Criopreservación de espermatozoides de llama obtenidos del conducto deferente utilizando tres curvas de congelamiento

The aim of this study was to determine the effect of three freezing curves on the post-thaw viability of spermatozoa collected from the vas deferens of llamas. Six male llamas with surgical diversion of the vas deferens were used. The samples of the six males were mixed for processing (pools) and diluted with Tris-egg yolk. Cooling was carried out to 5 °C where the dilution was completed (dilutor with glycerol) and was maintained for 30 minutes (equilibrium phase). Samples in 0.25 ml straws were subjected to freezing using a temperature drop rate of -20°C/min until reaching -80°C (TI), -100°C (TII) and -120°C (TIII) in 4, 5 and 6 minutes, respectively, to finally store them in liquid nitrogen. The samples were collected for three months (n=19 pools). Total motility (progressive, circular, oscillatory motility), viability and membrane functionality were determined after collection, in the equilibrium phase and upon thawing. A significant decrease (p<0.05) was observed in all the sperm characteristics evaluated in the samples after the equilibrium compared to the samples after collection. A high positive correlation was obtained between viability and total motility (r2=0.78) and in the equilibrium phase between membrane functionality and total motility (r2=0.881). In thawed samples, total motility and viability were significantly higher in frozen samples. with the freezing curve TIII with respect to TI (p=0.041 and p=0.003, respectively). No significant differences in membrane functionality were observed between the three freezing curves (p>0.27). In conclusion, the temperature drop curve down to -120 °C using a rate of -20 °C/min would be the most suitable for cryopreserving llama spermatozoa obtained from the deviation of the vas deferens.El objetivo del estudio fue determinar el efecto de tres curvas de congelación sobre la viabilidad pos-descongelación de espermatozoides colectados del conducto deferente de llamas. Se utilizaron seis llamas machos con desviación quirúrgica de los conductos deferentes. Las muestras de los seis machos se mezclaron para su procesamiento (pools) y se diluyeron con Tris-yema de huevo. Se procedió al enfriamiento hasta los 5 °C donde se completó la dilución (dilutor con glicerol) y se mantuvo por media hora (fase de equilibrio). Las muestras en pajillas de 0.25 ml fueron sometidas a congelamiento utilizando una tasa de descenso de temperatura de -20°C/min hasta llegar a -80°C (TI), a -100 °C (TII) y a -120 °C (TIII) en 4, 5 y 6 minutos, respectivamente, para finalmente almacenarlas en nitrógeno líquido. Las muestras fueron colectadas durante tres meses (n=19 pooles). Se determinó la motilidad total (motilidad progresiva, circular, oscilatoria), viabilidad y funcionalidad de membrana luego de la colecta, en la fase de equilibrio y al descongelamiento. Se observó una disminución significativa (p˂0.05) en todas las características espermáticas evaluadas en las muestras equilibradas respecto a las muestras luego de la colecta. Se obtuvo una correlación alta positiva entre viabilidad y motilidad total (r2=0.78) y en fase de equilibrio entre funcionalidad de membrana y motilidad total (r2=0.881) En las muestras descongeladas, la motilidad total y viabilidad fueron significativamente mayores en las muestras congeladas con la curva de congelamiento TIII respecto al TI (p=0.041 y p=0.003, respectivamente). No se observaron diferencias significativas en la funcionalidad de membrana entre las tres curvas de congelamiento (p˃0.27). En conclusión, la curva de descenso de la temperatura hasta los -120 °C utilizando una tasa de -20 °C/min sería la más adecuada para criopreservar espermatozoides de llama obtenidos a partir de la desviación de los conductos deferentes

DETERMINACIÓN DE RESISTENCIA ANTIHELMÍNTICA (Fasciola hepatica) EN OVINOS FRENTE A ALBENDAZOL Y TRICLABENDAZOL, LA PAZ – BOLIVIA

