REFLEXIONES SOBRE EL DISEÑO DE UNA POLÍTICA INSTITUCIONAL DE OPEN SCIENCE E IMPLICACIONES DE SU IMPLANTACIÓN

El presente trabajo tiene como objetivo exponer las reflexiones realizadas para definir las principales guías en el diseño e implantación de la política institucional de Open Science -OS- en la Universidad de Castilla-La Mancha -UCLM- para que ésta sea congruente con los compromisos adquiridos con la adhesión a la Declaración de Berlín y, en los últimos meses, con la asunción de los compromisos CRUE ante la OS. Partiendo del marco de obligaciones actual, derivado de los mandatos de ámbito nacional y europeo, y del diagnóstico de la situación de Open Access a partir de las estadísticas del repositorio institucional de la UCLM RUIdeRA (exclusivamente depósitos de publicaciones de investigación: artículos y tesis doctorales) intentaremos determinar la efectividad de los mandatos externos y la necesidad y conveniencia de diseñar nuestra política institucional de OS en términos de recomendación o requerimiento. Así mismo, se analizarán las implicaciones organizativas y económicas que podrían derivarse de la implantación y cuya anticipación modela la política de Ciencia Abierta a desarrollar. The present paper exposes some reflections made to define the main guidelines in the design and implementation of the Open Science institutional policy at the University of Castilla-La Mancha -UCLM-. This policy should be consistent with the commitments made with the adherence to the Berlin Declaration and, recently, with the assumption of CRUE commitments to the Open Science. Starting from the framework of current obligations, based on the mandates of National and European level, and on the diagnosis of the Open Access situation from the statistics of the institutional repository of the UCLM RUIdeRA (deposits of research publications exclusively: articles and doctoral theses), this paper tries to determine the success of those external mandates and the need and convenience of designing our own Open Science Institutional Policy in terms of recommendation or in terms of requirement. Furthermore, the organizational and economic implications that could be developed from its implementation are analysed

Catalase supplementation on thawed bull spermatozoa abolishes the detrimental effect of oxidative stress on motility and DNA integrity

P. 353-359The potential protective effect of catalase supplementation during in vitro culture of frozen/thawed bull spermatozoa was investigated. Frozen/thawed semen collected from three fighting bulls was diluted in phosphate buffered saline (PBS) and incubated at 37 °C under different experimental conditions: Control, Catalase (CAT) (200 U/mL), Oxidant (OXI) (100 μm Fe2+/1 mm ascorbate), and Catalase + Oxidant (CAT/OXI). We assessed sperm motility, acrosomal integrity, viability and chromatin status (SCSA®) at 0, 2 and 6 h of incubation. Our results showed that catalase abolished the effect of the oxidant, protecting spermatozoa against reactive oxygen species, and improving both sperm motility and chromatin status during incubation. The OXI treatment significantly reduced the percentage of motile sperm after 6 h of incubation. The statistical model also showed that there were differences in sperm motility between CAT/OXI (20.8 ± 2.9%) and OXI (11.6 ± 7.6%) (p < 0.001). There were no significant effects of OXI on sperm viability, acrosomal status or proportion of abnormal tails. %DFI (spermatozoa with moderate or high DNA Fragmentation Index) was significantly higher on OXI (p < 0.001). Catalase prevented DNA fragmentation even in the presence of the oxidant (%DFI: 30.3 ± 0.8% OXI vs. 17.4 ± 0.7% CAT/OXI). We conclude that catalase supplementation after thawing could protect bull spermatozoa against oxidative stress, and it could improve media used for processing thawed spermatozoa

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

P. 1073-1084The 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 × 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, 1 mM of the antioxidant Trolox, 100 μM of the oxidant Fe (with ascorbate), or both. The 3 × 4 treatments were incubated at 37°C and assessed each hour up to 3 h 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 4 h 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.S

