Percepción de la validez del cuestionario Migraine Disability Assessment por una población de pacientes con migraña crónica / Perception of the validity of the Migraine Disability Assessment questionnaire in a population of patients with chronic migraine

Objetivos: El cuestionario Migraine Disability Assessment (MIDAS) es el instrumento más empleado para valorar el grado de discapacidad en los estudios de migraña. El objetivo del estudio es determinar el nivel de cumplimentación del cuestionario, valorar su facilidad de uso y conocer la percepción subjetiva del paciente sobre la capacidad del cuestionario para medir realmente su discapacidad.Material y métodosEstudio prospectivo sobre una población de 78 pacientes con migraña crónica. Se determina el nivel educativo y la situación laboral. En la visita basal se adiestra a los pacientes sobre la correcta cumplimentación del cuestionario. A los 3 meses se determina la puntuación total y el nivel de cumplimentación. Además los pacientes contestan una encuesta que mide: facilidad de uso y percepción del paciente sobre si la escala refleja su propia discapacidad.ResultadosSolo el 46% rellena completamente el cuestionario. El 69% de los pacientes indica que el cuestionario no les resulta fácil de cumplimentar (resultado no influido por el nivel educativo, pero sí por la situación laboral de los pacientes). El 62% de los encuestados opina que el cuestionario no refleja completamente su propia percepción de discapacidad.ConclusionesAunque está más que demostrada la validez y consistencia del cuestionario MIDAS, un porcentaje elevado de nuestra población reconoce que el cuestionario no es fácil de rellenar y además es percibido por muchos de nuestros pacientes como un cuestionario que no refleja adecuadamente su discapacidad. Conocer la opinión de los pacientes sobre la idoneidad de los cuestionarios administrados en las consultas es crucial para mejorar su cumplimentación. (AU)

Objectives: The Migraine Disability Assessment (MIDAS) questionnaire is the most frequently used instrument for assessing the level of disability in studies into migraine. This study aims to determine the level of completion of the questionnaire, assess the ease of use, and understand patients’ subjective perception of the questionnaire's actual ability to measure disability.Material and methodsWe performed a prospective study of a sample of 78 patients with chronic migraine, determining their level of education and employment status. In a baseline visit, patients were trained to properly complete the questionnaire. At 3 months, we determined the total score and level of completion. Patients also completed a survey measuring ease of use of the questionnaire and patients’ perception of whether the score accurately reflected their disability.ResultsOnly 46% of patients fully completed the questionnaire. Sixty-nine percent reported finding it difficult to complete (this was influenced by patient's employment status but not by educational level). Sixty-two percent of respondents believed that the questionnaire did not fully reflect their own perception of their disability.ConclusionsAlthough the validity and consistence of the MIDAS questionnaire are well documented, a high percentage of the study population reported finding it difficult to complete; many patients also considered that the questionnaire did not accurately reflect their disability. Understanding patients’ opinions of the suitability of questionnaires used in consultation is crucial to improving completion. (AU)

Perception of the validity of the Migraine Disability Assessment questionnaire in a population of patients with chronic migraine.

OBJECTIVES: The Migraine Disability Assessment (MIDAS) questionnaire is the most frequently used instrument for assessing the level of disability in studies into migraine. This study aims to determine the level of completion of the questionnaire, assess the ease of use, and understand patients' subjective perception of the questionnaire's actual ability to measure disability. MATERIAL AND METHODS: We performed a prospective study of a sample of 78 patients with chronic migraine, determining their level of education and employment status. In a baseline visit, patients were trained to properly complete the questionnaire. At 3 months, we determined the total score and level of completion. Patients also completed a survey measuring ease of use of the questionnaire and patients' perception of whether the score accurately reflected their disability. RESULTS: Only 46% of patients fully completed the questionnaire. Sixty-nine percent reported finding it difficult to complete (this was influenced by patient's employment status but not by educational level). Sixty-two percent of respondents believed that the questionnaire did not fully reflect their own perception of their disability. CONCLUSIONS: Although the validity and consistence of the MIDAS questionnaire are well documented, a high percentage of the study population reported finding it difficult to complete; many patients also considered that the questionnaire did not accurately reflect their disability. Understanding patients' opinions of the suitability of questionnaires used in consultation is crucial to improving completion.

