Ammonia and methane gas emissions from pig slurry: variability induced through feeding strategies

Esta Tesis doctoral fue desarrollada para estudiar las emisiones de amoniaco (NH3) y metano (CH4) en purines de cerdos, y los efectos ocasionados por cambios en la formulación de la dieta. Con este propósito, fueron llevados a cabo tres estudios. El experimento 1 fue realizado con el objetivo de analizar los factores de variación de la composición de purines y establecer ecuaciones de predicción para emisiones potenciales de NH3 y CH4. Fueron recogidas setenta y nueve muestras de piensos y purines durante dos estaciones del año (verano y invierno) de granjas comerciales situadas en dos regiones de España (Centro y Mediterráneo). Se muestrearon granjas de gestación, maternidad, lactación y cebo. Se determinó la composición de piensos y purines, y la emisión potencial de NH3 y CH4. El contenido de nutrientes de los piensos fue usado como covariable en el análisis. La espectroscopia de reflectancia del infrarrojo cercano (NIRS) se evaluó como herramienta de predicción de la composición y potencial emisión de gases del purín. Se encontró una amplia variabilidad en la composición de piensos y purines. Las granjas del Mediterráneo tenían mayor pH (P<0,001) y concentración de cenizas (P =0,02) en el purín que las del Centro. El tipo de granja también afectó al contenido de extracto etéreo (EE) del purín (P =0,02), observando los valores más elevados en las instalaciones de animales jóvenes. Los resultados sugieren un efecto tampón de la fibra de la dieta en el pH del purín y una relación directa (P<0,05) con el contenido de fibra fecal. El contenido de proteína del pienso no afectó al contenido de nitrógeno del purín, pero disminuyó (P=0,003) la concentración de sólidos totales (ST) y de sólidos volátiles (SV). Se obtuvieron modelos de predicción de la emisión potencial de NH3 (R2=0,89) y CH4 (R2=0,61) partir de la composición del purín. Los espectros NIRS mostraron una buena precisión para la estimación de la mayor parte de los constituyentes, con coeficientes de determinación de validación cruzada (R2cv) superiores a 0,90, así como para la predicción del potencial de emisiones de NH3 y CH4 (R2cv=0,84 y 0,68, respectivamente). El experimento 2 fue realizado para investigar los efectos del nivel de inclusión de dos fuentes de sub-productos fibrosos: pulpa de naranja (PN) y pulpa de algarroba (PA), en dietas iso-fibrosas de cerdos de cebo, sobre la composición del purín y las emisiones potenciales de NH3 y CH4. Treinta cerdos (85,4±12,3 kg) fueron alimentados con cinco dietas iso-nutritivas: control comercial trigo/cebada (C) y cuatro dietas experimentales incluyendo las dos fuentes de sub-productos a dos niveles (75 y 150 g/kg) en una estructura 2 × 2 factorial. Después de 14 días de periodo de adaptación, heces y orina fueron recogidas separadamente durante 7 días para medir la digestibilidad de los nutrientes y el nitrógeno (N) excretado (6 réplicas por dieta) en cerdos alojados individualmente en jaulas metabólicas. Las emisiones de NH3 y CH4 fueron medidas después de la recogida de los purínes durante 11 y 100 días respectivamente. La fuente y el nivel de subproductos fibrosos afectó a la eficiencia digestiva de diferentes formas, ya que los coeficientes de digestibilidad total aparente (CDTA) para la materia seca (MS), materia orgánica (MO), fracciones fibrosas y energía bruta (EB) aumentaron con la PN pero disminuyeron con la inclusión de PA (P<0,05). El CDTA de proteína bruta (PB) disminuyó con la inclusión de las dos fuentes de fibra, siendo más bajo al mayor nivel de inclusión. La concentración fecal de fracciones fibrosas aumentó (P<0,05) con el nivel de inclusión de PA pero disminuyó con el de PN (P<0,01). El nivel más alto de las dos fuentes de fibra en el pienso aumentó (P<0,02) el contenido de PB fecal pero disminuyó el contenido de N de la orina (de 205 para 168 g/kg MS, P<0,05) en todas las dietas suplementadas comparadas con la dieta C. Adicionalmente, las proporciones de nitrógeno indigerido, nitrógeno soluble en agua, nitrógeno bacteriano y endógeno excretado en heces no fueron afectados por los tratamientos. Las características iniciales del purín no difirieron entre las diferentes fuentes y niveles de fibra, excepto para el pH que disminuyó con la inclusión de altos niveles de sub-productos. La emisión de NH3 por kg de purín fue más baja en todas las dietas suplementadas con fibras que en la dieta C (2,44 vs.1,81g de promedio, P<0,05). Además, purines de dietas suplementadas con alto nivel de sub-productos tendieron (P<0,06) a emitir menos NH3 por kg de nitrógeno total y mostraron un potencial más bajo para emitir CH4, independientemente de la fuente de fibra. El experimento 3 investigó los efectos de la fuente de proteína en dietas prácticas. Tres piensos experimentales fueron diseñados para sustituir una mescla de harina y cascarilla de soja (SOJ) por harina de girasol (GIR) o por DDGS del trigo (DDGST). La proporción de otros ingredientes