperm cell populations for fertilizing ability. These findings should prove useful in further investigations of mechanisms involved in achievement of fertilizing ability by rabbit spermatozoa.     

Use of triple stain technique for simultaneous assessment of vitality and acrosomal status in boar spermatozoa

The object of this study was to adapt the triple stain technique to diluted and incubated boar spermatozoa. Freshly ejaculated semen was resuspended in MR-A diluent to contain 3x10(7) cells/ml (diluted spermatozoa) and was subsequently capacitated (incubated spermatozoa). Experiments were conducted to show the conditions required for optimal staining quality and validation of triple stain technique. The most satisfactory staining solutions for diluted spermatozoa were 2% Trypan blue at 37 degrees C for 15 minutes, 0.8% Bismarck brown in 30% ethyl alcohol (pH 2.8) at 40 degrees C for 10 minutes and 0.8% rose Bengal in 0.1 M of Tris (pH 4.3) at 21 degrees C for 20 minutes. Satisfactory results were obtained for incubated spermatozoa with rose Bengal when the staining time was 10 minutes. Triple stain technique seemed to be a useful method for the simultaneous assessment of sperm vitality and acrosomal status; consequently, it should be valuable tool, for use in porcine in vitro fertilization systems.     

Horn quality and postmortem sperm parameters in Spanish ibex (Capra pyrenaica hispanica)

The horns are secondary sexual characteristics used by males of many ungulate species for intra-sexual fights during the rut. Thus, the dominant males with most developed horns are naturally selected for reproduction. Several studies have suggested that the quality of the horn, in many wild ruminants, may be correlated with semen quality. The aim of the present study was to determine whether inter-individual differences in levels of horn asymmetry and horn size are related to differences in sperm quality in a wild population of Spanish ibex by the assay of epididymal spermatozoa collected postmortem. In order to test this hypothesis we collected morphometric horns data from a total of 59 mature males (9-15 years of age) that were legally hunted during rutting season. The testicles were recovered, and the collection of epididymal spermatozoa was done at different times after death (2-60 h). The percentage of motile spermatozoa, motility rate, plasma membrane integrity, sperm viability, sperm morphology, and acrosome integrity were evaluated. Our findings showed that viable epididymal spermatozoa may be retrieved from dead animals many hours after death. However, sperm parameters were affected by the elapsed time between the death of the animal and spermatozoa collection. The study revealed that the horn quality was firstly associated with sperm motility.     

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.     

Sperm treatment affects capacitation parameters and penetration ability of ejaculated and epididymal boar spermatozoa

This work was designed to study how this ability is affected by different sperm treatments routinely used for in vitro fertilization (IVF) assay. In this study, boar sperm samples from epididymal or ejaculated origin were processed by three different methods: left unwashed (NW group), washed in Dulbecco's phosphate-buffered saline supplemented with 0.1% BSA (BSA group), and washed on a Percoll^® gradient (PERCOLL group). After preparation of semen samples, changes in motility patterns were studied by CASA, calcium uptake by spectrofluorimetry, and ROS generation, spontaneous acrosome reaction, and lipid disorder by means of flow cytometry. Finally IVF assays were also performed with the different semen samples and penetrability results evaluated at 2 and 4 h post insemination (hpi). Independently of the sperm treatment, epididymal spermatozoa showed higher values of progressive motility, percentage of live cells with low lipid disorder, and penetration ability at 4 hpi than the corresponding ejaculated spermatozoa. Ejaculated spermatozoa showed higher levels of calcium uptake, ROS generation and percentage of spontaneous acrosome reaction than epididymal sperm. Regarding sperm treatments, PERCOLL group showed the highest values for some motility parameters (linearity of the curvilinear trajectory, straightness, and average path velocity/curvilinear velocity), ROS generation and penetration ability at 2 and 4 hpi; however this same group showed the lowest values for sperm curvilinear velocity and lateral head displacement. From all experimental groups, ejaculated-PERCOLL-treated spermatozoa showed the highest fertilization ability after 2 hpi. Results suggest that capacitation pathways can be regulated by suitable treatments making the ejaculated sperm able to reach capacitation and fertilize oocytes in similar levels than epididymal spermatozoa, although most of the studied capacitation-associated changes do not correlate with this ability.     

Full-term development of rats from oocytes fertilized in vitro using cryopreserved ejaculated sperm

For preservation of rat spermatozoa, the general-purpose method requires that the male be sacrificed for collection of spermatozoa from the epididymides. However, it would be highly useful if the ejaculated spermatozoa could be successfully cryopreserved and the frozen–thawed spermatozoa used for in vitro fertilization, since this would allow the genetically valuable rats to be maintained alive rather than sacrificed. The aim of the present study was to clarify whether ejaculated rat spermatozoa could be successfully cryopreserved and fertilized in vitro. The motility and viability of frozen–thawed ejaculated spermatozoa were similar to those of frozen–thawed epididymal spermatozoa (around 10%). The percentage of acrosomal integrity in epididymal spermatozoa was significantly higher than that in ejaculated spermatozoa after freezing/thawing. The level of capacitation-associated protein tyrosine phosphorylation in frozen–thawed ejaculated sperm was slightly increased at 5 h. When the frozen–thawed ejaculated spermatozoa were used for in vitro fertilization, the percentages of fertilization, pronuclear formation, and development to the 2-cell stage (26.5%, 23.0%, and 91.0%, respectively) were similar to those of frozen–thawed epididymal spermatozoa (19.4%, 15.0%, and 84.1%, respectively). However, the rate of blastocyst formation in the ejaculated group was significantly lower than that in the epididymal group (12.0% vs 43.2%). Results from the embryo transfer experiment showed that the proportions of embryos developed to term were similar between the ejaculated (47.7%) and epididymal groups (53.7%). We showed here for the first time that ejaculated spermatozoa can be cryopreserved and the frozen–thawed sperm could be developed to term via in vitro fertilization in rats.     

Boar sperm plasma membrane protein profile: Correlation with the zona-free hamster ova bioassay

This study was designed to compare differences among porcine sperm plasma membrane proteins with the ability of spermatozoa to interact with zona-free hamster ova. Sperm plasma membrane vesicles were recovered from 24 ejaculates from 10 fertile boars, and from cauda epididymal spermatozoa from 3 fertile and 1 very subfertile boar. Solubilized sperm plasma membrane proteins were run on 1D SDS-PAGE gels, transferred to western blots, stained, and analyzed for quantity of protein per band by scanning laser densitometry. Variation in the quantities of individual sperm plasma membrane proteins in the 20 identified bands were statistically compared with the ability of spermatozoa from the same ejaculate to penetrate zona-free hamster ova. The percentages of plasma membrane protein present in 3 bands (90, 84 and 60 kD) were positively correlated with the ability of spermatozoa from the same ejaculate to fuse with zona-free hamster ova (P = 0.002, 0.01, 0.04; R = 0.53, 0.40, 0.38, respectively). The quantities of protein in 2 other bands (69 and 35 kD) were significantly but negatively correlated with the results of the zona-free hamster ova bioassay (P = 0.02, 0.01; R = -0.42, -0.37, respectively). The sperm plasma membrane profiles were quantitatively similar between the ejaculated samples and the fertile epididymal samples. Six epididymal sperm plasma membrane proteins were present in statistically different quantities in the subfertile boar sample and the 3 fertile controls. The 90 kD band positively correlated with the hamster ova bioassay in the ejaculated samples was not detected in the subfertile epididymal sperm plasma membrane sample. These results suggest that protein(s) in one or more of the 3 positively correlated ejaculated sperm plasma membrane protein bands may be involved in sperm-oocyte interaction.