Diversity of N2-fixing cyanobacteria from Andalusian paddy fields and analysis of their potential as bioinoculants

The marshes of the Guadalquivir River contain the largest area of rice cultivation in Spain, where more than 40,000 ha are used every year for rice production. These wetland areas provide a perfect place for rice cultivation, and represent a unique aquaticterrestrial habitat that hold more wintering waterfowl than any other European wetland. Paddies require large amounts nitrogen and phosphorus for their growth, development and production. Though, flooded conditions used for rice cultivation drastically diminish efficiency inorganic nitrogen fertilizers, being only 30–40% used by the plant, and in some cases even less. Large amounts of nitrogen fertilizers are dissolved in the surface water and lost, causing environmental pollution and health problems due to losses through N2O and NO volatilization, denitrification, and leaching (Ishii et al., 2011)

DNA integrity and viability of testicular cells from diverse wild species after slow freezing or vitrification

Introduction and objectiveCryopreservation of testicular tissues offers new possibilities to protect endangered species, genetically valuable individuals or even the fertility potential of prepubertal individuals who have died unexpectedly. However, the use of this technique still remains a challenge. In this study, slow freezing and vitrification of testicular tissue was investigated to find out which cryopreservation method could better preserve the viability and DNA integrity of testicular germ cells in diverse wild species.MethodsTestes were obtained post-mortem from 18 artiodactyls (wild boar, roe deer, dwarf goat, mhor gazelle, European mouflon, African forest buffalo, Malayan tapir, dorcas gazelle, Iberian ibex, gnu, red river hog), 5 primates (colobus monkey, capuchin monkey, mandrill), 8 carnivores (gray wolf, Persian leopard, binturong, European mink, American black bear, suricata), and 2 rodents (Patagonian mara). The testicles belonged to adult individuals and were cut into small pieces and cryopreserved by needle immersed vitrification or uncontrolled slow freezing using a passive cooling device. After warming or thawing, testicular tissues were enzymatically digested and two germ cell types were differentiated based on their morphology: rounded cells (spermatogonia, spermatocytes, and early spermatids) and elongated cells (elongated spermatids and spermatozoa). Cell viability was assessed by SYBR-14/propidium iodide while DNA fragmentation by TUNEL assay with fluorescence microscope.Results and discussionOur preliminary results revealed that our uncontrolled slow freezing method better preserved the viability and DNA integrity of elongated cells than vitrification. Such trend was observed in all species, being significant in artiodactyls, carnivores, and primates. Similarly, the viability and DNA integrity of rounded cells was also better maintained in primates by uncontrolled slow freezing, while in carnivores, vitrification by needle immersion showed better results in this type of cells. In artiodactyls and rodents both techniques preserved the viability of rounded cells in a similar manner, although the DNA integrity of these cells was greater after needle immersed vitrification in artiodactyls.ConclusionsIn conclusion, the effectiveness of each cryopreservation method is affected by the phylogenetic diversity between species and cell type

Effectiveness of ultra-rapid cryopreservation of sperm from endangered species, examined by morphometric means