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

P. 225–235Fe2+/ascorbate, hydrogen peroxide (H2O2), 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 μM Fe2+ (hydroxyl radical generator); 1 mM, 100, and 10 μM H2O2; and 100, 10, and 1 mU/ml XOD (superoxide and H2O2 generator), incubated at 37 °C for 180 min. Intracellular reactive oxygen species (ROS; H2DCFDA) increased with dose and time similarly for the three systems at each concentration level. Motility and mitochondrial membrane potential (Δψm) were considerably decreased by H2O2 (1 mM and 100 μM) and XOD (100 and 10 mU/ml). Only 1 mM H2O2 reduced viability. The antioxidant Trolox (10 μM) reduced intracellular ROS, but could not prevent the H2O2 or XOD effects. In a second experiment, YO-PRO-1 and M540 were used as apoptotic and membrane stability markers respectively. Only H2O2 increased the proportion of apoptotic and membrane-destabilized spermatozoa. Catalase added to XOD prevented Δψm loss, confirming that H2O2 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 H2O2 increased their proportion after 60 min. There were important differences between ROS generators, H2O2 being the most cytotoxic. Although H2O2 and XOD caused Δψm dissipation, this was not reflected in increasing apoptotic markers.S

Quality, oxidative markers and DNA damage (DNA) fragmentation of red deer thawed spermatozoa after incubation at 37 °C in presence of several antioxidants

P. 1005-1019Antioxidants may be useful for supplementing sperm extenders. We have tested dehydroascorbic acid (DHA), TEMPOL, N-acetyl-cysteine (NAC) and rutin on epididymal spermatozoa from red deer, during incubation at 37 °C. Cryopreserved spermatozoa were thawed, washed and incubated with 1 mm or 0.1 mm of each antioxidant, including oxidative stress (Fe2+/ascorbate). Motility (CASA and clustering of subpopulations), viability, mitochondrial membrane potential, and acrosomal status were assessed at 2 and 4 h. Lipoperoxidation, intracellular reactive oxygen species (ROS) and DNA damage (DNA) status (TUNEL) were checked at 4 h. Oxidative stress increased ROS, lipoperoxidation and DNA damage. Overall, antioxidants negatively affected motility and physiological parameters. Only DHA 1 mm protected motility, increasing the fast and progressive subpopulation. However, it had a detrimental effect on acrosomal and DNA status, in absence of oxidative stress. Tempol and rutin efficiently reduced lipoperoxidation, ROS, and DNA damage in presence of oxidative stress. NAC was not as efficient as TEMPOL or rutin reducing lipoperoxidation or protecting DNA, and did not reduce ROS, but its negative effects were lower than the other antioxidants when used at 1 mm, increasing the subpopulation of hyperactivated-like spermatozoa at 2 h. Our results show that these antioxidants have mixed effects when spermatozoa are incubated at physiological temperatures. DHA may not be suitable because of prooxidant effects, but TEMPOL, NAC and rutin may be considered for cryopreservation trials. In general, exposure of red deer spermatozoa to these antioxidants should be limited to low temperatures, when only protective effects may develop.S

Effect of Several Antioxidants on Thawed Ram Spermatozoa Submitted to 37°C up to Four Hours

P. 907-914Thawed ram spermatozoa were incubated at 37°C in the presence of dehydroascorbic acid (DHA), TEMPOL (TPL), N‐acetyl‐cysteine (NAC) and rutin (RUT), at 0.1 and 1 mm, in order to test their effects on sperm physiology. Cryopreserved spermatozoa from four rams were thawed, pooled, washed and incubated in TALP‐Hepes with 1 mm or 0.1 mm of each antioxidant, performing a replicate with induced oxidative stress (Fe2+/ascorbate). Motility (CASA), viability and mitochondrial membrane potential (flow cytometry) were analysed at 2 and 4 h. Lipoperoxidation (MDA production), intracellular reactive oxygen species (ROS) and DNA status (TUNEL) were analysed at 4 h. Antioxidants, except DHA 0.1 mm, decreased motility and kinematic parameters, but had little effect on viability or mitochondrial activity. Except 1 mm DHA, the antioxidants reduced ROS at 4 h. Moreover, NAC 1 mm, rutin and TEMPOL reduced ROS and DNA damage in the presence of oxidative stress. N‐acetyl‐cysteine, rutin 1 mm and TEMPOL reduced lipoperoxidation in the presence of oxidative stress. However, DHA did not affect lipoperoxidation. At 1 mm, DHA increased DNA damage in the absence of oxidative stress. Dehydroascorbic acid effects could arise from spermatozoa having a low capacity for reducing it to ascorbic acid, and it may be tested in the presence of other antioxidants or reducing power. Future research should focus in testing whether the inhibition of motility observed for NAC, rutin and TEMPOL is reversible. These antioxidants might be useful at lower temperatures (refrigerated storage or cryopreservation) when their protective effects could be advantageous.S

