Alejandro Maroto-Morales

Reactive oxygen species generators affect quality parameters and apoptosis markers differently in red deer spermatozoa

Fe(2)(+)/ascorbate, hydrogen peroxide (H(2)O(2)), and hypoxanthine/xanthine oxidase (XOD) are commonly used for inducing oxidative stress on spermatozoa. A comparative study of these agents was carried out on thawed spermatozoa from red deer. First, we tested a high, medium, and low concentration of each agent: 100, 10, and 1 microM Fe(2)(+) (hydroxyl radical generator); 1 mM, 100, and 10 microM H(2)O(2); and 100, 10, and 1 mU/ml XOD (superoxide and H(2)O(2) generator), incubated at 37 degrees C for 180 min. Intracellular reactive oxygen species (ROS; H(2)DCFDA) increased with dose and time similarly for the three systems at each concentration level. Motility and mitochondrial membrane potential (Deltapsi(m)) were considerably decreased by H(2)O(2) (1 mM and 100 microM) and XOD (100 and 10 mU/ml). Only 1 mM H(2)O(2) reduced viability. The antioxidant Trolox (10 microM) reduced intracellular ROS, but could not prevent the H(2)O(2) or XOD effects. In a second experiment, YO-PRO-1 and M540 were used as apoptotic and membrane stability markers respectively. Only H(2)O(2) increased the proportion of apoptotic and membrane-destabilized spermatozoa. Catalase added to XOD prevented Deltapsi(m) loss, confirming that H(2)O(2) was the causative agent, not superoxide. In a third experiment, caspase activation was tested using the (FAM-VAD-FMK) probe. Viable spermatozoa with activated caspases could be detected in untreated samples, and only H(2)O(2) increased their proportion after 60 min. There were important differences between ROS generators, H(2)O(2) being the most cytotoxic. Although H(2)O(2) and XOD caused Deltapsi(m) dissipation, this was not reflected in increasing apoptotic markers.

Heterologous in vitro fertilization is a good procedure to assess the fertility of thawed ram spermatozoa

A heterologous in vitro fertilization (IVF) test using calf oocytes with zona pellucida was employed to assess the fertility of thawed ram sperm samples. Six males with significant differences in fertility (P=0.003) were used. The males were classified as having high fertility (>or=42%) and low fertility (<or=41%). Male fertility was not influenced by number of inseminated ewes (P=0.584), insemination technician (P=0.156), insemination date (P=0.323) or farm (P=0.207). Thawed sperm samples were employed to assess several sperm parameters for each male: motility, acrosomal integrity, viability, membrane stability, membrane phospholipid disorder, mitochondrial membrane potential and chromatin stability. These samples were used to carry out a heterologous in vitro fertilization. In vitro-matured calf oocytes (n=716) were inseminated with thawed ram semen and in vitro cultured for 40 h. Overall, at thawing, variability among males respect to sperm quality was high. Despite this variability, there were not differences (P<0.05) between fertility groups. Yield of hybrid embryos ranged from 31 to 59% between males. There were not differences between males (P=0.340). However, there were differences between fertility groups (high fertility: 55%; low fertility: 39%; P=0.020). Multiple regression analysis showed that the heterologous in vitro fertility was the only predictive parameter for in vivo male fertility. Correlation between both parameters was fair (r(2)=0.760; P=0.025). These results indicate that heterologous in vitro fertilization tests can be useful to predict the fertility of ram spermatozoa using calf oocytes with intact-zona pellucida.

Cryopreservation of Iberian red deer (Cervus elaphus hispanicus) spermatozoa obtained by electroejaculation.

