RESUMEN
Capacitation-like changes affect sperm of several species, such as ram, reducing cell survival and fertilizing competence. Proteins from seminal plasma stabilize sperm plasma membranes, being an interesting focus to develop strategies for improving cryopreserved ram semen performance. To date, biotechnologies are focused to reduce damage in frozen-thawed ram spermatozoa through the addition of bioactives. Serine Protease Inhibitor Kazal-type 3 (SPINK3) is a little protein synthesized by mouse seminal vesicle and secreted to seminal plasma. While attached to the sperm, this protein binds to non-capacitated sperm and blocks calcium entry, avoiding a premature physiological capacitation and consequently, acrosome reaction. Due to these characteristics, SPINK3 has been proposed as a decapacitating factor. The aim of this work was to assess whether heterologous SPINK3 is able to protect ram sperm from the well-known cell damages produced by freezing/thawing and to understand the mechanisms by which it is acting. Sperm were supplemented with 13 µM SPINK3 before freezing in an egg yolk-based extender or after thawing and selection. Under both conditions, SPINK3 decreased intracellular calcium content (p < 0.05) and reduced the 25 kDa tyrosine phosphorylated protein demonstrating a decapacitating effect, although the addition of the protein before cryopreservation was not enough to improve other sperm parameters. However, the addition of SPINK3 post thawing was able to significantly ameliorate viability, motility, mitochondrial status and to avoid the increase of lipid peroxidation (p < 0.05). Moreover, sperm treated with SPINK3 was not only still capable to fertilize, but also improved it, as evidenced by an increase in the oocyte cleavage rate (p < 0.05) although, the embryo development and embryo quality were not affected. Our findings would contribute to develop a strategy for improving sperm quality by using decapacitating proteins. In fact, the outcomes of this work demonstrate that SPINK3 is able to reduce sperm cryo-injuries when is added after thawing, improving functionality and thus in vitro fertilization results.
Asunto(s)
Criopreservación/veterinaria , Preservación de Semen/veterinaria , Inhibidores de Serinpeptidasas Tipo Kazal/farmacología , Ovinos/fisiología , Espermatozoides/fisiología , Animales , Técnicas de Cultivo de Embriones , Embrión de Mamíferos , Fertilización In Vitro/veterinaria , Regulación de la Expresión Génica , Peroxidación de Lípido/efectos de los fármacos , Masculino , Análisis de Semen , Inhibidores de Serinpeptidasas Tipo Kazal/genética , Inhibidores de Serinpeptidasas Tipo Kazal/metabolismo , Motilidad EspermáticaRESUMEN
This work offers researchers the first version of an open-source sperm tracker software (Sperm Motility Tracker, V1.0) containing a novel suit of algorithms to analyze sperm motility using ram and buck sperm as models. The computer-assisted semen analysis is used in several publications with increasing trend worldwide in the last years, showing the importance of objective methodologies to evaluate semen quality. However, commercial systems are costly and versatility is constrained. In the proposed method, segmentation is applied and the tracking stage is performed by using individual Kalman filters and a simplified occlusion handling method. The tracking performance in terms of precision (number of true tracks), the percentage of fragmented paths and percentage of correctly detected particles were manually validated by three experts and compared with the performance of a commercial motility analyzer (Microptic's SCA). The precision obtained with our sperm motility tracker was higher than the one obtained with a commercial software at the current acquisition frame rate of 25 fps (P < 0.0001), concomitantly with a similar percentage of fragmentized tracks (P = 0.0709) at sperm concentrations ranging 25-37 × 106 cells/mL. Moreover, our tracker was able to detect trajectories that were unseen by SCA. Kinetic values obtained by using both methods were contrasted. The higher values found were explained based on the better performance of our sperm tracker to report speed parameters for very fast motile sperm. To standardize results, acquisition conditions are suggested. This open-source sperm tracker software has a good plasticity allowing researchers to upgrade according requirements and to apply the tool for sperm from a variety of species.
Asunto(s)
Análisis de Semen/métodos , Motilidad Espermática/fisiología , Animales , Cabras , Masculino , Ovinos , Programas Informáticos , Recuento de EspermatozoidesRESUMEN
Assisted sperm morphometry analysis (ASMA) was used in this study to determine the effects of cryopreservation on bull spermatozoa distribution in morphometrically distinct subpopulations. Ejaculates were collected from five bulls and were divided. One portion was diluted at 30 degrees C in a skim milk-egg yolk medium, containing glycerol. A microscope slide was prepared from single extended sperm samples prior to freezing. The remainder of each sample was frozen in nitrogen vapours. After thawing, sperm smears were prepared as described above. All slides were air dried and stained with Hemacolor. The sperm-head dimensions for a minimum of 200 sperm heads were analysed from each sample by means of the Sperm-Class Analyser (SCA), and the mean measurements recorded. Our results showed that applying the ASMA technology and multivariate cluster analyses, it was possible to determine that three separate subpopulations of spermatozoa with different morphometric characteristics coexist in bull ejaculates (large, average and small spermatozoa). The mean values of each sperm head dimension among the three subpopulations of spermatozoa were significantly different (p < 0.001). Besides, there were significant (p < 0.001) differences in the distribution of these three sperm subpopulations between fresh and thawed samples. Thus, the percentage of representation of the subpopulation that includes those spermatozoa whose dimensions are the biggest, decreased from 52.06% in extended fresh samples to 15.51% in the thawed ones. Contrarily, the percent of representation of the subpopulation containing the smallest spermatozoa, increased from 8.70% in extended fresh samples to 34.04% in the thawed ones. In conclusion, the present study confirms the heterogeneity of sperm head dimensions in bull semen, heterogeneity that vary through the cryopreservation procedure.
