Effects of Different Diluents and Freezing Methods on Cryopreservation of Hu Ram Semen.

The purpose of this study was to investigate the effects of different diluents and freezing methods on the quality of thawed sperm after cryopreservation and find an inexpensive and practical method for freezing Hu ram semen for use in inseminations under farm conditions. Ejaculates were collected from five Hu rams. In experiment I, ejaculates were diluted with eight different freezing diluents (basic diluents A, B, C, D, E, F, G, and H). After dilution and cooling, the samples were loaded into 0.25 mL straws and frozen using the liquid nitrogen fumigation method. In experiment II, diluent C was used as the basic diluent and the semen was frozen using liquid nitrogen fumigation and two program-controlled cooling methods. For analysis, frozen samples were evaluated in terms of motility parameters (total motility (TM), progressive motility (PM)), biokinetic characteristics (straight-line velocity (VSL), average path velocity (VAP), curvilinear velocity (VCL), amplitude of lateral head displacement (ALH), wobble movement coefficient (WOB), average motion degree (MAD)), reactive oxygen species (ROS) level, and membrane and acrosome integrity. In experiment I, diluent C had higher TM, PM, and acrosome and membrane integrity and lower ROS compared to other extenders (p < 0.05) except diluent A. Diluent C exhibited higher (p < 0.05) VCL, VAP, ALH, WOB, and MAD compared to diluents B, D, E, and F. In experiment II, TM and all biokinetic characteristics did not show significant differences (p > 0.05) amongst the three freezing methods. Liquid nitrogen fumigation resulted in higher (p < 0.05) PM, membrane integrity, acrosome integrity, and lower ROS level compared to the program. In conclusion, the thawed semen diluted with diluent C had higher quality compared to other diluents. The liquid nitrogen fumigation demonstrated superior semen cryopreservation effects compared to the program-controlled cooling method using diluent C.

Effect of fumigation height and time on cryopreservation of ram semen.

The cooling rate is a crucial factor in the process of freezing semen, influencing the overall freezing effectiveness. The height and time of fumigation can significantly impact the rate of cooling. Appropriate cooling rates can help minimize the formation of ice crystals in spermatozoa and reduce potential damage to them. Therefore, the aim of this study was to evaluate the effect of different fumigation heights and time for the cryopreservation of Hu ram semen. Experiments I-IV assessed the effect of semen cryopreservation by testing the post-thawed spermatozoa total motility (TM), progressive motility (PM) and kinetic parameters fumigated at distances of 2, 4, 6 and 8 cm for durations of 5, 10, 15 and 20 min, respectively. Based on the results of experiments I to IV, experiment V evaluated the effect of semen cryopreservation by testing the post-thawed spermatozoa TM, PM, kinetic parameters, plasma membrane integrity, acrosome integrity and reactive oxygen species (ROS) level fumigated at distances of 2, 4, 6 and 8 cm for duration of 20 min. The results indicated that fumigation at 2 cm for 20 min significantly (P < 0.05) improved spermatozoa TM, PM, mean angular displacement (MAD), plasma membrane integrity and acrosome integrity compared to other groups. Additionally, it significantly (P < 0.05) reduced spermatozoa ROS level compared to the 6 and 8 cm groups. In conclusion, fumigation for 20 min at a distance of 2 cm from the liquid nitrogen surface is the most suitable cooling method for the cryopreservation of Hu ram semen.

Effect of Different Dilution Methods and Ratios of Ram Semen on Sperm Parameters after Cryopreservation.