Fasciola hepatica resistance in sheep against albendazole and triclabendazole was evaluated in two farms in La Paz, Bolivia: one located near Tambillo (farm 1) and the other near Batallas (farm 2). Thirty male lambs of 7 to 8 months of age were selected in each farm. They were treated with 10 mg/kg of albendazole or 10 mg/kg of triclabendazole, and one group remained as non-treated. The gama glutamyl transpeptidase enzyme (GGT) profile and faecal eggs count reduction test (FECRT) were conducted. Lambs were slaughtered 8 weeks after treatment and the number of flukes in liver was counted. Minor reduction of GGT levels and faecal eggs count were observed in animals treated with albendazole. In the group treated with triclabendazole in farm 1 was observed a reduction of the GGT levels and faecal eggs count throughout the study, but not in farm 2. The reduction of flukes in liver lambs was 13.6 and 2.3% for albendazol in farms 1 and 2 respectively, and 98.0 and 36.3% for triclabendazol. It was concluded that anthelminthic resistance of F. hepatica to albendazol occurred in both farms, while in farm 2 resistance was gradually appearing to triclabendazole.Se determinó la resistencia de Fasciola hepatica frente a albendazol y triclabendazol en ovinos de dos rebaños en La Paz, Bolivia: uno cercano a Tambillo (rebaño 1) y otro cercano a Batallas (rebaño 2). Se eligió al azar 30 corderos de 8 a 9 meses de edad por rebaño, y se trataron con 10 mg/kg de albendazol ó 10 mg/kg de triclabendazol, quedando un grupo como control no tratado. Se midió la enzima gama glutamil transpeptidasa (GGT) y el número de huevos por gramo de heces (hpg) para la prueba de reducción de la oviposición. Los corderos se sacrificaron 8 semanas después del tratamiento y se contó el número de fasciolas. Se presentó una baja reducción de los niveles de GGT y de hpg en los grupos tratados con albendazol. En el grupo tratado con triclabendazol en el rebaño 1 se observó una de reducción de los niveles de GGT y hpg durante todo el estudio, no así en el rebaño 2. El porcentaje de reducción en los hígados fue de 13.6 y 2.3% para albendazol en los rebaños 1 y 2, respectivamente, y de 98.0 y 36.3% para triclabendazol. Se concluye que existe resistencia de F. hepatica frente a albendazol en los dos rebaños y que la resistencia está en proceso de establecimiento para triclabendazol en el rebaño 2

Estandarización y validación de una prueba de PCR para el diagnóstico precoz de leptospirosis humana

Objectives: To standardize and optimize a PCR assay to detect rrs gene (rRNA 16S) from Leptospira spp. to use it with samples of suspected patients of febrile syndrome, in endemic areas from Peru. Methods: We standardized and validated a PCR assay for rapid diagnostic of leptospirosis from blood and urine samples. The PCR assay was optimized with DNA samples from different species of Leptospira . Sensitivity and specificity was determined in comparison with serological test: MAT and IgM ELISA in 180 clinical samples of patients with suspect of leptopirosis. Results: The PCR assay amplified DNA of twenty five serovars of six pathogenic species of Leptospira spp. No amplification was detected with DNA from other pathogens. Sensitivity and specificity was 100% in vitro (culture samples). In blood samples, sensitivity was 100% (95CI: 97,9 - 100) in patients on the first 8 days of disease; and 30% (95CI: 16,3 - 43,7) when the time of disease was greater. In urine samples the PCR assay showed a low sensitivity. Conclusions: The standardized PCR assay for Leptospira was more sensitive than serological tests in the first days of disease. On the other hand, it has low sensitivity when bacterial population is poor.Objetivos: Estandarizar y optimizar la prueba de PCR dirigida al gen rrs (16S) de Leptospira spp., para luego validar su uso en muestras de pacientes con síndrome febril captados en zonas endémicas del Perú. Material y métodos: Se estandarizó y validó una prueba de PCR para el diagnostico rápido de leptospirosis en muestras de sangre y orina. Se optimizó la prueba con muestras de ADN de Leptospira de diferentes especies, y se determinó en 180 muestras clínicas de pacientes con sospecha de leptospirosis su sensibilidad y especificidad en comparación con la prueba de aglutinación microscópica (MAT) y ELISA IgM. Resultados: El PCR estandarizado amplificó el ADN de 25 serovares de seis especies de Leptospira spp. No amplificó las muestras de ADN de otros microorganismos patógenos. s. La sensibilidad y especificidaddel método fue 100%in vitro. Su sensibilidad fue de 100% (IC95: 97,9 - 100%) en muestras de sangre antes de los ochoprimeros días de enfermedad y de 30% (IC95: 16,3 - 43-,7) cuando el tiempo de enfermedad fue mayor. El PCR en orinatiene una baja sensibilidad.Conclusiones:El PCR estandarizado es más sensible que las pruebas serológicas en losprimeros días de enfermedad y es poco sensible cuando la carga bacteriana es baja en sangre

Performance of the SUBSTOR-potato model across contrasting growing conditions.