We tested extenders and freezing protocols for Iberian red deer semen. Samples were obtained by electroejaculation (10 stags), and analyzed for motility (CASA), viability (propidium ioide), acrosomal (PNA-FITC) and mitochondrial status (JC-1). Samples were diluted 1+1 in extender, cooled and adjusted for glycerol (extender with higher glycerol concentration), brought to 160 x 10(6)mL(-1) and frozen. Four experiments were carried out, repeating sperm analysis after thawing to compare treatments. In a first experiment, seven samples were frozen using Triladyl (20% egg yolk) and UL extender (Tes-Tris-fructose, 15% egg yolk, 4% glycerol). Triladyl yielded higher motility after thawing. In a second trial, 17 samples were frozen using Triladyl, Andromed, Bioxcell, and UL with 8% LDL (low-density lipoproteins). Triladyl, and Andromed performed better than Bioxcell on motility, and than UL-LDL on viability and acrosomal status. In a third experiment, the performance of freezing the sperm-rich ejaculate fraction versus the whole ejaculate was tested on nine samples. The sperm-rich ejaculate fraction not only rendered more motile and viable spermatozoa but also showed higher freezability (higher motile spermatozoa recovery). In a fourth experiment, we tried three modifications of the freezing protocol, for improving the freezability of low concentration samples: prior removal of seminal plasma; replacing extender (second fraction) for pure glycerol to reduce dilution; and performing only the 1+1 dilution, not the second dilution. No differences were found, although only three samples could be used. Both Triladyl and Andromed were deemed appropriate for freezing Iberian red deer semen, and the rich fraction should be selected for freezing.

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.

Washing increases the susceptibility to exogenous oxidative stress in red deer spermatozoa

The effects of routine sperm work are often overlooked. We assessed the effect of washing cryopreserved epididymal spermatozoa from red deer (Cervus elaphus hispanicus, Helzheimer 1909). After thawing, epididymal samples (four stags) were diluted in TALP-HEPES. A split was left untouched, another was centrifuged (300 x g, 5 min) and resuspended, and a third was centrifuged and the supernatant substituted by fresh TALP-HEPES (washing). Each split was supplemented either with nothing, 1mM of the antioxidant Trolox, 100 microM of the oxidant Fe (with ascorbate), or both. The 3x4 treatments were incubated at 37 degrees C and assessed each hour up to 3h for motility (computer-aided sperm assessment) and viability/apoptosis plus mitochondrial status (YO-PRO-1, propidium iodide, Mitotracker Deep Red; flow cytometry). DNA damage at 4h was assessed using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay. Centrifugation alone affected neither sperm quality nor DNA, and the oxidant had no effect in control or centrifuged samples. Washed samples were not different than control, but oxidant decreased motility, mitochondrial status and viability, and altered the motility subpopulation pattern, being partially suppressed by Trolox. Spermatozoa with damaged DNA dramatically increased in the washed-oxidized sample (from 22.30+/-3.52% to 67.94+/-5.07%), but not when antioxidant was present. Although samples from different males behaved similarly, male-to-male variability was detected regarding susceptibility to oxidative damage after washing. We concluded that, although red deer thawed spermatozoa seemed resilient to centrifugation, the vulnerability to oxidative stress after washing makes it advisable to supplement manipulation media with antioxidants, especially taking into account male-to-male variability.

Characterization of ram (Ovis aries) sperm head morphometry using the Sperm-Class Analyzer.

Sperm morphology has been identified as a characteristic that can be useful in the prediction of fertilizing capacity. The aim of the current study was to characterize ram sperm heads morphometrically as a basis for future studies on the relationship between sperm quality and male fertility. For this purpose, ejaculates from 241 mature rams (Ovis aries) belonging to 36 different dairy herds were used to evaluate sperm head morphometry by means of the Sperm-Class Analyzer. Sperm samples, collected by artificial vagina, were diluted in phosphate buffered saline (PBS) for the analysis. A microscope slide was prepared from single-diluted fresh sperm samples. Slides were air-dried and stained with Hemacolor. A minimum of 115 sperm heads were analyzed from each male. Each sperm head was measured for four primary parameters (area, perimeter, length, width), and four derived parameters of head shape were obtained. Significant differences in sperm head morphometry were found between rams (CV for morphometric parameters ranging from 0.9 to 10.1), and there were marked differences in the sperm morphometric composition of the ejaculates. For all parameters, within-animal CVs were greater than between-animal CVs. Within-animal CVs ranged from 4.2 to 10.6, showing the high degree of sperm polymorphism present in the sheep ejaculate. Significant differences in sperm head morphometry were found between rams belonging to the different herds (i.e., origin). An important part of the variability observed on morphometric parameters was due to the male itself, with an explained variance ranging from 3.6% for regularity to 34.0% for p2a (perimeter(2)/[4xpixarea]). The explained variance by the herd of origin of the males ranged from 0.6% for regularity to 10.8% for area. Our results suggest that a genetic component might be responsible for the observed sperm head morphometry differences between herds.