The dilution method and ratio were tested to assess their effects on the Hu ram semen after cryopreservation. Experiment I aimed to explore the effect of various dilution ratios (1:1, 1:2, 1:3, 1:4) of diluent I (Tris-based and egg yolk) under the condition of 1:1 dilution of diluent II (diluent I and glycerol) on the Hu ram semen preserved in liquid nitrogen regarding spermatozoa motility and kinetic parameters. Experiment II aimed to investigate the effect of various dilution ratios (1:1, 1:2, 1:3, 1:4) of diluent I under the condition of 1:2 dilution of diluent II to the Hu ram semen for cryopreservation on spermatozoa motility and kinetic parameters. The purpose of experiment III is to assess the effect of various dilution methods and ratios on the cryopreservation of Hu ram semen by detecting spermatozoa motility, kinetic parameters, plasma membrane integrity, acrosome integrity and reactive oxygen species (ROS) level. Experiment III includes four groups: one-step dilution method and two-step dilution method. The two-step dilution method includes two groups: 1:2, 1:1 and 1:3, 1:2, and the one-step dilution method includes two groups: 1:5 and 1:11. The results indicated that the post-thawed spermatozoa total motility (TM), progressive motility (PM) and average motion degree (MAD) were highest in the 1:2 group and significantly higher (p < 0.05) than those in the 1:1 and 1:4 groups under the condition of 1:1 dilution of diluent II. The post-thawed spermatozoa TM and PM of the 1:3 group were significantly higher (p < 0.05) than those of the other groups under the condition of 1:2 dilution of diluent II. The post-thawed spermatozoa TM, PM, plasma membrane integrity and acrosome integrity of the two-step group (1:3, 1:2) were the highest and significantly higher (p < 0.05) than those in the other groups. Additionally, the post-thawed spermatozoa ROS level of the two-step group (1:3, 1:2) was significantly lower (p < 0.05) than that in the one-step groups (1:5 and 1:11). Therefore, a two-step dilution (1:3, 1:2) was found to be the most suitable method and ratio for diluting the Hu ram semen after cryopreservation.

Recurrent Neural Circuits Overcome Partial Inactivation by Compensation and Re-learning.

Technical advances in artificial manipulation of neural activity have precipitated a surge in studying the causal contribution of brain circuits to cognition and behavior. However, complexities of neural circuits challenge interpretation of experimental results, necessitating new theoretical frameworks for reasoning about causal effects. Here, we take a step in this direction, through the lens of recurrent neural networks trained to perform perceptual decisions. We show that understanding the dynamical system structure that underlies network solutions provides a precise account for the magnitude of behavioral effects due to perturbations. Our framework explains past empirical observations by clarifying the most sensitive features of behavior, and how complex circuits compensate and adapt to perturbations. In the process, we also identify strategies that can improve the interpretability of inactivation experiments.

Impact of X-Ray Exposure From Computed Tomography on Wearable Insulin Delivery Devices.

BACKGROUND: To investigate the impact of radiation exposure from a computed tomography (CT) scanner on the functional integrity of a wearable insulin delivery system. METHODS: A total of 160 Omnipods and four personal diabetes managers (PDMs) were evenly divided into four groups: (1) control group (no radiation exposure), (2) typical radiation exposure group, (3) 4× typical radiation exposure group, and (4) scatter radiation group. Pods were attached to an anthropomorphic torso phantom on the abdomen (direct irradiation) or shoulder (scatter radiation) region. A third-generation dual-source CT scanner was used to scan the pods using either a typical exposure (used for routine CT abdominal study of a median size patient) or 4× typical exposure. A manufacturer-recommended 20-step functionality test was performed for all 160 Omnipods. RESULTS: The radiation dose (measured in volume CT Dose index) was 16 mGy for a typical exposure, and 64 mGy for 4× typical exposure. The scatter radiation is less than 0.1 mGy. All Pods passed the functionality test except one pod in the scatter radiation group, which sounded an alarm due to occlusion. The blockage to the fluid was due to a kink in the soft cannula, a mechanical issue not caused by the radiation exposure. CONCLUSIONS: This study suggests X-ray exposure levels used in radiological imaging procedures do not negatively impact the functional integrity of Omnipods. This finding may support the potential for the manufacturer to remove the warning that patients should remove the Pod for X-ray imaging procedures, which will have a huge impact on patient care.

Integration of transcriptomics and metabolomics provides metabolic and functional insights into reduced insulin secretion in MIN6 ß-cells exposed to deficient and excessive arginine.