Reversible Homonymous Inferior Quadrantanopia in a Nonketotic Hyperglycemic Patient.

Homonymous hemianopia is frequently associated with retrochiasmal lesions. Vascular etiology is the most common and usually evident on magnetic resonance imaging. When the results of neuroimaging are normal, there are other etiologies that we should consider, like nonketotic hyperglycemia (NKH). We report a 62-year-old female diabetic patient with headache, colour vision and sudden homonymous inferior quadrantanopia and elevated blood sugar levels with normal pH. The neuroimaging was normal and the visual lost improved after the correction of the hyperglycemia. NKH should be considered in patients with sudden and transient hemianopia and normal neuroimaging.

Metformin inhibits human spermatozoa motility and signalling pathways mediated by protein kinase A and tyrosine phosphorylation without affecting mitochondrial function.

Metformin is a leading antidiabetic drug that is used worldwide in the treatment of diabetes mellitus. This biguanide exerts metabolic and pleiotropic effects in somatic cells, although its invitro actions on human spermatozoa remain unknown. The present study investigated the effects of metformin on human sperm function. Human spermatozoa were incubated in the presence or absence of 10mM metformin for 8 or 20h, and motility was measured by computer-aided sperm analysis (CASA); other parameters were evaluated by flow cytometry. Metformin significantly reduced the percentage of motile, progressive and rapid spermatozoa and significantly decreased sperm velocity. Metformin did not affect viability, mitochondrial membrane potential (MMP) or mitochondrial superoxide anion generation of human spermatozoa at any time studied. However, metformin clearly inhibited the protein kinase (PK) A pathway and protein tyrosine phosphorylation at 8 and 20h, key regulatory pathways for correct sperm function. In summary, metformin treatment of human spermatozoa had a detrimental effect on motility and inhibited essential sperm signalling pathways, namely PKA and protein tyrosine phosphorylation, without affecting physiological parameters (viability, MMP, mitochondrial superoxide anion generation). Given the growing clinical use of metformin in different pathologies in addition to diabetes, this study highlights an adverse effect of metformin on spermatozoa and its relevance in terms of human fertility in patients who potentially could be treated with metformin in the future.

Perception of the validity of the Migraine Disability Assessment questionnaire in a population of patients with chronic migraine. / Percepción de la validez del cuestionario Migraine Disability Assessment por una población de pacientes con migraña crónica.

OBJECTIVES: The Migraine Disability Assessment (MIDAS) questionnaire is the most frequently used instrument for assessing the level of disability in studies into migraine. This study aims to determine the level of completion of the questionnaire, assess the ease of use, and understand patients' subjective perception of the questionnaire's actual ability to measure disability. MATERIAL AND METHODS: We performed a prospective study of a sample of 78 patients with chronic migraine, determining their level of education and employment status. In a baseline visit, patients were trained to properly complete the questionnaire. At 3 months, we determined the total score and level of completion. Patients also completed a survey measuring ease of use of the questionnaire and patients' perception of whether the score accurately reflected their disability. RESULTS: Only 46% of patients fully completed the questionnaire. Sixty-nine percent reported finding it difficult to complete (this was influenced by patient's employment status but not by educational level). Sixty-two percent of respondents believed that the questionnaire did not fully reflect their own perception of their disability. CONCLUSIONS: Although the validity and consistence of the MIDAS questionnaire are well documented, a high percentage of the study population reported finding it difficult to complete; many patients also considered that the questionnaire did not accurately reflect their disability. Understanding patients' opinions of the suitability of questionnaires used in consultation is crucial to improving completion.

Expanded equine cumulus-oocyte complexes exhibit higher meiotic competence and lower glucose consumption than compact cumulus-oocyte complexes.