Sperm characteristics and in vitro fertilization ability of thawed spermatozoa from Black Manchega ram: Electroejaculation and postmortem collection

The aim of this study was to assess two models of sperm collection on the quality and fertility of thawed spermatozoa from Black Manchega rams, a threatened breed. Sperm samples were collected by electroejaculation and postmortem from each male. Samples were diluted with Biladyl and frozen. Motility (subjective and objective by means of computer-assisted semen analysis), membrane integrity, and acrosomal status (microscopy) were assessed on fresh and thawed semen; plasmalemma integrity, mitochondrial membrane potential, DNA integrity, and acrosomal status were evaluated by flow cytometry on thawed semen. Thawed spermatozoa were used in a heterologous in vitro fertilization test. After thawing, the proportion of live spermatozoa with intact membrane (YO-PRO-1-/PI-) was higher for postmortem samples (P<0.001), although the ratio of YO-PRO-1- spermatozoa within the PI- population was higher for ejaculated samples (P=0.007). Likewise, the proportion of live spermatozoa having high mitochondrial membrane potential (MitoTracker+) and intact acrosomes (PNA-) was higher for postmortem samples (P<0.001 and P<0.001, respectively). Considering only live spermatozoa, the ratio of MitoTracker+/PNA- cells was higher for electroejaculated samples (P=0.026 and P=0.003). Both electroejaculated and postmortem samples fertilized oocytes. Nevertheless, electroejaculated samples yielded a higher percentage of hybrid embryos (P=0.041). In conclusion, although postmortem spermatozoa had better sperm quality after thawing, electroejaculated spermatozoa showed higher ratios for sperm quality when only the live population was considered. Electroejaculated and postmortem samples might be used for germplasm banking of this threatened breed, but the fertility of postmortem spermatozoa might be lower.

Analysis of selected sperm by density gradient centrifugation might aid in the estimation of in vivo fertility of thawed ram spermatozoa

The aim of the present study was to evaluate the effect of selecting a sperm subpopulation by means of a discontinuous density gradient centrifugation (DGC) on the quality of ram thawed semen, and the relationships between sperm parameters assessed in unselected and in selected sperm samples with in vivo fertility after intrauterine artificial insemination (IUI) using unselected sperm samples. Semen samples from twenty males were collected by artificial vagina and cryopreserved following a standard protocol. After thawing, unselected sperm samples were used in an in vivo fertility trial and sperm motility (subjective and objective, assessed by means of CASA) and membrane and acrosomal integrities (microscopy) were evaluated on unselected and selected sperm samples. In addition, plasmalemma integrity (YO-PRO-1/PI), membrane fluidity (Merocyanine 540/YO-PRO-1), mitochondrial activity (Mitotracker Deep Red/YO-PRO-1), and DNA fragmentation index (%DFI) assessed by Sperm Chromatin Structure Assay (SCSA) were evaluated by flow cytometry before and after sperm processing using DGC. Results showed that DGC improved all sperm parameters significantly, except the %DFI, which increased after the selection procedure. No relationships were found between sperm parameters evaluated in unselected sperm samples and in vivo fertility. However, we found a positive correlation between spermatozoa with high membrane fluidity within the viable sperm population (VIABMerocyanine+) evaluated in selected sperm samples and in vivo fertility (r = 0.370, P = 0.019). In conclusion, our results suggest that selected spermatozoa represent a sperm subpopulation different to the unselected one that could be related with the in vivo fertility.

