Paulino de Paz

Sperm Subpopulations in Iberian Red Deer Epididymal Sperm and Their Changes Through the Cryopreservation Process

Abstract We have applied a statistical protocol based on principal component analysis, clustering methods, and discriminant analysis for the identification of sperm subpopulations in computer-assisted sperm analysis (CASA) data. Samples were obtained from the cauda epididymis of 11 Iberian red deer and cryopreserved following a standard protocol. Motility by CASA was analyzed just after sperm recovery, just before freezing, and after thawing, and eight motility descriptors for each individual spermatozoon were recorded. Sperm viability and acrosomal status were also assessed. Subpopulation analysis was performed in four sequential steps: principal component analysis using the eight motility descriptors; nonhierarchical clustering analysis (k-means) using the first two principal components; hierarchical clustering analysis (UPGMA); and selection of the final number of clusters. Three clusters were obtained for each motility analysis: slow and nonlinear; rapid and linear; and rapid, high ALH, nonlinear. We detected variations in the clusters between treatments (initial, prefreezing and postthawed). Indeed, motility increased and linearity decreased in the prefreezing analysis. A discriminant analysis isolated three descriptors that were used again in the same statistical analysis, giving four clusters that resembled the pattern found in the first classification. We also performed a clustering analysis of the males according to prefreezing/postthawed variation of total motility, viability, and acrosomal status. The proportion of the linear subpopulations in the prefreezing treatment, in both clustering analyses, correlated positively with postthawed viability recovery. Our results show that clustering analysis of CASA data gives useful and practical information that is not obtained by conventional sperm analysis.

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 animals 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 animals 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 animals 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 animals death.

Statistical Series: Opportunities and challenges of sperm motility subpopulation analysis.

Computer-assisted sperm analysis (CASA) allows assessing the motility of individual spermatozoa, generating huge datasets. These datasets can be analyzed using data mining techniques such as cluster analysis, to group the spermatozoa in subpopulations with biological meaning. This review considers the use of statistical techniques for clustering CASA data, their challenges and possibilities. There are many clustering approaches potentially useful for grouping sperm motility data, but some options may be more appropriate than others. Future development should focus not only in improvements of subpopulation analysis, but also in finding consistent biological meanings for these subpopulations.

Reduced glutathione and Trolox (vitamin E) as extender supplements in cryopreservation of red deer epididymal spermatozoa

The use of assisted reproductive techniques in cervids is increasing as the commercial use of these species increase. We have tested the suitability of the antioxidants Trolox and reduced glutathione (GSH) for freezing red deer epididymal spermatozoa, aiming at improving post-thawing quality. Samples from 19 stags were frozen in a TES-Tris-fructose extender (20% egg yolk, 8% glycerol), with 1 or 5 mM of antioxidant. Motility (CASA), lipoperoxidation (malondialdehyde -MDA- production), membrane status, mitochondrial activity, acrosomal status (flow cytometry) and chromatin status (SCSA: %DFI and %HDS; flow cytometry) were assessed after thawing and after 6 h at 39°C. There were few differences between treatments after thawing, with Trolox reducing MDA production in a dose-response manner. After the incubation, sperm quality decreased and %DFI increased moderately, with no change for MDA. GSH improved motility, kinematic parameters and mitochondrial status, with a slight increase in %HDS. GSH 5 mM also increased moderately MDA production and %DFI, possibly due to enhanced metabolic activity and reducing power. Trolox maintained MDA low, but was detrimental to sperm quality. Trolox might not be appropriate for the cryopreservation of red deer epididymal spermatozoa, at least at the millimolar range. GSH results are promising, especially regarding motility improvement after the post-thawing incubation, and should be selected for future fertility trials.

The relationship between ram sperm head morphometry and fertility depends on the procedures of acquisition and analysis used.