Equine cumulus-oocyte complexes (COCs) are classified as compact (cCOC) or expanded (eCOC) and vary in their meiotic competence. This difference could be related to divergent glucose metabolism. To test this hypothesis in the present study, eCOCs, cCOCs and expanded or compact mural granulosa cells (EC and CC respectively) were matured in vitro for 30h, at which time maturation rate, glucose metabolism and the expression of genes involved in glucose transport, glycolysis, apoptosis and meiotic competence were determined. There were significant differences between eCOCs and cCOCs in maturation rate (50% vs 21.7% (n=192 and 46) respectively; P<0.001), as well as mean (±s.e.m.) glucose consumption (1.8±0.5 vs 27.9±5.9 nmol per COC respectively) and pyruvate (0.09±0.01 vs 2.4±0.8 nmol per COC respectively) and lactate (4.7±1.3 vs 64.1±20.6 nmol per COC respectively; P<0.05 for all) production. Glucose consumption in EC and CC did not differ significantly. Expression of hyaluronan-binding protein (tumour necrosis factor alpha induced protein 6; TNFAIP6) was increased in eCOCs and EC, and solute carrier family 2 member 1 (SLC2A1) expression was increased in eCOCs, but there were no differences in the expression of glycolysis-related enzymes and solute carrier family 2 member 3 (SLC2A3) between the COC or mural granulosa cell types. The findings of the present study demonstrate that metabolic and genomic differences exist between eCOCs and cCOCs and mural granulosa cells in the horse.

Oocyte holding in the Iberian red deer (Cervus elaphus hispanicus): Effect of initial oocyte quality and epidermal growth factor addition on in vitro maturation.

Current in vitro embryo production protocols in the Iberian red deer (Cervus elaphus hispanicus) need to be optimized; oocyte harvesting in situ followed by overnight holding could reduce the human effort and shipping costs. In our work, post-mortem ovaries were retrieved, and the oocytes were harvested and allocated to G1 group (good quality) or G2 + G3 group (low quality). The oocytes were separately subjected to immediate in vitro maturation (IVM) or held overnight in a holding medium composed of 40% of TCM 199 with Earle's salts, 40% TCM 199 with Hanks' salts and 20% fetal bovine serum (FBS), at room temperature (16 hr). In vitro maturation was carried out in a basal medium supplemented or not with 50 ng/ml of epidermal growth factor (EGF). Our data showed that addition of EGF to the maturation medium increases the percentage of G1 oocytes reaching metaphase II (3.9% vs. 50%, basal vs. EGF; p < .001) and decreased their degeneration rate (69.9% vs. 22.2%, basal vs. EGF; p < .01) when oocytes were immediately matured. Overnight holding increased the meiotic competence of G1 oocytes (37.5% matured in basal medium) and EGF increased prophase arrest in G2 + G3 oocytes (16.1% vs. 38.8% in germinal vesicle [GV] stage in basal medium vs. EGF added medium; p < .05). Our data demonstrate that oocyte holding can be used in Iberian red deer oocytes. Interestingly, EGF addition increases the oocytes' meiotic competence in immediately matured oocytes but not after oocyte holding depending upon initial oocyte quality.

Human sperm motility is downregulated by the AMPK activator A769662.

AMP-activated kinase (AMPK) plays a key function in maintaining cellular energy homeostasis. We recently identified and localized AMPK protein in human spermatozoa and showed that inhibition of AMPK activity significantly modified human sperm motility. Recently, AMPK has gained great relevance as prime target for pharmacological approaches in several energy-related pathologies and therefore pharmacological research is aimed to develop direct AMPK-activating compounds such as A769662. Our aim was to investigate the effect of A769662 in essential functional processes of human spermatozoa. Human spermatozoa were incubated in the presence or absence of the AMPK activator A769662 for different incubation times (0-20 h) and motility was evaluated by CASA system whereas other functional parameters were evaluated by flow cytometry. A769662 treatment significantly reduces the percentages of motile, progressive, and rapid spermatozoa starting at 2 h. Moreover, AMPK activator in human spermatozoa causes a significant reduction in any velocity measured, which is concomitant to a significant decrease in the percentage of rapid spermatozoa, both at short- (2-3 h) and long-time treatment (20 h). Treatment of human spermatozoa with A769662 does not significantly alter any of the following functional parameters: sperm viability, mitochondrial membrane potential, phosphatidylserine translocation to the outer leaf of plasma membrane, acrosome membrane integrity, or mitochondrial superoxide anion production. In summary, our results suggest that AMPK in human spermatozoa contributes to the regulation of sperm motility, without affecting basic physiological parameters of human spermatozoa (viability, mitochondrial membrane potential or reactive oxygen species production, acrosome membrane integrity, phosphatidylserine exposure at plasma membrane). As sperm motility is required in the female reproductive tract to achieve fertilization, we conclude that AMPK is an essential regulatory kinase of the human spermatozoa function. This conclusion needs to be taken into account when AMPK is elected as prime target in pharmacological approaches for several energy-related pathologies.

