Redox cycling induces spermptosis and necrosis in stallion spermatozoa while the hydroxyl radical (OH•) only induces spermptosis.

Oxidative stress is a major factor explaining sperm dysfunction of spermatozoa surviving freezing and thawing and is also considered a major inducer of a special form of apoptosis, visible after thawing, in cryopreserved spermatozoa. To obtain further insights into the link between oxidative stress and the induction of apoptotic changes, stallion spermatozoa were induced to oxidative stress through redox cycling after exposure to 2-methyl-1,4-naphthoquinone (menadione), or hydroxyl radical formation after FeSO4 exposure. Either exposure induced significant increases (p < 0.05) in two markers of lipid peroxidation: 8-iso-PGF2α and 4-hydroxynonenal (4-HNE). While both treatments induced changes indicative of spermptosis (caspase-3 activation and decreased mitochondrial membrane potential) (p < 0.01), menadione induced sperm necrosis and a dramatic reduction in motility and thiol content in stallion spermatozoa. Thus, we provided evidence that oxidative stress underlies spermptosis, and thiol content is a key factor for stallion sperm function.

Computational flow cytometry reveals that cryopreservation induces spermptosis but subpopulations of spermatozoa may experience capacitation-like changes.

The reduced lifespan of cryopreserved spermatozoa in the mare reproductive tract has been attributed to both capacitative and apoptotic changes. However, there is a lack of studies investigating both phenomena simultaneously. In order to improve our knowledge in this particular point, we studied in raw and frozen-thawed samples apoptotic and capacitative markers using a wide battery of test based in flow cytometry. Apoptotic markers evaluated were caspase 3 activity, externalization of phosphatidylserine (PS), and mitochondrial membrane potential. Markers of changes resembling capacitation were membrane fluidity, tyrosine phosphorylation, and intracellular sodium. Conventional and computational flow cytometry using nonlinear dimensionally reduction techniques (t-distributed stochastic neighbor embedding (t-SNE)) and automatic classification of cellular expression by nonlinear stochastic embedding (ACCENSE) were used. Most of the changes induced by cryopreservation were apoptotic, with increase in caspase 3 activation (P < 0.01), PS translocation to the outer membrane (P < 0.001), loss of mitochondrial membrane potential (P < 0.05), and increase in intracellular Na+ (P < 0.01). Average values of markers of capacitative changes were not affected by cryopreservation; however, the analysis of the phenotype of individual spermatozoa using computational flow cytometry revealed the presence of subpopulations of spermatozoa experiencing capacitative changes. For the first time advanced computational techniques were applied to the analysis of spermatozoa, and these techniques were able to disclose relevant information of the ejaculate that remained hidden using conventional flow cytometry.

Improving sperm banking efficiency in endangered species through the use of a sperm selection method in brown bear (Ursus arctos) thawed sperm.

BACKGROUND: Sperm selection methods such as Single Layer Centrifugation (SLC) have been demonstrated to be a useful tool to improve the quality of sperm samples and therefore to increase the efficiency of other artificial reproductive techniques in several species. This procedure could help to improve the quality of genetic resource banks, which is essential for endangered species. In contrast, these sperm selection methods are optimized and focused on farm animals, where the recovery task is not as important as in endangered species because of their higher sperm availability. The aim of this study was to evaluate two centrifugation methods (300 x g/20 min and 600 x g/10 min) and three concentrations of SLC media (Androcoll-Bear -80, 65 and 50%) to optimise the procedure in order to recover as many sperm with the highest quality as possible. Sperm morphology could be important in the hydrodynamic relationship between the cell and centrifugation medium and thus the effect of sperm head morphometry on sperm yield and its hydrodynamic relationship were studied. RESULTS: The samples selected with Androcoll-Bear 65% showed a very good yield (53.1 ± 2.9) although the yield from Androcoll-Bear 80% was lower (19.3 ± 3.3). The latter showed higher values of motility than the control immediately after post-thawing selection. However, both concentrations of colloid (65 and 80%) showed higher values of viable sperm and viable sperm with intact acrosome than the control. After an incubation of 2 h at 37 °C, the samples from Androcoll-Bear 80% had higher kinematics and proportion of viable sperm with intact acrosome. In the morphometric analysis, the sperm selected by the Androcoll-Bear 80% showed a head with a bigger area which was more elongated than the sperm from other treatments. CONCLUSIONS: We conclude that sperm selection with Androcoll-Bear at either 65% or 80% is a suitable technique that allows a sperm population with better quality than the initial sample to be obtained. We recommend the use of Androcoll-Bear 65% since the yield is better than Androcoll-Bear 80%. Our findings pave the way for further research on application of sperm selection techniques to sperm banking in the brown bear.