fue modificada para mantener los contenidos de nutrientes similares a través de las dietas. El cambio en la fuente de proteína dio lugar a diferencias en el contenido de fibra neutro detergente ligada a proteína bruta (FNDPB), fibra soluble (FS) y lignina ácido detergente (LAD) en la dieta. Veinticuatro cerdos (ocho por dieta), con 52,3 o 60,8 kg en la primera y segunda tanda respectivamente, fueron alojados individualmente en jaulas metabólicas. Durante un periodo de 7 días fue determinado el balance de MS, el CDTA de los nutrientes y la composición de heces y orina. Se realizó el mismo procedimiento del experimento 2 para medir las emisiones de NH3 y CH4 de los purines de cada animal. Ni la ingestión de MS ni el CDTA de la MS o de la energía fueron diferentes entre las dietas experimentales, pero el tipo de pienso afectó (P<0.001) la digestibilidad de la PB, que fue mayor para GIR (0,846) que para SOJ (0,775), mientras que la dieta DDGST mostró un valor intermedio (0,794). La concentración fecal de PB fue por tanto influenciada (P<0,001) por el tratamiento, observándose la menor concentración de PB en la dieta GIR y la mayor en la dieta SOJ. La proporción de N excretado en orina o heces disminuyó de 1,63 en la dieta GIR hasta 0,650 en la dieta SOJ, como consecuencia de perdidas más bajas en orina y más altas en heces, con todas las fracciones de nitrógeno fecales creciendo en paralelo a la excreción total. Este resultado fue paralelo a una disminución de la emisión potencial de NH3 (g/kg purín) en la dieta SOJ con respecto a la dieta GIR (desde 1,82 a 1,12, P<0,05), dando valores intermedios (1,58) para los purines de la dieta DDGST. Por otro lado, el CDTA de la FS y de la fibra neutro detergente (FND) fueron afectados (P<0,001 y 0,002, respectivamente) por el tipo de dieta, siendo más bajas en la dieta GIR que en la dieta SOJ; además, se observó un contenido más alto de FND (491 vs. 361g/kg) en la MS fecal para la dieta GIR que en la dieta SOJ, presentando la dieta DDGST valores intermedios. El grado de lignificación de la FND (FAD/FND x 100) de las heces disminuyó en el orden GIR>DDGST>SOJ (desde 0,171 hasta 0,109 y 0,086, respectivamente) en paralelo a la disminución del potencial de emisión de CH4 por g de SV del purín (desde 301 a 269 y 256 mL, respectivamente). Todos los purines obtenidos en estos tres experimentos y Antezana et al. (2015) fueron usados para desarrollar nuevas calibraciones con la tecnología NIRS, para predecir la composición del purín y el potencial de las emisiones de gases. Se observó una buena precisión (R2cv superior a 0,92) de las calibraciones cuando muestras de los ensayos controlados (2, 3 y Antezana et al., 2015) fueron añadidas, aumentando el rango de variación. Una menor exactitud fue observada para TAN y emisiones de NH3 y CH4, lo que podría explicarse por una menor homogeneidad en la distribución de las muestras cuando se amplía el rango de variación del estudio. ABSTRACT This PhD thesis was developed to study the emissions of ammonia (NH3) and methane (CH4) from pig slurry and the effects caused by changes on diet formulation. For these proposes three studies were conducted. Experiment 1 aimed to analyse several factors of variation of slurry composition and to establish prediction equations for potential CH4 and NH3 emissions. Seventy-nine feed and slurry samples were collected at two seasons (summer and winter) from commercial pig farms sited at two Spanish regions (Centre and Mediterranean). Nursery, growing-fattening, gestating and lactating facilities were sampled. Feed and slurry composition were determined, and potential CH4 and NH3 emissions measured. Feed nutrient contents were used as covariates in the analysis. Near infrared reflectance spectroscopy (NIRS) was evaluated as a predicting tool for slurry composition and potential gaseous emissions. A wide variability was found both in feed and slurry composition. Mediterranean farms had a higher pH (P<0.001) and ash (P=0.02) concentration than those located at the centre of Spain. Also, type of farm affected ether extract (EE) content of the slurry (P=0.02), with highest values obtained for the youngest animal facilities. Results suggested a buffer effect of dietary fibre on slurry pH and a direct relationship (P<0.05) with fibre constituents of manure. Dietary protein content did not affect slurry nitrogen content (N) but decreased (P=0.003) in total solid (TS) and volatile solids (VS) concentration. Prediction models of potential NH3 emissions (R2=0.89) and biochemical CH4 potential (B0) (R2=0.61) were obtained from slurry composition. Predictions from NIRS showed a high accuracy for most slurry constituents with coefficient of determination of cross validation (R2cv) above 0.90 and a similar accuracy of prediction of potential NH3 and CH4 emissions (R2cv=0.84 and 0.68, respectively) thus models based on slurry composition from commercial farms. Experiment 2 was conducted to investigate the effects of increasing the level of two sources of fibrous