HSP90 maintains boar spermatozoa motility and mitochondrial membrane potential during heat stress.

Heat Shock Proteins (HSP) is a family of proteins that protects cells from high temperatures. The present work aimed to elucidate the role that HSP90 exerts on boar sperm incubated under heat stress conditions on viability, total motility (TM), progressive motility (PM), acrosome status, mitochondrial membrane potential and plasma membrane lipid organization. Sperm were incubated in non-capacitating conditions (Tyrode's basal medium or TBM) for 3, 8 and 24h or in capacitating conditions (Tyrode's complete medium or TCM) for 4h at 38.5°C or 40°C (Heat stress) in the presence or absence of 5 or 20µM of 17-AAG, a specific HSP90 inhibitor. Sperm viability was not affected by the presence of 17-AAG in any condition tested compared with its own control (at the same temperature and incubation time). In non-capacitating conditions TM (22.7±4.1 vs. 1.9±1.1; % mean±SEM), PM (3.1±0.9 vs. 0) and high mitochondrial membrane potential (19.5±2.2 vs. 11.8±0.8) decreased significantly in sperm incubated at 40°C for 24h in the presence of 20µM 17-AAG (control vs. 20µM 17-AAG, respectively; p<0.05). In sperm incubated at 38.5°C only a mild decrease in TM was observed (48.7±3.1 vs. 32.1±4.8; control vs. 20µM 17-AAG, respectively; p<0.05). However, under capacitating conditions none of the sperm parameters studied were affected by 17-AAG after 4h of incubation. These results demonstrate for the first time the role of HSP90 in the maintenance of boar sperm motility and mitochondrial membrane potential during prolonged heat stress in non-capacitating conditions.

Effect of Different Media and Protein Source on Equine Gametes: Potential Impact During In Vitro Fertilization.

Equine in vitro fertilization (IVF) is still inconsistent. In the present work, we studied how modified Whitten's (MW) medium and Tissue Culture Medium 199 (TCM) added with Foetal Bovine Serum (FBS; 10% v/v) or Bovine Serum Albumin (BSA; 7 mg/ml) affected equine gametes to subsequently run IVF trials. Compact (Cp) and expanded (Ex) cumuli equine oocytes were matured and placed in TCM or MW supplemented with BSA or FBS for 18-20 h (no sperm added). In Ex oocytes, TCM-199 added with FBS or BSA resulted in higher metaphase II (MII) rates (75.7% and 62.7%, respectively) than MW added with BSA (54%) or FBS (52.2%; p < 0.05); this was not observed for Cp oocytes. Equine sperm were capacitated in the same media at 10 × 10(6) sperm/ml for 4 h at 37°C; total motility and protein tyrosine phosphorylation (PY) were evaluated. While motility remained unchanged, TCM or MW added with FBS enhanced the number of sperm showing PY-stained tails (25 ± 4.8% and 31 ± 6.6%; mean ± SEM, respectively) over BSA supplemented media (3 ± 1.2% and 11.7 ± 1.1%) for TCM and MW (p < 0.05). In view of the previous results, sperm were capacitated in TCM + FBS and MW + BSA (control); IVF trials were run in the same media supplemented with 200 ng/ml of progesterone, but no fertilization occurred. Our results show that TCM + FBS enhances Ex equine oocyte's meiotic competence over MW + BSA and TCM or MW added with FBS successfully induce equine PY over media supplemented with BSA.

Effect of calcium, bicarbonate, and albumin on capacitation-related events in equine sperm.