TRUST trial: BAY 86-6150 use in haemophilia with inhibitors and assessment for immunogenicity.

INTRODUCTION: The most serious and challenging complication of haemophilia treatment is development of inhibitors to replacement factors VIII or IX. Innovative therapies currently being explored for patients with haemophilia and inhibitors include BAY 86-6150, a modified recombinant activated factor VII (FVIIa). Immunogenicity remains a substantial barrier in this endeavour. AIM: To present safety and efficacy results of the BAY 86-6150 study in patients with inhibitors and report detailed analysis of epitope mapping in a patient who developed anti-BAY 86-6150 antibodies. METHODS: Patients aged 12-62 years with moderate or severe haemophilia A or B were eligible for the phase 3 TRUST trial if they had a history of high-titre inhibitors. Four escalating doses of BAY 86-6150 (6.5, 20, 50, 90 µg kg-1 ) were planned with ≥10 patients per dose level. Bleeding episodes were treated with BAY 86-6150. Development of anti-BAY 86-6150 antibodies was considered a serious adverse event. RESULTS: TRUST was discontinued after one patient in the 6.5-µg kg-1 cohort developed anti-BAY 86-6150 neutralizing antibodies following three exposures. The anti-BAY 86-6150 antibodies cross-reacted with and neutralized wild-type FVIIa (WT-FVIIa). Post hoc epitope mapping using peripheral blood mononuclear cells from the responding patient found that none of the 14 peptides unique to BAY 86-6150 were recognized by the patient's T cells, but strong responses were detected against 2 WT-FVIIa peptides. CONCLUSION: In the single patient with haemophilia A who developed anti-BAY 86-6150 antibodies, results of T-cell epitope mapping indicated BAY 86-6150 was no more immunogenic than WT-FVIIa.

The use of gelatine in long-term storage (up to 48 hr) at 5°C preserves the pre-freezing and post-thawing quality of brown bear sperm.

Sedimentation of spermatozoa occurs during long-term liquid storage and this may produce deleterious changes. Our aim was to apply gelatine supplementation during long-term pre-freezing storage of bear sperm, applying final dilution and 6% glycerol at room temperature and cool in straws. We tested four models of sperm storage using a 1:1 dilution in TTF-ULE-Bear extender (TesT-fructose-egg yolk-glycerol 6%): (i) second 1:1 dilution at room temperature (RT), cooling at 5°C in a tube and final dilution (100 × 10(6)  sperm ml(-1) ) (Standard); (ii) final dilution at RT and cooling in a tube (FD-Tube); (iii) final dilution at RT and cooling in 0.25 ml plastic straw (FD-Straw); and (iv) final dilution at RT in extender supplemented with 1.5% gelatine (Gelatine) and cooling in a 0.25 ml plastic straw. A Standard sample was stored at 5°C for 1 hr (Control); the rest of the samples (Standard, FD-Tube, FD-Straw, Gelatine) were stored for 24 or 48 hrs before freezing (100 × 10(6)  sperm ml(-1) , glycerol 6%). The quality of the samples was assessed for motility by CASA, and viability (SYBR-14/propidium iodide-PI-; VIAB), acrosomal status (PNA-FITC/PI; iACR) and apoptotic status (YO-PRO-1/PI; YOPRO-) by flow cytometry. At pre-freezing, after 48 hr, Gelatine showed significantly higher viability (for VIAB and YOPRO-) and progressiveness (PM, LIN and STR). At 48 hr, Gelatine showed similar YOPRO-, iACR, LIN, STR and ALH respect to Control. At both 24 and 48 h post-thawing, Gelatine sample had similar scores for YOPRO-, iACR, LIN, STR, WOB and VIAB (only 24 hr) when compared with Control, and lower for TM, PM, rapidPM, VAP and ALH. No differences were found among others experimental groups with respect to Control. In conclusion, gelatine could be a suitable alternative to preserve the viability and progressive motility of brown bear ejaculates during long-term pre-freezing storage at 5°C.