Asunto(s)
Criopreservación/veterinaria , Preservación de Semen/veterinaria , Espermatozoides/fisiología , Animales , Bovinos , MasculinoRESUMEN
Venous thromoboembolism is one of the most common complications in cancer patients and may have serious consequences. At present, most clinical oncologists report using thromboprophylaxis in less than 5% of patients. One of the possible reasons for this limited use is the lack of oncology specific guidelines. In effect, while there are excellent guidelines for optimising the use of antithrombotic agents to prevent and treat thromboembolism, they must be adapted to the concrete context of cancer patients. The present review explores how different situations affect cancer patients and their risk of developing venous thromboembolism (VTE), and evaluates the situations in which antithrombotic agents should be administered to treat and prevent VTE.
Asunto(s)
Fibrinolíticos/uso terapéutico , Neoplasias/sangre , Tromboembolia/prevención & control , Trombofilia/etiología , Trombosis de la Vena/prevención & control , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Antineoplásicos Hormonales/efectos adversos , Antitrombina III/análisis , Cateterismo Venoso Central/efectos adversos , Ensayos Clínicos como Asunto/estadística & datos numéricos , Método Doble Ciego , Femenino , Fibrinolíticos/administración & dosificación , Fibrinolíticos/efectos adversos , Hemorragia/inducido químicamente , Hemorragia/prevención & control , Heparina de Bajo-Peso-Molecular/administración & dosificación , Heparina de Bajo-Peso-Molecular/uso terapéutico , Humanos , Inmovilización/efectos adversos , Masculino , Péptido Hidrolasas/análisis , Complicaciones Posoperatorias/etiología , Complicaciones Posoperatorias/prevención & control , Estudios Prospectivos , Embolia Pulmonar/tratamiento farmacológico , Embolia Pulmonar/etiología , Embolia Pulmonar/prevención & control , Riesgo , Tromboembolia/etiología , Trombofilia/tratamiento farmacológico , Factor A de Crecimiento Endotelial Vascular/sangre , Trombosis de la Vena/etiologíaRESUMEN
BACKGROUND: Although the treatment of asthma has been addressed in several guidelines, the management of the first acute wheezing episode in infants has not often been evaluated. We surveyed practicing pediatricians in Spain about the treatment they would provide in a simulated case. MATERIAL AND METHODS: A random sample of 3000 pediatricians and physicians who normally treated children was surveyed. The questionnaire inquired about how they would treat a first mild-to-moderate wheezing attack in a 5-month-old boy with a personal and family history of allergy. Pediatricians were asked about their professional background. RESULTS: A total of 2347 questionnaires were returned with useful data (78.2%). Most (90.4%) of the pediatricians would use a short-acting beta2 agonist (SABA) via a metered-dose inhaler with a spacer and a face mask or nebulizer. However, only 34.5% chose a SABA alone: 31.3% added an oral steroid and 27.6% added an inhaled corticosteroid (ICS). The factors associated with the use of ICS in the acute attack were: (1) lack of specific training in pediatrics (OR 1.45; 1.12-1.85) and (2) primary care health center setting (OR 1.31; 1.01-1.69) or rural setting (OR 1.28; 1.01-1.66). Forty-four percent did not recommend any follow-up treatment while 20.7% prescribed ICS as maintenance therapy. The factors related to this decision were the same as those described above. CONCLUSIONS: The management of a first wheezing episode seems to meet published guidelines among Spanish pediatricians with formal training in pediatrics and in those who work in a hospital setting or in urban areas.
Asunto(s)
Manejo de Caso/estadística & datos numéricos , Hipersensibilidad Inmediata/tratamiento farmacológico , Pediatría/estadística & datos numéricos , Pautas de la Práctica en Medicina/estadística & datos numéricos , Ruidos Respiratorios , Enfermedad Aguda , Administración por Inhalación , Administración Oral , Corticoesteroides/administración & dosificación , Corticoesteroides/uso terapéutico , Antagonistas de Receptores Adrenérgicos beta 2 , Antagonistas Adrenérgicos beta/administración & dosificación , Antagonistas Adrenérgicos beta/uso terapéutico , Antiasmáticos/administración & dosificación , Antiasmáticos/uso terapéutico , Asma/complicaciones , Asma/diagnóstico , Asma/tratamiento farmacológico , Espasmo Bronquial/diagnóstico , Espasmo Bronquial/tratamiento farmacológico , Espasmo Bronquial/etiología , Preescolar , Recolección de Datos , Quimioterapia Combinada , Adhesión a Directriz , Humanos , Hipersensibilidad Inmediata/diagnóstico , Hipersensibilidad Inmediata/etiología , Lactante , Espaciadores de Inhalación , Máscaras , Inhaladores de Dosis Medida , Guías de Práctica Clínica como Asunto , Recurrencia , España , Encuestas y CuestionariosRESUMEN
To obtain better ram semen extenders for artificial insemination (AI), we developed effective trehalose-containing hypertonic diluents. The cryoprotective action of trehalose has been explained by its dehydrating activity and interaction with cell membranes. Accordingly, we tested the cryopreserving capacity of different combinations of a Salamon's modified plus trehalose extender with EDTA. Evaluations were based on the percentage of motile spermatozoa and acrosome integrity, measured after thawing and after a 4-h post-thaw resistance test at 37 degrees C. We conclude that the combination of trehalose plus EDTA confers the highest cryopreserving activity tested, not only for freeze-thawing but also for post-thawing resistance, possibly by removing calcium from the medium thereby preventing cation competition with trehalose for membrane-binding sites.