8 Pág.This study compares the effectiveness of the ultra-rapid and conventional freezing of sperm from captive bovids, giraffids, cervids, ursids, a cercopithecid, a delphinid and a phascolarctid. The relationship between sperm head dimensions and cryosurvival was also examined. Compared to conventional freezing, the ultra-rapid freezing of epididymal sperm from the dama gazelle, giraffe and brown bear returned higher cryoresistance ratios (CR, the ratio, in percentage, between the value of the variable after thawing/value before thawing) for sperm viability and motility. In the remaining species, the conventional freezing of epididymal sperm returned better CR values. The conventional freezing method also returned better CR values for ejaculated samples from all species. The head dimensions of both fresh epididymal and ejaculated sperm differed widely among species: for epididymal sperm, dolphin sperm heads were the smallest (7.189 ± 0.049 μm2) and dama gazelle sperm heads the largest (43.746 ± 0.291 μm2), while for ejaculated sperm, giant panda sperm heads were the smallest (15.926 ± 0.150 μm2) and mouflon sperm heads the largest (38.258 ± 0.104 μm2). However, no significant correlations were detected between the CR for motility, viability, membrane functional integrity or acrosome integrity and the sperm head area, either for epididymal or ejaculated sperm. In conclusion, ultra-rapid freezing is especially recommended for the cryopreservation of dama gazelle, giraffe and brown bear epididymal sperm. Sperm head dimensions appear not to be useful predictors of how well sperm might survive freezing.This work was funded by MINECO/AEI/FEDER, UE, Spain grants AGL2014-52081-R and AGL2017-85753-R, and by the Fundación Parques Reunidos - INIA, Spain agreement CC16-001. The authors thank DG Biodiversidad (Principado de Asturias), and the Consejería de Medio Ambiente (Junta de Andalucía), for their unfailing help in the provision of animals from the mentioned wildlife reserves and in implementing the projects proposed. The authors thank the Ayuntamiento de Sedella (Málaga) for its help in the provision of a field laboratory close to the Tejeda and Almijara mountains. Thanks are also due to the Parque Zoólogico del Ayuntamiento de Córdoba, the Parque Zoológico del Ayuntamiento de Guadalajara and the Zoo-Aquarium, Madrid, for their help in the execution of this project.Peer reviewe

Inflammatory cytokines and organ dysfunction associate with the aberrant DNA methylome of monocytes in sepsis

Sepsis, a life-threatening organ dysfunction caused by a dysregulated systemic immune response to infection, associates with reduced responsiveness to subsequent infections. How such tolerance is acquired is not well understood but is known to involve epigenetic and transcriptional dysregulation. Bead arrays were used to compare global DNA methylation changes in patients with sepsis, non-infectious systemic inflammatory response syndrome, and healthy controls. Bioinformatic analyses were performed to dissect functional reprogramming and signaling pathways related to the acquisition of these specific DNA methylation alterations. Finally, in vitro experiments using human monocytes were performed to test the induction of similar DNA methylation reprogramming. Here, we focused on DNA methylation changes associated with sepsis, given their potential role in stabilizing altered phenotypes. Tolerized monocytes from patients with sepsis display changes in their DNA methylomes with respect to those from healthy controls, affecting critical monocyte-related genes. DNA methylation profiles correlate with IL-10 and IL-6 levels, significantly increased in monocytes in sepsis, as well as with the Sequential Organ Failure Assessment score; the observed changes associate with TFs and pathways downstream to toll-like receptors and inflammatory cytokines. In fact, in vitro stimulation of toll-like receptors in monocytes results in similar gains and losses of methylation together with the acquisition of tolerance. We have identified a DNA methylation signature associated with sepsis that is downstream to the response of monocytes to inflammatory signals associated with the acquisition of a tolerized phenotype and organic dysfunction

Slow and ultra-rapid freezing protocols for cryopreserving roe deer (Capreolus capreolus) epididymal sperm collected at different times of year

10 Pág. Centro de Investigación en Sanidad Animal (CISA) / Departamento de Reproducción animalThe roe deer is a monoestrous species with a very short rutting season. The present work reports the most suitable period for collecting epididymal sperm and describes the effect of two cooling rates on the post-thaw quality of sperm. Testes were collected 24–48 h after death. Samples of sperm flushed from the epididymis were subjected to either (1) dilution in a Tris-citric acid-glucose-egg yolk-based medium with glycerol, and slow freezing in straws, or (2) dilution in the same extender but replacing the glycerol with 100 mM of sucrose, and ultra-rapid freezing in pellets. Sperm motility, acrosome and membrane integrity, morphometry and morphological abnormalities were analysed before and after cryopreservation. Spermatogenic activity was investigated via histological examination of testis sections. Several testes collected between April, May and September showed no spermatogenic activity. All those collected in June–August showed spermatogenic activity. No significant difference was detected in the cryoresistance ratios associated with the conventional slow freezing, between sperm collected during the pre-rutting (April–May) and rutting (June–August) periods. No significant differences were seen between the slow-frozen-thawed and the ultra-rapid-frozen-thawed sperm in terms of percentage of viable sperm or the percentage of sperm with morphological abnormalities. Slow freezing returned significantly better (P<0.05) values for post-thaw acrosome integrity (43.3% vs. 25.0%) and straight-line velocity (19 μm/s vs. 4 μm/s). For both freezing methods, sperm heads were smaller post-thawing than pre-freezing (P<0.001). In conclusion, both the pre-rutting and rutting season are suitable periods for freezing roe deer sperm. Ultra-rapid freezing did not provide suitable results.This research was funded by MINECO/AEI/FEDER and EU grant AGL2017-85753-R. P. Bóveda was the recipient of a grant for pre-doctoral researchers from MINECO (AEI/FSE, UE). Octavio Mejía was the recipient of a research fellowship from the PASPA-DGAPA-UNAM (México). V.N. Flores-Gil was funded by FONDECYT-CONCYTEC (grant contract number 000245-2015-FONDECYT).Peer reviewe