Alternative procedures for the cryopreservation of brown bear ejaculates depending on the flexibility of the "in cooling" period (5°C).

The adaptability of cryopreservation protocols for brown bear spermatozoa collected under field conditions and frozen in a nearby laboratory (transported for a few hours) or shipped to a reference laboratory for sex sorting (transported for a few days) was evaluated. Forty-nine electroejaculates from 15 mature brown bears were extended to 100×10(6) sperm/mL in a TES-Tris-Fructose based extender and cryopreserved (-20°C/min to -100°C and stored at -196°C). After thawing, the quality of the seminal samples was assessed for total (TM), progressive (PM) motility and kinetic parameters - by CASA -, and viability (VIAB), viable and non-apoptotic status (YOPRO-), high membrane mitochondrial potential (MIT) and intact acrosomes (iACR) - by flow cytometry -. In Experiment 1, we assessed different storage times (0, 0.5, 1 - control -, 4-5, 7-8 and 11-12 h) at 5°C from final dilution to freezing. After thawing, non-equilibrated samples (0 h) showed lower values of iACR, TM and PM. No significant differences were found for the different periods of equilibration tested. In Experiment 2, we evaluated three long-term storage times (24, 48 and 72 h) at 5°C before freezing using storage for 1h as control. The post-thawing quality of brown bear spermatozoa declined markedly after 48-72 h of pre-freezing. In conclusion, our findings suggest the possibility of extending the pre-freezing cooling period up to 24h post-collection without freezing. This knowledge should enable the adaptation of the freezing protocols for when a special handling conditions are required such as the shipment of seminal samples to technological centers for the pre-freezing application of enhancer spermatic biotechnologies.

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.

Factors influencing intention to plagiarize among nursing students in the Philippines.

Background: Plagiarism is an ethical concern among students but is insufficiently discussed and acknowledged in some educational institutions. Determining what factors influence plagiarism can help the academic community manage its incidence. Objective: This study determined the factors affecting the intention to plagiarize among Filipino nursing students. Methods: Utilizing a cross-sectional research design, data were gathered from 304 nursing students last June 2021 using valid instruments administered online. Regression analysis identified influencing factors of intention to plagiarize. Results: Findings indicated a generally low intention (M = 1.47, SD = 0.74) to plagiarize among nursing students. Internet literacy (B = -0.314, p <0.001), attitude (B = 0.257, p <0.001), moral obligation (B = -0.236, p <0.001), past behavior (B = 0.139, p <0.001), and subjective norm (B = -0.095, p = -0.001) influenced nursing students' plagiarism intention. Conclusion: Several interrelated factors play major roles in nursing students' tendency to plagiarize. Creating an educational environment that does not favor the unethical practice of plagiarism is recommended for nursing schools.

Effect of several antioxidants on thawed ram spermatozoa submitted to 37°C up to four hours.

Thawed 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 (Fe(2+) /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.

Optimization of glycerol concentration and freezing rate in the cryopreservation of ejaculate from brown bear (Ursus arctos).

In order to establish a semen bank for the endangered Cantabrian brown bear, we tested five glycerol concentrations and three freezing rates for electroejaculated semen. Electroejaculation was performed on nine males. Semen was diluted in TES-Tris-Fructose (20% egg yolk, 2% EDTA, 1% Equex) with 2%, 4%, 6%, 8% or 10% glycerol and frozen at -10, -20 or -40°C/min. Before and after cryopreservation, samples were analysed for motility (CASA), viability and acrosomal status (flow cytometry). Pre-freezing results showed that glycerol concentration had no significant effect on total motility or progressive motility, but it significantly decreased VCL, ALH, viability and acrosomal status (p < 0.05). After thawing, sperm motility was higher at extender with 4%, 6% and 8% glycerol, but only at 4% and 6% glycerol for viability and acrosomal status. For 4% and 6% glycerol, freezing rates did not have significant effects. The curve fitting gave an estimate of the optimal glycerol concentration, with all the optimal values for every parameter between 6% and 7% glycerol falling. We propose using 6% glycerol and a freezing velocity of -20°C/min for freezing brown bear ejaculated spermatozoa.