Previous work demonstrated that arginine is one of the strongest insulin secretagogues. However, knowledge of the mechanisms linking chronic arginine metabolism with ß-cell function and insulin secretion is relatively limited. After preliminary selection of concentration according to the cell proliferation, the MIN6 pancreatic ß-cells were randomly assigned to culture in 0.04 mM (low-arginine, LA), 0.4 mM (standard-arginine, SA), or 8 mM arginine (high-arginine, HA) for 24 h. Following the treatment, a combination of transcriptomics and metabolomics, together with a series of molecular biological tests were performed to investigate the responses of ß-cells to varied arginine availability. Our results showed that HA treatment reduced the chronic insulin releases, and LA and HA treatments decreased the glucose-stimulated insulin secretions (GSIS) of ß-cells relative to the SA group (p < .05). Transcriptomics analysis indicated that LA administration significantly inhibited oxidative phosphorylation and ATP metabolic process but promoted DNA repair and mRNA processing in ß-cells, while HA administration affected ammonium ion metabolic process and mRNA export (p < .05). Both LA and HA regulated the expressions of genes involved in DNA replication, cell-cycle phase transition, and response to oxidative stress (p < .05). Protein-protein interaction and transcription factor analyses suggested that Trp53 and Nr4a2 genes may play key roles during arginine stimulation. On the contrary, metabolomics analysis demonstrated that the differentially expressed metabolites (DEM) of MIN6 ß-cells induced by LA were mainly enriched in glycerophospholipid metabolism, linoleic acid metabolism, and purine metabolism, while most DEMs between LA vs. SA comparison belonged to amino acid metabolism. When combined the three groups, co-expression analysis suggested that insulin secretions had strong associations with L-pyroglutamic acid, L-glutamate, and creatine concentrations, while intracellular insulin contents were mainly correlated to L-arginine, argininosuccinic acid, and phosphorylcholine. At last, integrated analysis of transcriptomics and metabolomics showed that glycerophospholipid metabolism, biosynthesis of unsaturated fatty acids, and amino acid metabolism were the most relevant pathways in ß-cells exposed to abnormal arginine supply. This descriptive bioinformatics analysis suggested that the disturbed carbohydrate, lipid, and amino acid metabolisms, as well as the increased apoptosis and elevated oxidative stress, contributed to the reduced insulin secretion and lower GSIS in ß-cells induced by LA or HA treatments, while some underlying mechanisms need to be further explored.

Long-term consumption of alcohol exacerbates neural lesions by destroying the functional integrity of the blood-brain barrier.

Recently, increasing numbers of studies have shown that the consumption of large amounts of alcohol is a major risk factor for dementias, which has led to widespread concern about the harmful effects of alcohol consumption on health. However, the pathological changes in the brain caused by this habit are not clear. This study aimed to investigate the possible causes by determining the permeability of the blood-brain barrier (BBB), pathomorphological changes, the mRNA, and protein expressions of adhesion proteins and the concentrations of ß-amyloid (Aß) and some related functional proteins in the brains of C57BL/6 and APPswe/PS1dE9 mice before and after intragastric administration of alcohol for 2 months. The results showed that long-term consumption of alcohol aggravated cognitive decline, increased the permeability of the BBB, led to pathomorphological changes and downregulated some related structural proteins (zonula occludens-1, VE-cadherin, and occludin) and functional proteins (major facilitator superfamily domain-containing protein-2a (Mfsd2a), low-density lipoprotein receptor-related protein-1 (LRP1), receptor for advanced glycation end products (RAGE), and aquaporin-4 (AQP4)) in the BBB but did not increase the concentration of Aß1-42. These novel findings suggested that long-term consumption of alcohol induces neural lesions, which is related to the destruction of the integrity of the BBB.

Organic-matter decomposition as a bioassessment tool of stream functioning: A comparison of eight decomposition-based indicators exposed to different environmental changes.

Organic-matter decomposition has long been proposed as a tool to assess stream functional integrity, but this indicator largely depends on organic-matter selection. We assessed eight decomposition-based indicators along two well-known environmental gradients, a nutrient-enrichment gradient (0.2-1.4 mg DIN/L) in central Portugal and an acidification gradient (pH: 4.69-7.33) in north-eastern France to identify the most effective organic-matter indicator for assessing stream functional integrity. Functional indicators included natural leaf litter (alder and oak) in 10-mm and 0.5-mm mesh bags, commercial tea (Lipton green and rooibos teas in 0.25-mm mesh bags), wood sticks (wood tongue depressors) and cotton strips. Biotic indices based on benthic macroinvertebrates (IPtIN for Portugal and IBGN for France) were calculated to compare the effectiveness of structural and functional indicators in detecting stream impairment and to assess the relationship between both types of indicators. The effectiveness of organic-matter decomposition rates as a functional indicator depended on the stressor considered and the substrate used. Decomposition rates generally identified nutrient enrichment and acidification in the most acidic streams. Decomposition rates of alder and oak leaves in coarse-mesh bags, green and rooibos teas and wood sticks were positively related with pH. Only decomposition rates of rooibos tea and wood sticks were related with DIN concentration; decomposition rates along the nutrient-enrichment gradient were confounded by differences in shredder abundance and temperature among streams. Stream structural integrity was good to excellent across streams; the IPtIN index was unrelated to DIN concentration, while the IBGN index was positively related with pH. The relationships between decomposition rates and biotic indices were loose in most cases, and only decomposition rates of alder leaves in coarse-mesh bags and green tea were positively related with the IBGN. Commercial substrates may be a good alternative to leaf litter to assess stream functional integrity, especially in the case of nutrient enrichment.