Vitrification of Iberian wolf (Canis lupus signatus) sperm: A possible alternative to conventional cryopreservation

9 Pág. Departamento de Reproducción animal. (INIA)Sperm vitrification is a simple, inexpensive method that allows the cryopreservation of sperm in the field and for endangered species is a useful alternative to conventional freezing. The study, therefore, is focused on the suitability of vitrification for cryopreserving Iberian wolf sperm and utilizing plasma testosterone concentration as a marker for procedure efficacy. Sperm and blood samples were collected from 17 wolves. There were 14 samples suitable for cryopreservation (12 ejaculated and two epididymal). Immediately after collection, these samples were proportioned into two aliquots for conventional freezing using a Tris-citric acid-glucose based extender (TCG) or vitrification utilizing an animal protein free extender (HTF®). Vitrification occurred by directly plunging a sperm suspension into liquid nitrogen. Sperm were assessed for motility, membrane integrity, acrosomal status and DNA integrity before and after cryopreservation. With both techniques, there were similar post-thaw/warming results (P > 0.05) with respect to progressive motility, kinetic variables VCL, VSL, VAP and BCF, DNA fragmentation, sperm membrane functionality and morphological abnormalities. Total motile sperm, progression ratios LIN, STR, and WOB, the ALH, sperm viability and sperm with intact membrane and acrosome were greater (P < 0.05) in the conventional frozen-thawed sperm than vitrified-warmed sperm. Plasma testosterone concentrations varied from 0.0 ng/mL to 7.7 ng/mL. For epididymal sperm, sperm motility and viability following thawing were greater in vitrified-warmed samples than conventionally-frozen samples; however, small sample numbers precluded statistical analysis. When considered together, these results indicate vitrification may be a possible alternative for wolf sperm cryopreservation.This research was funded by MCINN/AEI/FEDER grant AGL2017-85753-R and by the Fundación Parques Reunidos INIA agreement CC19-096.Peer reviewe

Cooling rate modifies the location of aquaporin 3 in spermatozoa of sheep and goat

30 Pág.The freeze-thawing process induces osmotic changes that may affect the membrane domain location of aquaporins’ (AQP) in spermatozoa. Recent studies suggest that changes in AQP3 localization allows better sperm osmo-adaptation, improving the cryoresistance. Ultra-rapid freezing is an alternative cryopreservation technique that requires less equipment than conventional freezing, and it is faster, simpler and can be used in the field. This study aimed to determine the influence of freezing-thawing rates (slow (control) vs. ultra-rapid) on AQP3 expression and location in the spermatozoa from small ruminants (sheep and goats) and its relationship with sperm cryo-damage. Spermatozoa were collected from 10 Merino rams and 10 Murciano-Granadina bucks. The presence and distribution of AQP3 were assessed by Western blotting and immunocytochemistry (ICC), employing a commercial rabbit polyclonal antibody. Sperm motility was CASA system-analyzed, and membrane and acrosome integrity assessed by fluorescence (PI/PNA-FITC). Western blotting did not detect a significant effect of freezing-thawing rate on the amount of AQP3 while ICC found freezing-thawing rate affecting AQP3 location (P<0.05). In both species, the percentages of spermatozoa showing AQP3 in the post-acrosome region, mid-piece, and principal piece of the tail were greater in samples cryopreserved by slow freezing-thawing (control) than ultra-rapid freezing-thawing rates (P<0.05). Spermatozoa cryopreserved using ultra-rapid freezing-thawing showed decrease motility, plasma membrane, and acrosome integrity (P<0.05), which might be related, at least in part, to a lower expression of AQP3. In conclusion, the cooling rate modifies the location of AQP3 in spermatozoa of sheep and goat, which might be associated with sperm cryosurvival.This study was supported by the project PID2020-113288RB-I00/AEI/10.13039/501100011033. B. Pequeño was the recipient of a grant for pre-doctoral researchers from AEI (PRE2018-085637).Peer reviewe