The challenges of managing a complex stoma with NPWT.

This paper reports the clinical experience of a 71-year-old female with resection of the colon, who subsequently developed postoperative complications. A colostomy was carried out; necrosis of the colostomy edges and stoma retraction complicated optimal stoma appliance placement. It was possible to treat the resulting cavity with negative pressure wound therapy (NPWT) by isolating the stoma and treating the peristomal wound area. The wound was treated with NPWT for 30 days; at day 20, the patient was discharged to home to continue with NPWT for a further 10 days by community nurses, with regular follow-up visits in the outpatient clinic. The patient improved steadily and achieved complete closure of the wound.

Evaluation of three different extenders for use in emergency salvaging of epididymal spermatozoa from a Cantabric brown bear.

The Cantabrian brown bear (Ursus arctos) constitutes an endangered subpopulation of the European brown bear in the north of Spain. We have carried out a post-mortem recovery of epididymal spermatozoa from a Cantabrian brown bear (7 years old, 170 kg; 30 min post-mortem), cryopreserving those recovered from the cauda epididymis (929 × 10(6) spermatozoa, 54% motile, 82% cytoplasmic droplets). For freezing, three extenders based on Test-Tris-Fructose + 4% glycerol were used: (1) 325 mOsm/kg and 10% egg yolk; (2) 430 mOsm/kg and 15% egg yolk; (3) 300 mOsm/kg, Equex-EDTA and 20% egg yolk. After thawing, we obtained higher motility for extender 3 (31%), but extender 2 yielded the highest viability (66.9%) and mitochondrial activity (67.1%). Caffeine stimulation showed that extender two rendered the highest recovery values of post-thawing motility with respect to the fresh sample. In conclusion, epididymal spermatozoa of brown bear can be frozen applying an extender with osmolality similar to epididymal environment.

The acidic probe LysoSensor is not useful for acrosome evaluation of cryopreserved ram spermatozoa.

To try new acrosomal probes for assessing ram spermatozoa, we compared the LysoSensor probe, which labels acidic organelles, with the frequently used peanut agglutinin acrosomal probe (PNA-PE; phycoerythrin as fluorescent moiety). The previous microscopic observations showed a lack of relationship of LysoSensor with acrosomal status. Semen was obtained from five rams and frozen in four pools. Each pool was analysed carrying out a triple staining propidium ioide/PNA-PE/LysoSensor Green DND-189 to test acrosome labelling, and a double staining SYBR-14/PI, to assess sperm viability. Stained samples were analysed by flow cytometry. All measurements were replicated. Data were processed using agreement and repeatability tests. LysoSensor labelling did not agree with PNA (mean of differences: 30.8%; coefficient of agreement: 22.6%), confirming microscopic observations. Nevertheless, when LysoSensor was compared with SYBR-14/PI, the agreement was high (mean of differences: -0.05%; coefficient of agreement: 5.07%). Repeatability of both methods was high and similar. LysoSensor did not seem to specifically stain the acrosome, but it may accumulate in the cytoplasm and label viable spermatozoa. Therefore, LysoSensor might not be used as an acrosomal probe in ram spermatozoa, but it could be used in other kind of studies, taking advantage of its pH sensitivity.

Probes and techniques for sperm evaluation by flow cytometry.

CONTENTS: Flow cytometry has become an important technique in sperm evaluation and is increasingly used both for routine assessment and for research in veterinary science. We have revised the literature, describing fluorescent probes that have been used for analysing spermatozoa by flow cytometry, regarding: viability, acrosomal status, capacitation, mitochondrial status, apoptotic markers, oxidative stress markers, DNA damage, sperm counting and sperm sizing. Details and problems of some techniques are reviewed, with special attention to the occurrence of non-sperm particles in the samples ('debris'). New and promising aspects of flow cytometry, such as sperm sorting using viability markers as selection criteria, are highlighted. The relationship between flow cytometry analyses and fertility and their future improvements are considered.

Unconventional diagnostic tests for Lyme borreliosis: a systematic review.