Sperm head morphometry is a parameter in the evaluation of semen that has been associated with fertility in two ways: comparing morphometric measures between predefined groups of fertility; or analyzing morphometric data by multivariate techniques to identify cell populations. We analyzed the morphometry of ram sperm head by three procedures and checked its relationship with male fertility. A Computer-Aided Sperm Morphometric Assessment procedure (CASMA), an image analysis software (NIS-Elements) in combination with an optical microscope (MO-NIS) and this image analysis software in combination with a scanning electron microscope (SEM-NIS) were used. Eight morphometric parameters were assessed: length, width, area, perimeter, ellipticity, form factor, elongation and regularity. We observed significant differences between the morphometric data of sperm head obtained with three study procedures. The CASMA procedure shows the highest values for all parameters and the SEM-NIS procedure the lowest. The analysis of a semen sample, when only the mean of morphometric parameters is used to describe the cell population, is too limited to interpret their fertilizing capacity. It is essential to analyze the complex structure of the samples by defining subpopulations by multivariate methods. With few exceptions, the means of each morphometric parameter differ between the three subpopulations analyzed in each procedure. Only the subpopulations obtained with the MO-NIS procedure showed a significant correlation with male fertility. In short, it is necessary to establish an instrumental standard for the analysis of sperm morphometry to obtain reliable results and we believe that the MO-NIS system presents these basic requirements.

Refrigerated storage of ram sperm in presence of Trolox and GSH antioxidants: Effect of temperature, extender and storage time

Antioxidants have a potential to improve the quality and fertility of refrigerated-stored ram semen. Reduced glutathione (GSH) and Trolox (0.2, 1 and 5mM) were evaluated in ram semen preserved at 15 and 5°C up to 48 and 96h, respectively. Extenders were also evaluated (15°C: Tris-citrate-fructose, TCF, without lipids, and TES-Tris-fructose 10% egg yolk, TTF-EY; 5°C: TTF-EY and 3.5% soybean lecithin, TTF-SL; INRA96 at both temperatures). Storage at 5°C resulted in poorer quality than 15°C up to 48h, while allowing acceptable quality at 96h. Antioxidants had few effects on sperm quality, with use of Trolox resulting in reduced motility and viability in TCF. Storage at 15°C in the TCF extender resulted in decreased motility, viability and mitochondrial activity compared with use of TTF-EY. Sperm quality when storage was at 5°C was similar, but storage in TTF-SL resulted in decreased motility and mitochondrial activity. Acrosomal status was only slightly affected by extender and antioxidant. Mitochondrial activity was improved by antioxidants in TTF-SL, and GSH at 5mM when semen was stored at 5°C in TTF-EY. A preliminary artificial insemination trial indicated that supplementation with GSH has the potential for improving lambing (P<0.1). In conclusion, use of antioxidants resulted in lesser effects than extender composition or storage time on quality of ram semen. Use of Trolox negatively impacted sperm quality and GSH had some positive impacts. The use of soybean lecithin requires further research to assess its impact on mitochondria.

POST-MORTEM SPERMATOZOA RECOVERY AND FREEZING IN A CANTABRIC BROWN BEAR (URSUS ARCTOS) : A PRELIMINARY REPORT

At present the Cantabric Brown Bear (Ursus arctos), which probably constitutes the last pure breed aggregate of brown bear in the world, is a seriously endangered population (~80 animals are living in a fragmentary area in the North of Spain). The harvesting of gametes obtained post-mortem could be a useful tool in creating a genetic resource bank. The present work is a preliminary report about post-mortem spermatozoa (spz) recovery in a Cantabric Brown Bear (7 years old, 170 kg), died 8 days after an accident in the wilds (3 rd May 1998). The testis was extracted 70 min post-mortem and the epididymis was dissected at 5°C. A sample of gametes was obtained from each epididymis region and deferens ductus (reference of the preejaculatory spermatic cells). The percentage and position of the cytoplasmic droplets (CD) were evaluated as a gamete maturity index (viability prediction).