Repeatable methods for IVF have not been established in the horse, reflecting the failure of standard capacitating media to induce changes required for fertilization capacity in equine sperm. One important step in capacitation is membrane cholesterol efflux, which in other species is triggered by cholesterol oxidation and is typically enhanced using albumin as a sterol acceptor. We incubated equine sperm in the presence of calcium, BSA, and bicarbonate, alone or in combination. Bicarbonate induced an increase in reactive oxygen species (ROS) that was abolished by the addition of calcium or BSA. Bicarbonate induced protein tyrosine phosphorylation (PY), even in the presence of calcium or BSA. Incubation at high pH enhanced PY but did not increase ROS production. Notably, no combination of these factors was associated with significant cholesterol efflux, as assessed by fluorescent quantitative cholesterol assay and confirmed by filipin staining. By contrast, sperm treated with methyl-ß-cyclodextrin showed a significant reduction in cholesterol levels, but no significant increase in PY or ROS. Presence of BSA increased sperm binding to bovine zonae pellucidae in all three stallions. These results show that presence of serum albumin is not associated with a reduction in membrane cholesterol levels in equine sperm, highlighting the failure of equine sperm to exhibit core capacitation-related changes in a standard capacitating medium. These data indicate an atypical relationship among cholesterol efflux, ROS production, and PY in equine sperm. Our findings may help to elucidate factors affecting failure of equine IVF under standard conditions.

Calcium-calmodulin and pH regulate protein tyrosine phosphorylation in stallion sperm.

The mechanisms leading to capacitation in stallion sperm are poorly understood. The objective of our study was to define factors associated with regulation of protein tyrosine phosphorylation in stallion sperm. Stallion sperm were incubated for 4 h in modified Whitten's media with or without bicarbonate, calcium, or BSA. When sperm were incubated in air at 30×106/ml at initial pH 7.25, protein tyrosine phosphorylation was detected only in medium containing 25 mM bicarbonate alone; calcium and BSA inhibited phosphorylation. Surprisingly, this inhibition did not occur when sperm were incubated at 10×106/ml. The final pH values after incubation at 30×106 and 10×106 sperm/ml were 7.43 ± 0.04 and 7.83 ± 0.07 (mean ± s.e.m.) respectively. Sperm were then incubated at initial pH values of 7.25, 7.90, or 8.50 in either air or 5% CO2. Protein tyrosine phosphorylation increased with increasing final medium pH, regardless of the addition of bicarbonate or BSA. An increase in environmental pH was observed when raw semen was instilled into the uteri of estrous mares and retrieved after 30 min (from 7.47 ± 0.10 to 7.85 ± 0.08), demonstrating a potential physiological role for pH regulation of capacitation. Sperm incubated in the presence of the calmodulin (CaM) inhibitor W-7 exhibited a dose-dependent increase in protein tyrosine phosphorylation, suggesting that the inhibitory effect of calcium was CaM mediated. These results show for the first time a major regulatory role of external pH, calcium, and CaM in stallion sperm protein tyrosine phosphorylation.

Osmotic shock induces structural damage on equine spermatozoa plasmalemma and mitochondria.

The present study aimed to elucidate the effects that osmotic shock exerts on equine spermatozoa. To achieve this goal, a retrospective study of the cellular volume of 40 equine ejaculates subjected to osmolarities ranging from 75 to 900 mOsm in Biggers-Whitten-Whittingham (BWW) media was performed using a Multisizer3 Coulter Counter®. The 300 mOsm BWW solution was used as control. The sperm volume ranged between 37.93±0.6 (mean±Standard Error of the Mean (SEM)) in 75 mOsm BWW to 21.61±0.27 (mean±SEM) for 900 mOsm BWW. Thus the spermatozoa behaved as linear osmometers when adjusted to the Boyle Van't Hoff equation (R2=0.9808). After the different osmotic challenges, spermatozoa were returned to 300 mOsm BWW and the cellular volume was measured again. The results showed that the spermatozoa were able to retrieve the isosmolar volume (20.81±0.34; mean±SEM). Also, an ultrastructural study of spermatozoa membrane and mitochondria was accomplished using Transmission Electron Microscopy (TEM) after the osmotic challenges in 2 ejaculates. As observed by TEM, sperm plasmalemma swelled and detached from the sperm head in hypotonic conditions (75 mOsm), with blebbing on return to isosmolarity. When subjected to 900 mOsm, the sperm plasmalemma shrank, with disarrangement and blebbing when returned to isosmolarity. Mitochondria were also found to change their volume; the main pathologic change was irreversible vacuolization and changes in their arrangement for all the osmotic challenges tested. The present work leads to a better understanding of how osmotic shock adversely affects equine spermatozoa structure.