BACKGROUND: Lyme borreliosis (LB) diagnosis currently relies mainly on serological tests and sometimes PCR or culture. However, other biological assays are being developed to try to improve Borrelia-infection diagnosis and/or monitoring. OBJECTIVES: To analyse available data on these unconventional LB diagnostic assays through a systematic literature review. METHODS: We searched PubMed and Cochrane Library databases according to the PRISMA-DTA method and the Cochrane Handbook for Systematic Reviews of Interventions. We analysed controlled and uncontrolled studies (published 1983-2018) on biological tests for adults to diagnose LB according to the European Study Group for Lyme Borreliosis or the Infectious Diseases Society of America definitions, or identify strongly suspected LB. Two independent readers evaluated study eligibility and extracted data from relevant study reports; a third reader analysed full texts of papers to resolve disagreements. The quality of each included study was assessed with the QUADAS-2 evaluation scale. RESULTS: Forty studies were included: two meta-analyses, 25 prospective controlled studies, five prospective uncontrolled studies, six retrospective controlled studies and two case reports. These biological tests assessed can be classified as: (i) proven to be effective at diagnosing LB and already in use (CXCL-13 for neuroborreliosis), but not enough to be standardized; (ii) not yet used routinely, requiring further clinical evaluation (CCL-19, OspA and interferon-α); (iii) uncertain LB diagnostic efficacy because of controversial results and/or poor methodological quality of studies evaluating them (lymphocyte transformation test, interferon-γ, ELISPOT); (iv) unacceptably low sensitivity and/or specificity (CD57+ natural killer cells and rapid diagnostic tests); and (v) possible only for research purposes (microscopy and xenodiagnoses). DISCUSSION: QUADAS-2 quality assessment demonstrated high risk of bias in 25/40 studies and uncertainty regarding applicability for 32/40, showing that in addition to PCR and serology, several other LB diagnostic assays have been developed but their sensitivities and specificities are heterogeneous and/or under-evaluated or unassessed. More studies are warranted to evaluate their performance parameters. The development of active infection biomarkers would greatly advance LB diagnosis and monitoring.

Evaluation of oxidative DNA damage promoted by storage in sperm from sex-reversed rainbow trout.

Short-term storage and cryopreservation of sperm are two common procedures in aquaculture, used for routine practices in artificial insemination reproduction and gene banking, respectively. Nevertheless, both procedures cause injuries affecting sperm motility, viability, cell structure and DNA stability, which diminish reproductive success. DNA modification is considered extremely important, especially when sperm storage is carried out with gene banking purposes. DNA damage caused by sperm storage is not well characterized and previous studies have reported simple and double strand breaks that have been attributed to oxidative events promoted by the generation of free radicals during storage. The objective of this study was to reveal DNA fragmentation and to explore the presence of oxidized bases that could be produced by oxidative events during short-term storage and cryopreservation in sex-reversed rainbow trout (Oncorhynchus mykiss) spermatozoa. Sperm from six males was analyzed separately. Different aliquots of the samples were stored 2h (fresh) or 5 days at 4 degrees C or were cryopreserved. Then spermatozoa were analyzed using the Comet assay, as well as combining this method with digestion with two endonucleases from Escherichia coli (Endonuclease III, that cut in oxidized cytosines, and FPG, cutting in oxidized guanosines). Both storage procedures yielded DNA fragmentation, but only short-term storage oxidative events were clearly detected, showing that oxidative processes affect guanosines rather than cytosines. Cryopreservation increases DNA fragmentation but the presence of oxidized bases was not noticed, suggesting that mechanisms other than oxidative stress could be involved in DNA fragmentation promoted by freezing.

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.

Comprehensive geriatric assessment in older patients suffering from infective endocarditis. A prospective multicentric cohort study.