The antioxidant effects of soybean lecithin- or low-density lipoprotein-based extenders for the cryopreservation of brown-bear (Ursus arctos) spermatozoa

Egg yolk low-density lipoproteins (LDL) and soybean lecithin were evaluated as replacements for egg yolk in extenders used for the cryopreservation of brown-bear spermatozoa. The motility, viability and acrosomal status of post-thawed spermatozoa were analysed, and an egg-yolk extender was used as a control. The total antioxidant capacity of these extenders was tested. Soybean lecithin showed an effect that was dependent on the soybean concentration (2%, 3.5% and 5%) and source (Type A: 24% L-α-phosphatidylcholine, and Type B: 14-23% L-α-phosphatidylcholine). Only semen cryopreserved with 5% Type A soybean exhibited a sperm motility similar to that of semen cryopreserved in egg-yolk-based extender after thawing, although the sperm viability and acrosome status were not as high. Semen frozen in an extender containing LDL (10-15%) exhibited improved sperm viability in comparison with the control, but sperm motility was lower. The LDL-based extender exhibited a higher anti-oxidant activity than the egg-yolk extender and soy lecithin-based extenders. The extenders with higher anti-oxidant activity showed improvements in frozen sperm viability but lower semen motility. These results indicate that soybean lecithin did not have the same protective effect as egg yolk during the freezing of brown-bear spermatozoa but suggest that LDL (10-15%) could be a useful substitute for egg yolk in these extenders.

Brown bear sperm double freezing: Effect of elapsed time and use of PureSperm gradient between freeze-thaw cycles

The use of sexed spermatozoa has great potential to captive population management in endangered wildlife. The problem is that the sex-sorting facility is a long distance from the semen collection place and to overcome this difficulty two freeze-thaw cycles may be necessary. In this study, effects of refreezing on brown bear electroejaculated spermatozoa were analyzed. We carried out two experiments: (1) to assess the effects of the two freezing-thawing cycles on sperm quality and to analyze three different elapsed times between freezing-thawing cycles (30, 90 and 180 min), and (2) to analyze the use of PureSperm between freezing-thawing cycles to select a more motile and viable sperm subpopulation which better survived first freezing. The motility, viability and undamaged acrosomes were significantly reduced after the second thawing respect to first thawing into each elapsed time group, but the elapsed times did not significantly affect the viability and acrosome status although motility was damaged. Our results with the PureSperm gradient showed higher values of viability in freezability of select sample (pellet) respect to the rest of the groups and it also showed a significant decrease in the number of acrosome damaged. In summary, the double freezing of bear semen selected by gradient centrifugation is qualitatively efficient, and thus could be useful to carry out a sex-sorting of frozen-thawed bear spermatozoa before to send the cryopreserved sample to a biobank. Given the low recovery of spermatozoa after applying a selection gradient, further studies will be needed to increase the recovery rate without damaging of the cell quality.

Ram spermatozoa migrating through artificial mucus in vitro have reduced mitochondrial membrane potential but retain their viability.

Sperm motility in vitro is one of the most common predictors of fertility in male screening. We propose that a mucus-penetration assay can isolate a cellular subpopulation critical to reproductive success. To this end, a device was designed with three modules (sample, test and collection) and its conditions of use evaluated (length of mucus, incubation time, mucus medium, sperm concentration and position in relation to the horizontal). The number of spermatozoa migrating and the viability and acrosomal status of the spermatozoa not migrating were calculated. The second objective was to evaluate the qualitative parameters of the spermatozoa migrating in 1.6% polyacrylamide for 30min. The number of spermatozoa migrating and the sperm motility, viability and the acrosomal and mitochondrial status of three sperm populations (fresh, not migrating and migrating) were determined. A higher number of migrating spermatozoa were observed after 60min of incubation, but this situation adversely affected sperm quality. The methylcellulose-based test showed a significantly lower number of migrating spermatozoa than the polyacrylamide test. The position at an angle of 45° resulted in a higher number of migrating spermatozoa in the polyacrylamide-based test. The sperm counts for three consecutive assays indicated an acceptable repeatability of the method. The viability and acrosomal status of the migrating spermatozoa showed no significant changes with regard to the control when the device was placed at 45°, whereas these parameters showed lower values at 0°. The percentage of high mitochondrial membrane potential spermatozoa was significantly reduced in the population of migrating spermatozoa.