Agriculture impairs stream ecosystem functioning in a tropical catchment.

The expansion of agriculture is particularly worrying in tropical regions of the world, where native forests are being replaced by crops at alarming rates, with severe consequences for biodiversity and ecosystems. However, there is little information about the potential effects of agriculture on the functioning of tropical streams, which is essential if we are to assess the condition and ecological integrity of these ecosystems. We conducted a litter decomposition experiment in streams within a tropical catchment, which were subjected to different degrees of agricultural influence: low (protected area, PA), medium (buffer area, BA) and high (agricultural area, AA). We quantified decomposition rates of litter enclosed within coarse-mesh and fine-mesh bags, which allowed the distinction of microbial and detritivore-mediated decomposition pathways. We used litter of three riparian species representing a gradient in litter quality (Alnus acuminata > Ficus insipida > Quercus bumelioides), and examined detritivore assemblages through the contents of litterbags and benthic samples. We found that the increasing agricultural influence promoted microbial decomposition, probably due to nutrient-mediated stimulation; and inhibited detritivore-mediated and total decomposition because of reduced detritivore numbers, most likely caused by pesticides and sedimentation. Effects were evident for Alnus and Ficus, but not for Quercus, which was barely decomposed across the gradient. Our study provides key evidence about the impact of agriculture on tropical stream ecosystem functioning, which is associated to changes in stream assemblages and may have far-reaching repercussions for global biochemical cycles.

Melatonin and Mesenchymal Stem Cells as a Key for Functional Integrity for Liver Cancer Treatment.

Hepatocellular carcinoma (HCC) is the most common hepatobiliary malignancy with limited therapeutic options. On the other hand, melatonin is an indoleamine that modulates a variety of potential therapeutic effects. In addition to its important role in the regulation of sleep-wake rhythms, several previous studies linked the biologic effects of melatonin to various substantial endocrine, neural, immune and antioxidant functions, among others. Furthermore, the effects of melatonin could be influenced through receptor dependent and receptor independent manner. Among the other numerous physiological and therapeutic effects of melatonin, controlling the survival and differentiation of mesenchymal stem cells (MSCs) has been recently discussed. Given its controversial interaction, several previous reports revealed the therapeutic potential of MSCs in controlling the hepatocellular carcinoma (HCC). Taken together, the intention of the present review is to highlight the effects of melatonin and mesenchymal stem cells as a key for functional integrity for liver cancer treatment. We hope to provide solid piece of information that may be helpful in designing novel drug targets to control HCC.

La descomposición de la hojarasca como herramienta para evaluar la integridad funcional de ríos altoandinos del sur del Ecuador / Leaf-litter breakdown as a tool to assess the functional integrity of high Andean streams of Southern Ecuador