OBJECTIVES: The aim was to describe the impact of infective endocarditis (IE) on functional, cognitive and nutritional statuses, and to estimate the influence of these parameters on surgical management and mortality. METHOD: This was a prospective study over 13 months in 14 French hospitals, including patients ≥75 years of age with definite or possible IE. A comprehensive geriatric assessment (CGA) was performed during the first week of hospitalization, including a retrospective estimation of functional status 2 months before hospitalization, and 3 months after. RESULTS: A total of 120 patients were included (mean age 83.1 ± 5.0 (75-101) years). IE was associated with a dramatic impairment of functional status between 2 months prior hospitalization and the first geriatric evaluation (90.8% able to walk vs. 35.5% (p < 0.0001), ADL (Activities in Daily Living) 5.0 ± 1.7 vs. 3.1 ± 2.1 (p < 0.0001)). The 19 operated patients (15.8%) had less comorbidities (cumulative illness rating scale geriatric 10.8 ± 8.2 vs. 15.3 ± 7.1 (p 0.0176)), better functional (ADL 5.9 ± 0.4 vs. 4.9 ± 1.8 (p 0.0171) and nutritional (mini nutritional assessment 20.4 ± 5.0 vs. 17.3 ± 6.2 (p 0.0501)) statuses than non-operated patients. Among all infectious, cardiac and geriatric parameters, body mass index (HR 0.9, range 0.8-1, p 0.05) and ADL at the time of the first evaluation (HR 0.7, range 0.6-0.9, p 0.002) were the sole independent predictors of the 3-month (32.5%) and 1-year mortality (42.5%). Three months later, the 57 assessed patients only partially recovered their ADL (3.7 ± 1.9 vs. 5.3 ± 1.4 2 months prior hospitalization and 4.6 ± 1.9 at the first CGA; p < 0.0001). CONCLUSION: Functional and nutritional abilities are crucial components that can be accurately explored through a CGA when managing IE in oldest patients.

Diuretic activity of Smilax canariensis, an endemic Canary Island species.

ETHNOPHARMACOLOGICAL RELEVANCE: Smilax canariensis is an endemic species of the Canary Islands, popularly known as "Zarzaparrilla sin espinas". This species has wide use in folk-medicine practice on the islands, especially as diuretic. So the aim of our study is to evaluate the diuretic activity of an aqueous and a methanol extract of this species. MATERIAL AND METHODS: Three infusions doses (250, 500 and 750 mg/kg) and two methanol extract doses (100 and 200 mg/kg) were orally administered to laboratory rats. Water excretion rate, pH, density, conductivity, and content of Na(+) and K(+) were measured in the urine of saline-loaded rats. RESULTS: Water excretion rates were significantly increased in a dose-dependent manner by both hot water infusions and the alcohol extract. The electrolytic excretion was also dose-dependent, although potassium excretion was markedly reduced when using the alcohol extract compared with that observed for the infusion. CONCLUSIONS: Smilax canariensis presents a notable diuretic effect which appeared to be related both to its potassium content and to the presence of polar organic compounds. The present results provide a quantitative basis explaining the traditional folk-medicine use of Smilax canariensis as a diuretic agent by the Canary Island population.

Effects of cryopreservation on head morphometry and its relation with chromatin status in brown bear (Ursus arctos) spermatozoa.

The Cantabrian brown bear (Ursus arctos) is a highly endangered species in Spain and basic studies are necessary in order to bank its germplasm. Sperm heads are mainly made up of chromatin, thus their shape depends partly on chromatin structure. Thawed semen from 10 bears was used to analyze chromatin status by sperm chromatin structure assay (SCSA) and head morphometry by the computer-assisted sperm morphology assessment (CASMA) system. Morphometry was analyzed before and after freezing-thawing in order to evaluate the effects of cryopreservation on sperm heads. Each spermatozoon was measured for four primary parameters (length, L; width, W; area, A; perimeter, P) and derived parameters (ellipticity: L/W, circularity: 4piA/ P2, elongation: (L-W)/(L+W), regularity: piLW/ 4A). All the derived parameters significantly differed between bears. Likewise, cryopreservation affected head morphometry by reducing its size. Clustering based on morphometric parameters separated three subpopulations, one of them being significantly more influenced by the cryopreservation process. We obtained high correlations between head morphometry and SCSA parameters: standard deviation of DNA fragmentation index (SD-DFI) was correlated with perimeter and area (r=0.75 and r=0.62, respectively) and DFIm and DFIt (moderate and total DNA fragmentation index) were correlated with perimeter (r=0.65 and r=0.67, respectively). Nevertheless, classification of males according to SCSA or head morphometry did not completely agree so the two assays might explain male variability differently. We conclude that cryopreservation affected morphometry at least in a subset of spermatozoa. These results might improve future application of sperm banking techniques in this species.