Androcoll-E large selects a subset of live stallion spermatozoa capable of producing ROS.

The aim of this study was to elucidate if SLC after 24 h storage selects the subpopulation of spermatozoa that better withstands osmotic shock. To test this hypothesis, viability, mitochondrial membrane potential (MMP) and superoxide anion (O(2)(·-)) production of uncentrifuged (UC) and single layer centrifugation (SLC) - selected spermatozoa were analyzed following SLC after storage of the semen. An aliquot of the extended ejaculate (100×10(6) spermatozoa/mL) was centrifuged through a single layer of a silane-coated silica based colloid formulation optimized for equine spermatozoa (Androcoll-E large, SLU, Sweden) and the rest was used as control. UC and SLC-sperm samples were subjected to osmotic challenges (75 and 900 mOsm) with a subsequent return to isosmolarity (300 mOsm) using Biggers-Whitten-Whittingham (BWW) medium. Viability and MMP decreased after the different osmotic stress in UC and SLC spermatozoa, and return to isosmolarity did not reverse these effects. O(2)(·-) production was enhanced after SLC in all osmolarities tested. Interestingly, the percentage of living spermatozoa showing O(2)(·-) production was increased after 900 mOsm stress in UC spermatozoa, this increase being more evident in SLC spermatozoa. Returning spermatozoa to 300 mOsm enhanced this percentage in UC viable cells but not in SLC spermatozoa. The scenario observed for UC spermatozoa shows that O(2)(·-) is produced in response to isolated hyperosmolarities and subsequent osmotic excursions. As the viability, MMP and cell volume remained the same between SLC and UC spermatozoa, we conclude that Androcoll-E large is likely selecting a higher percentage of physiologically O(2)(·-) producing spermatozoa.

Toxicity of glycerol for the stallion spermatozoa: effects on membrane integrity and cytoskeleton, lipid peroxidation and mitochondrial membrane potential.

Glycerol is, to date, the most widely used cryoprotectant to freeze stallion spermatozoa at concentrations between 2% and 5%. Cryoprotectant toxicity has been claimed to be the single most limiting factor for the success of cryopreservation. In order to evaluate the toxic effects of the concentrations of glycerol used in practice, stallion spermatozoa were incubated in Biggers Whitten and Whittingham (BWW) media supplemented with 0%, 0.5%, 1.5%, 2.5%, 3.5%, and 5% glycerol. In two additional experiments, a hyposmotic (75 mOsm/kg) and a hyperosmotic (900 mOsm/kg) control media were included. Sperm parameters evaluated included cell volume, membrane integrity, lipid peroxidation, caspase 3, 7, and 8 activation, mitochondrial membrane potential, and integrity of the cytoskeleton. Glycerol exerted toxicity at concentrations ≥ 3.5% and the maximal toxicity was observed at 5%. The actin cytoskeleton was especially sensitive to glycerol presence, inducing rapid F actin depolymerization at concentrations over 1.5%. The sperm membrane and the mitochondria were other structures affected. The toxicity of glycerol is apparently related to osmotic and nonosmotic effects. In view of our results the concentration of glycerol in the freezing media for stallion spermatozoa should not surpass 2.5%.

Fatty acids and plasmalogens of the phospholipids of the sperm membranes and their relation with the post-thaw quality of stallion spermatozoa.

Fatty acids and plasmalogens were extracted from the phospholipids of the plasma membrane of stallion spermatozoa, to determine their relation with sperm quality after freezing and thawing. Sperm quality was rated using a quality index that combined the results of the analysis of sperm motility and velocity (CASA analysis), membrane status and mitochondrial membrane potential (flow cytometry) post thaw. Receiving operating system (ROC) curves were used to evaluate the value of specific lipid components of the sperm membrane herein studied as forecast of potential freezeability. From all parameters studied the ratio of percentage of C16 plasmalogens related to total phospholipids was the one with the better diagnostic value. For potentially bad freezers, the significant area under the ROC-curve was 0.74, with 75% sensitivity and 79.9% specificity for a cut off value of 26.9. Also the percentage of plasmalogens respect to total phospholipids gave good diagnostic value for bad freezers. On the other hand, the percentage of C18 fatty aldehydes related to total phospholipids of the sperm membrane properly forecasted freezeability with an area under the ROC curve of 0.70 with 70% sensitivity and 62.5% specificity for a cut off value of 0.32.