Use of interactive response devices for the improvement of teaching in the degrees of the Faculty of Sciences

La implantación de los nuevos Grados dentro del EEES supone un nuevo enfoque metodológico, pero sobre todo evaluador. El profesor debe esforzarse en ser capaz de valorar no sólo el nivel de conocimientos del alumnado sobre los contenidos, ya que la sociedad actual demanda un perfil de los egresados más completo basado en la formación por competencias. La adquisición de ambas facetas por parte del alumnado se basa principalmente en actividades presenciales y metodologías docentes aplicadas en el desarrollo de las asignaturas, tanto en grupo reducido como en grupo completo. Por tanto, cualquier herramienta adicional que ayude al docente a facilitar esta doble tarea es bien recibida, pues permitirá evaluar el rendimiento académico global del alumnado. Recientemente la Facultad de Ciencias ha puesto en marcha una experiencia piloto mediante la cual un nutrido grupo de profesores ha incorporado en su docencia un sistema de mandos inalámbricos de respuesta. Actualmente, la Facultad de Ciencias dispone de un total de 82 mandos inalámbricos de respuesta y tres antenas receptoras. Esta nueva tecnología ha permitido al docente realizar, de forma interactiva, preguntas colectivas al alumnado y recoger las respuestas individuales de forma inmediata. Los resultados obtenidos en este proyecto nos permiten ser muy optimistas con el uso de esta herramienta y nos animan a continuar en la misma línea. El profesorado participante se ha mostrado dispuesto a continuar empleando esta TIC en su docencia, tratando de abarcar más asignaturas, diversificar el tipo de pruebas, y emplear los mandos de respuesta en grupo completo, cuando el número de alumnos lo permita. La puesta en común de los resultados revela que, de forma general, este tipo de sistemas de respuesta incrementa el nivel de atención del alumnado, ya que se les demanda de forma continuada su participación activa durante la sesión. Se potencia la retención de conceptos, al suministrar en el acto la respuesta correcta a cada pregunta realizada. Permite también al profesorado conocer el nivel previo de conocimientos sobre la materia a impartir, ya que se puede realizar este tipo de preguntas al comienzo de la sesión y enfocar entonces su atención en las debilidades manifestadas. Asimismo se puede testar el grado de adquisición de dichos conocimientos, realizando este tipo de cuestionarios después de la sesión. Además, el tratamiento posterior de los resultados, de forma individualizada, permite gestionar de forma ágil el nivel de conocimientos y adquisición de competencias en lo que es un sistema de evaluación continua, tan necesario en los nuevos enfoques metodológicos nacidos de la implantación del EEES. Por último destacar que el profesorado participante ha considerado que se trata de una herramienta de gran utilidad para la consecución del objetivo de excelencia en la docencia, que además es eficaz y de fácil manejo, y entiende que no debe ser una actividad puntual, sino un proceso transformador en la actividad docente e interacción alumno-profesor, configurándose como una herramienta habitual en los sistemas de enseñanza-aprendizaje.Implementation of the new degrees in the EHEA involve a new methodological approach, but above all evaluation. The teacher should strive to be able to assess not only the level of knowledge of the students on the content, as the current society demands a profile of the most complete graduates based on training by competencies. The acquisition of both facets by students is based mainly on face-to-face activities and teaching methodologies applied in the development of the subjects, both in small group and in whole group. Therefore, any additional tool that helps the teacher to facilitate this double task is well received, since it will allow to evaluate the overall academic performance of the students. Recently the Faculty of Sciences has launched a pilot experience through which a large group of teachers has incorporated into their teaching a system of interactive response devices. Currently, the Faculty of Sciences has a total of 82 wireless response controllers and three receiving antennas. This new technology has enabled the teacher to interact interactively with the students and collect the individual answers immediately. The results obtained in this project allow us to be very optimistic with the use of this tool and encourage us to continue in the same line. The participating teachers have been willing to continue using this ICT in their teaching, trying to cover more subjects, diversify the type of tests, and use the full group response commands, when the number of students allows. The sharing of results reveals that, in a general way, this type of response systems increases the level of attention of the students, since they are continuously demanded their active participation during the session. The retention of concepts is strengthened by providing the correct answer to every question asked. It also allows the faculty to know the previous level of knowledge about the subject to be taught, since this type of questions can be asked at the beginning of the session and then focus their attention on the weaknesses manifested. It is also possible to test the degree of acquisition of this knowledge, performing this type of questionnaire after the session. In addition, the subsequent treatment of the results, in an individualized way, allows to manage in an agile way the level of knowledge and acquisition of competences in what is a system of continuous evaluation, so necessary in the new methodological approaches born from the implantation of the EHEA. Finally, it should be pointed out that the participating teachers have considered that this is a very useful tool for achieving the objective of excellence in teaching, which is also efficient and easy to use and understands that it should not be a specific activity, but a transforming process in the teaching activity and student-teacher interaction, being configured as a habitual tool in the teaching-learning systems