Effect of semen collection method (artificial vagina vs. electroejaculation), extender and centrifugation on post-thaw sperm quality of Blanca-Celtibérica buck ejaculates.

The aim of this study was to evaluate the effect of semen collection method (artificial vagina compared to electroejaculation), season in which the semen was collected (breeding season compared to non-breeding season), freezing extender (Biladyl(®), Andromed(®) and skim milk based extender) and pre-treatment procedure (washing compared to non-washing) on post-thaw semen quality in buck. Ejaculates from seven bucks of the Blanca-Celtibérica breed were collected by artificial vagina and electroejaculation during the breeding (July to December) and non-breeding season (January to June). Samples were split in two aliquots and one of them was washed. Three freezing extenders were evaluated on washing and non-washing sperm samples. Ejaculates collected by artificial vagina had a greater sperm quality after thawing, with greater values (P≤0.05) for SM (sperm motility), NAR (acrosome intact), YO-PRO-1-/PI- (intact spermatozoa), and Mitotracker+/YO-PRO-1- (spermatozoa with active mitochondria) and lower % DFI (DNA fragmentation index). Thawed sperm samples which were collected during the breeding season had greater values (P≤0.05) for NAR, intact spermatozoa and spermatozoa with active mitochondria, than those semen samples obtained during the non-breeding season. Semen freezing with Biladyl(®) and Andromed(®) resulted in a greater sperm quality (P≤0.05) after thawing in relation to milk-based extender. Washing procedure had no effect on sperm parameters assessed at thawing. Results from the present study suggest that the success of semen cryopreservation in Blanca-Celtibérica goat depends on semen collection method and season, as well as on the extender used. Thus, the post-thaw sperm quality will be greater (P≤0.05) when samples are collected by artificial vagina during the breeding season and when Biladyl(®) or Andromed(®) are used as freezing extenders.

Single layer centrifugation (SLC) improves sperm quality of cryopreserved Blanca-Celtibérica buck semen

The aim of the present study was to evaluate the effect of sperm selection by means of single layer centrifugation (SLC) on sperm quality after cryopreservation, either when SLC is used before freezing or after thawing, using Blanca-Celtibérica buck semen collected by electroejaculation (EE). Ejaculates from six bucks were collected by EE and divided into two aliquots. One of them (unselected) was diluted with Biladyl(®) by the two-step method and frozen over nitrogen vapor. The other aliquot was selected by the SLC technique and subsequently frozen in the same way as the unselected samples (SLC before freezing). In a further treatment, two unselected straws were thawed and SLC was carried out (SLC after thawing). At thawing, sperm motility of all samples ((i) unselected; (ii) selected before freezing and (iii) selected after thawing) was evaluated by CASA. In addition, integrity of the plasma membrane, mitochondrial membrane potential, ROS production and DNA fragmentation index were assessed by flow cytometry. Most of the sperm parameters were improved (P≤0.001) in samples selected by SLC after thawing in relation to unselected or selected by SLC before freezing. The percentage of progressive motile spermatozoa was greater (86%) for sperm samples selected after thawing compared with unselected (58%) or selected before freezing (54%). Moreover, percentages of spermatozoa with intact plasma membrane and spermatozoa with high mitochondrial membrane potential (hMMP) were also greater for sperm samples selected after thawing compared to sperm samples unselected or selected before freezing (spermatozoa with intact plasma membrane: 80% vs. 32% vs. 12%; spermatozoa with hMMP: 54% vs. 1% vs. 15%; respectively). Therefore, sperm quality after cryopreservation is improved in Blanca-Celtibérica buck ejaculates collected by EE when a sperm selection technique such as SLC is carried out after thawing.