Crop models are essential tools in climate change impact assessments, but they often lack comprehensive field testing. In this study, we tested the SUBSTOR-potato model with 87 field experiments, including 204 treatments from 19 countries. The field experiments varied in potato species and cultivars, N fertilizer application, water supply, sowing dates, soil types, temperature environments, and atmospheric CO2 concentrations, and included open top chamber and Free-Air-CO2-Enrichment (FACE) experiments. Tuber yields were generally well simulated with the SUBSTOR-potato model across a wide range of current growing conditions and for diverse potato species and cultivars, including Solanum tuberosum, Solanum andigenum, Solanum juzepczukii species, as well as modern, traditional, early, medium, and late maturity-type cultivars, with a relative RMSE of 37.2% for tuber dry weight and 21.4% for tuber fresh weight. Cultivars ‘Desiree’ and ‘Atlantic’ were grown in experiments across the globe and well simulated using consistent cultivar parameters. However, the model underestimated the impact of elevated atmospheric CO2 concentrations and poorly simulated high temperature effects on crop growth. Other simulated crop variables, including leaf area, stem weight, crop N, and soil water, differed frequently from measurements; some of these variables had significant large measurement errors. The SUBSTOR-potato model was shown to be suitable to simulate tuber growth and yields over a wide range of current growing conditions and crop management practices across many geographic regions. However, before the model can be used effectively in climate change impact assessments, it requires improved model routines to capture the impacts of elevated atmospheric CO2 and high temperatures on crop growth

Selection of reference genes for qPCR in hairy root cultures of peanut

<p>Abstract</p> <p>Background</p> <p>Hairy root cultures produced via <it>Agrobacterium rhizogenes</it>-mediated transformation have emerged as practical biological models to elucidate the biosynthesis of specialized metabolites. To effectively understand the expression patterns of the genes involved in the metabolic pathways of these compounds, reference genes need to be systematically validated under specific experimental conditions as established by the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines. In the present report we describe the first validation of reference genes for RT-qPCR in hairy root cultures of peanut which produce stilbenoids upon elicitor treatments.</p> <p>Results</p> <p>A total of 21 candidate reference genes were evaluated. Nineteen genes were selected based on previous qPCR studies in plants and two were from the T-DNAs transferred from <it>A. rhizogenes</it>. Nucleotide sequences of peanut candidate genes were obtained using their homologous sequences in <it>Arabidopsis</it>. To identify the suitable primers, calibration curves were obtained for each candidate reference gene. After data analysis, 12 candidate genes meeting standard efficiency criteria were selected. The expression stability of these genes was analyzed using geNorm and NormFinder algorithms and a ranking was established based on expression stability of the genes. Candidate reference gene expression was shown to have less variation in methyl jasmonate (MeJA) treated root cultures than those treated with sodium acetate (NaOAc).</p> <p>Conclusions</p> <p>This work constitutes the first effort to validate reference genes for RT-qPCR in hairy roots. While these genes were selected under conditions of NaOAc and MeJA treatment, we anticipate these genes to provide good targets for reference genes for hairy roots under a variety of stress conditions. The lead reference genes were a gene encoding for a TATA box binding protein (<it>TBP2</it>) and a gene encoding a ribosomal protein (<it>RPL8C</it>). A commonly used reference gene <it>GAPDH </it>showed low stability of expression suggesting that its use may lead to inaccurate gene expression profiles when used for data normalization in stress-stimulated hairy roots. Likewise the <it>A. rhizogenes </it>transgene <it>rolC </it>showed less expression stability than <it>GAPDH</it>. This study proposes that a minimum of two reference genes should be used for a normalization procedure in gene expression profiling using elicited hairy roots.</p