Barrier crossings and winds shape daily travel schedules and speeds of a flight generalist

External factors such as geography and weather strongly affect bird migration influencing daily travel schedules and flight speeds. For strictly thermal-soaring migrants, weather explains most seasonal and regional differences in speed. Flight generalists, which alternate between soaring and flapping flight, are expected to be less dependent on weather, and daily travel schedules are likely to be strongly influenced by geography and internal factors such as sex. We GPS-tracked the migration of 70 lesser kestrels (Falco naumanni) to estimate the relative importance of external factors (wind, geography), internal factors (sex) and season, and the extent to which they explain variation in travel speed, distance, and duration. Our results show that geography and tailwind are important factors in explaining variation in daily travel schedules and speeds. We found that wind explained most of the seasonal differences in travel speed. In both seasons, lesser kestrels sprinted across ecological barriers and frequently migrated during the day and night. Conversely, they travelled at a slower pace and mainly during the day over non-barriers. Our results highlighted that external factors far outweighed internal factors and season in explaining variation in migratory behaviour of a flight generalist, despite its ability to switch between flight modes.Peer reviewe

Translocated LPS Might Cause Endotoxin Tolerance in Circulating Monocytes of Cystic Fibrosis Patients

Cystic Fibrosis (CF) is an inherited pleiotropic disease that results from abnormalities in the gene codes of a chloride channel. The lungs of CF patients are chronically infected by several pathogens but bacteraemia have rarely been reported in this pathology. Besides that, circulating monocytes in CF patients exhibit a patent Endotoxin Tolerance (ET) state since they show a significant reduction of the inflammatory response to bacterial stimulus. Despite a previous description of this phenomenon, the direct cause of ET in CF patients remains unknown. In this study we have researched the possible role of microbial/endotoxin translocation from a localized infection to the bloodstream as a potential cause of ET induction in CF patients. Plasma analysis of fourteen CF patients revealed high levels of LPS compared to healthy volunteers and patients who suffer from Chronic Obstructive Pulmonary Disease. Experiments in vitro showed that endotoxin concentrations found in plasma of CF patients were enough to induce an ET phenotype in monocytes from healthy controls. In agreement with clinical data, we failed to detect bacterial DNA in CF plasma. Our results suggest that soluble endotoxin present in bloodstream of CF patients causes endotoxin tolerance in their circulating monocytes