Mitochondrial activity and forward scatter vary in necrotic, apoptotic and membrane-intact spermatozoan subpopulations

P. 547-556In the present study, we have related mitochondrial membrane potential (ΔΨm) and forward scatter (FSC) to apoptotic-related changes in spermatozoa. Thawed red deer spermatozoa were incubated in synthetic oviductal fluid medium (37°C, 5% CO2), with or without antioxidant (100 μm Trolox). At 0, 3, 6 and 9 h, aliquots were assessed for motility and were stained with a combination of Hoechst 33342, propidium ioide (PI), YO-PRO-1 and Mitotracker Deep Red for flow cytometry. The proportion of spermatozoa YO-PRO-1+ and PI+ (indicating a damaged plasmalemma; DEAD) increased, whereas that of YO-PRO-1– and PI– (INTACT) spermatozoa decreased. The proportion of YO-PRO-1+ and PI– spermatozoa (altered plasmalemma; APOPTOTIC) did not change. Both DEAD and APOPTOTIC spermatozoa had low ΔΨm. Most high-ΔΨm spermatozoa were INTACT, and their proportion decreased with time. The FSC signal also differed between different groups of spermatozoa, in the order APOPTOTIC > DEAD > INTACT/low ΔΨm > INTACT/high ΔΨm; however, the actual meaning of this difference is not clear. APOPTOTIC spermatozoa seemed motile at 0 h, but lost motility with time. Trolox only slightly improved the percentage of INTACT spermatozoa (P < 0.05). The population of APOPTOTIC spermatozoa in the present study may be dying cells, possibly with activated cell death pathways (loss of ΔΨm). We propose that the sequence of spermatozoon death here would be: (1) loss of ΔΨm; (2) membrane changes (YO-PRO-1+ and PI–); and (3) membrane damage (PI+). INTACT spermatozoa with low ΔΨm or altered FSC may be compromised cells. The present study is the first that directly relates membrane integrity, apoptotic markers and mitochondrial status in spermatozoa. The results of the present study may help us understand the mechanisms leading to loss of spermatozoon viability after thawing.S

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

P. 37-46The 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.S

Sperm parameters on Iberian red deer: Electroejaculation and post-mortem collection

P. 216-226Artificial reproductive technologies (ART) for cervids have improved, but a need remains for the collection of basic data. We studied two models of sperm collection in Iberian red deer, post-mortem (PM) in a wild population (179 samples) and by electroejaculation (EE) in a farmed population (37 samples), recording: testicular and epididymal weight, testicular diameter, sperm quantity, pH and osmolality and spermatozoa quality (motility by CASA, abnormal forms, cytoplasmic droplets, viability and acrosomal status). We tested the relationship of these parameters with stag age and compared the two models (PM and EE; medians showed). Genitalia parameters were linearly related to stag age (testicular diameter: 31.5–50.5 mm for 2–9 years). Total number of spermatozoa collected were PM: 2.5 × 109 and EE: 3.6 × 109 (P > 0.05), increasing with age only for PM. We found a positive relationship between testicular size and spermatozoa collected for PM. Osmolality and pH were PM: 6.28 and 378 mOsm/kg; EE: 7.63 and 309 mOsm/kg (P < 0.05). The pH increased with age only for EE. Percentage of motile spermatozoa was similar for PM and EE, but motility quality was lower for PM. Abnormal forms, proximal and distal droplets were lower for EE (22%, 1.3%, 1.5% vs. PM: 23%, 4.3%, 83%). Viability was similar (74%) and intact acrosomes were higher for EE (97% vs. 89%). Both PM and EE samples could be used for germplasm banking. This study contributes with new data on red deer spermatology and for the development of ART in cervids.S