ResumenLas condiciones ecológicas de los ríos altoandinos tropicales estan amenazadas por numerosas actividades humanas que incluyen aquellas derivadas del cambio de la cobertura y uso del suelo de la cuenca. Para su evaluación se han propuesto protocolos que diagnostican el estado de la estructura pero no del funcionamiento de estos ecosistemas fluviales. En este trabajo se propone una herramienta de evaluación adaptada a las condiciones locales, incluyendo la metodología y los valores umbrales, utilizando el proceso de descomposición de la hojarasca como una medida del funcionamiento y salud de los ríos altoandinos del sur del Ecuador. Se seleccionaron 15 tributarios distribuidos en tres microcuencas (Mazán, Llaviuco y Matadero) dentro o en las adyacencias del Parque Nacional Cajas. En cada tributario se incubaron bolsas de descomposición elaboradas con dos tipos de malla (gruesa y fina) para separar la contribución de macroinvertebrados y microorganismos en el proceso. Como substrato vegetal se emplearon hojas de Alnus acuminata y Miconia bracteolata. Las bolsas fueron colocadas en los ríos y mantenidas durante 15, 28 y 64 días. Las tasas de descomposición (-K) fueron obtenidas a partir del período de tiempo en el cual se produjo la pérdida de aproximadamente el 50 % de la masa seca libre de ceniza. En cada período de recolección se determinaron parámetros físicos y químicos. Adicionalmente, se aplicaron los índices de calidad de ribera (QBR-And) y de habitat fluvial (IHF). Para la elaboración de los umbrales de condición funcional se utilizaron las métricas Ktotal y el cociente entre la tasa de descomposición en malla gruesa y malla fina (Kg/Kf). Para probar la sensibilidad de las métricas se emplearon métodos gráficos, Anova de una vía y se calculó la eficiencia discriminativa (ED) de las mismas. Los resultados del análisis de usos y coberturas del suelo revelaron un gradiente de intervención Matadero ≥ Llaviuco > Mazán. La composición de las variables ambientales de las corrientes de la microcuenca Mazán resultaron diferentes con respecto a las de Llaviuco y Matadero, cuyos valores parecen ser más semejantes entre sí. La evaluación de la calidad de ribera mediante el índice QBR-And muestra un gradiente importante desde valores muy bajos en las corrientes de Matadero (27.5), valores medios a altos en Llaviuco (66.5) y valores más altos en los tributarios de Mazán (87). Las tasas de descomposición de la hojarasca de los tributarios de la microcuenca del Río Mazán resultaron significativamente más rápidas que en los tributarios de los Ríos Matadero y Llaviuco para las hojas de A. acuminata pero no para las hojas de Miconia. La métrica Kg/Kf se consideró sensible y suficiente para discriminar los impactos en las áreas estudiadas. Los valores del cociente Kg/Kf resultaron significativamente mayores en Mazán en comparación a Llaviuco y Matadero, que no resultaron diferentes entre sí. Nuestros resultados apoyan la utilización del proceso de descomposición de la hojarasca como una herramienta adecuada para evaluar la condición ecológica funcional, complementando las evaluaciones estructurales, de los ríos altoandinos.

Abstract:The ecological condition of tropical Andean rivers are threatened by many human activities including changes in land use and cover in watersheds. Current protocols diagnose the structural condition of streams but not their function. In this study we proposed an assessment tool using the leaf-litter breakdown as a measure of the functional condition and health of high Andean streams in Southern Ecuador, including methodology and threshold values adapted to local conditions. We selected 15 streams in three micro-basins (Mazán, Llaviuco and Matadero) within or in the vicinity of Cajas National Park where we deployed litterbags for 15, 28 and 64 days. We used two types of mesh (coarse and fine) for the litterbags in order to separate the contribution of macro-invertebrates and microorganisms in the decomposition process and also tested two different leaf substrates: Alder (Alnus acuminata) and Miconia (Miconia bracteolata). In each collection period we determined physical and chemical parameters, applied the riparian quality index (QBR-And) and river habitat index (IHF). Decomposition rates (-k) were determined from the time period at which approximately 50 % ash-free mass would have been lost. We used Ktotal and the ratio of the rate of decomposition between coarse and fine mesh (Kg/Kf) to define the functional condition thresholds. To test the metrics sensitivity we used graphical methods, one-way ANOVA and discriminative efficiency (ED). The analysis of watershed land-use and cover showed a gradient of intervention: Matadero ≥ Llaviuco > Mazán. The composition of the environmental variables of Mazán streams were different with respect to Llaviuco and Matadero, whose values were similar to each other. The QBR-And index showed a significant gradient from very low levels in Matadero (27.5), moderately high values in Llaviuco (66.5) to high values in Mazán (87). Alder leaf-litter breakdown rates were significantly faster in Mazán than in Matadero and Llaviuco streams, while breakdown rates of Miconia were not significantly different among watersheds. The Kg/Kf ratio was significantly higher in Mazán compared to Llaviuco and Matadero, which did not differ. We consider the Kg/Kf metric sensitive enough to discriminate impacts in the studied areas. Our results support the use of the leaf-litter breakdown as an appropriate tool to assess functional ecological condition, complementing the structural assessments of these Andean rivers. Rev. Biol. Trop. 65 (1): 321-334. Epub 2017 March 01.