Calibration of a Shack-Hartmann wavefront sensor as an orthographic camera.

We demonstrate a method to calibrate a Shack-Hartmann sensor as an orthographic camera. This calibration method permits us to obtain the distance, the rotation matrix between the microlens array and CCD imaging planes, and the projection matrix, which models the projection of the incoming rays to the CCD imaging plane. The proposed calibration method introduces a very compact matrix notation and allows wavefront reconstruction without an explicit centroid search between the reference and distorted spot diagrams. We show a set of simulations in code V that prove the effectiveness of the proposed method.

Inhibition of the mitochondrial permeability transition pore reduces "apoptosis like" changes during cryopreservation of stallion spermatozoa.

In order to evaluate to what extent the changes that occur during cryopreservation involve the mitochondrial permeability transition pore (PT-pore), a specific inhibitor was used during the cryopreservation process of stallion spermatozoa. Four ejaculates from each of 7 stallions were frozen using a standard protocol. Before freezing, each ejaculate was split into three subsamples. The first was supplemented with 2.5 microM bongkrekic acid (BA) and the second with 5 microM BA. The third subsample served as control. The BA significantly reduced the percentage of spermatozoa depicting active caspases after thawing, reduced the percentage of spermatozoa with increased membrane permeability, and increased the mitochondrial membrane potential of thawed sperm. Sperm motility was reduced as a result of the treatment. It is concluded that the mitochondrial pathway of apoptosis seems to be an important factor involved in the sublethal damage that equine spermatozoa experience after freezing and thawing, and that sperm motility in the equine species is largely dependent on mitochondrial ATP produced by oxidative phosphorylation.

Does the microbial flora in the ejaculate affect the freezeability of stallion sperm?

In an attempt to evaluate the possible relationship between the microbial flora in the stallion ejaculate and its ability to freeze,three ejaculates from five stallions were frozen using a standard protocol. Before freezing, an aliquot was removed for bacteriological analysis. Bacterial growth was observed in all the ejaculates studied. The isolated microorganisms were:Staphylococcus spp. and Micrococcus spp. (in all the stallions), beta-haemolytic Streptococcus (in stallions 3 and 4), Corynebacterium spp. (in stallions 1, 3-5), Rhodococcus spp. (in stallion number 2), Pseudomonas spp. (in stallion number 1) and Klebsiella spp. (in stallions 1, 3 and 5). The presence and richness of Klebsiella and beta-haemolytic Streptococcus in the ejaculate were related to two sperm variables post-thaw,namely the proportion of dead spermatozoa (ethidium+ cells; r = 0.55, p < 0.05) and the amplitude of lateral displacement of the sperm head (ALH, microm; r = -0.56, p < 0.05), respectively.The degree of growth of Corynebacterium spp. in the ejaculate was positively correlated with the percentage of spermatozoa showing high caspase activity post-thaw(r = 0.62, p < 0.05). The presence and number of colonies of beta-haemolytic Streptococcus were negatively correlated (r = -0.55, p < 0.05) with low sperm caspase activity. It is concluded that the microbial flora of the equine ejaculate maybe responsible for some of the sublethal damage experimented by the spermatozoa during cryopreservation.

Effect of cryopreservation on nitric oxide production by stallion spermatozoa.

The ability of stallion spermatozoa to produce nitric oxide (NO) before (fresh) and after freezing and thawing (FT) was evaluated by means of flow cytometry after loading the sperm suspension with the probe, 4,5-diaminofluorescenin diacetate. The presence of NO synthase (NOS) was investigated by Western blotting using anti-NOS1, anti-NOS3, or anti-universal NOS antibodies (Abs). While NO was detected both in fresh and FT sperm suspensions, its production increased after cryopreservation only when egg yolk was removed from the extender. Anti-NOS1 Ab intensively labeled a single band with an apparent molecular mass of approximately 83 kDa. On the other hand, the Ab developed against the NOS3 showed a band of approximately 96 kDa in fresh and FT sperm lysates. NO production was positively correlated with sperm motility and velocity after thaw, suggesting an NO role for the functionality of cryopreserved stallion spermatozoa; but the production of NO is compromised in egg yolk-containing extenders.