by-products, orange pulp (OP) and carob meal (CM), in iso-fibrous diets for growing-finishing pig, slurry composition and potential NH3 and CH4 emissions. Thirty pigs (85.4±12.3 kg) were fed five iso-nutritive diets: a commercial control wheat/barley (C) and four experimental diets including two sources of fibrous by-products OP and CM and two dietary levels (75 and 150 g/kg) in a 2 × 2 factorial arrangement. After a 14-day adaptation period, faeces and urine were collected separately for 7 days to measure nutrient digestibility and the excretory patterns of N from pigs (6 replicates per diet) housed individually in metabolic pens. For each animal, the derived NH3 and CH4 emissions were measured in samples of slurry over an 11 and 100-day storage periods, respectively. Source and level of the fibrous by-products affected digestion efficiency in a different way as the coefficients of total tract apparent digestibility (CTTAD) for dry matter (DM), organic matter (OM), fibre fractions and gross energy (GE) increased with OP but decreased with CM (P<0.05). Crude protein CTTAD decreased with the inclusion of both sources of fibre, being lower at the highest dietary level. Faecal concentration of fibre fractions increased (P<0.05) with the level of inclusion of CM but decreased with that of OP (P<0.01). High dietary level for both sources of fibre increased (P<0.02) CP faecal content but urine N content decreased (from 205 to 168 g/kg DM, P<0.05) in all the fibre-supplemented compared to C diet. Additionally, the proportions of undigested dietary, water soluble, and bacterial and endogenous debris of faecal N excretion were not affected by treatments. The initial slurry characteristics did not differ among different fibre sources and dietary levels, except pH, which decreased at the highest by-product inclusion levels. Ammonia emission per kg of slurry was lower in all the fibre-supplemented diets than in C diet (2.44 vs. 1.81g as average, P<0.05). Additionally, slurries from the highest dietary level of by-products tended (P<0.06) to emit less NH3 per kg of initial total Kjeldahl nitrogen (TKN) and showed a lower biochemical CH4 potential , independently of the fibre source. Experiment 3 investigated the effects of protein source in practical diets. Three experimental feeds were designed to substitute a mixture of soybean meal and soybean hulls (SB diet) with sunflower meal (SFM) or wheat DDGS (WDDGS). The proportion of other ingredients was also modified in order to maintain similar nutrient contents across diets. Changes in protein source led to differences in dietary content of neutral detergent insoluble crude protein (NDICP), soluble fibre (SF) and acid detergent lignin (ADL). Twenty-four pigs (eight per diet), weighing 52.3 or 60.8 kg at the first and second batch respectively, were housed individually in metabolic pens to determine during a 7-day period DM balance, CTTAD of nutrients, and faecal and urine composition. Representative slurry samples from each animal were used to measure NH3 and CH4 emissions over an 11 and or 100-day storage period, respectively. Neither DM intake, nor DM or energy CTTAD differed among experimental diets, but type of feed affected (P<0.001) CP digestibility, which was highest for SFM (0.846) than for SB (0.775) diet, with WDDGS-based diet giving an intermediate value (0.794). Faecal DM composition was influenced (P<0.001) accordingly, with the lowest CP concentration found for diet SFM and the highest for SB. The ratio of N excreted in urine or faeces decreased from SFM (1.63) to SB diet (0.650), as a consequence of both lower urine and higher faecal losses, with all the faecal N fractions increasing in parallel to total excretion. This result was parallel to a decrease of potential NH3 emission (g/kg slurry) in diet SB with respect to diet SFM (from 1.82 to 1.12, P<0.05), giving slurry from WDDGS-based diet an intermediate value (1.58). Otherwise, SF and insoluble neutral detergent fibre (NDF) CTTAD were affected (P<0.001 and P=0.002, respectively) by type of diet, being lower for SFM than in SB-diet; besides, a higher content of NDF (491 vs. 361 g/kg) in faecal DM was observed for SFM with respect to SB based diet, with WDDGS diet being intermediate. Degree of lignification of NDF (ADL/NDF x 100) of faeces decreased in the order SFM>WDDGS>SB (from 0.171 to 0.109 and 0.086, respectively) in parallel to a decrease of biochemical CH4 potential per g of VS of slurry (from 301 to 269 and 256 ml, respectively). All slurry samples obtained from these three experiments and Antezana et al. (2015) were used to develop new calibrations with NIRS technology, to predict the slurry composition and potential gaseous emissions of samples with greater variability in comparison to experiment 1. Better accuracy (R2cv above 0.92) was observed for calibrations when samples from controlled trials experiments (2, 3 and Antezana et al., 2015) were included, increasing the range of variation. A lower accuracy was observed for TAN, NH3 and CH4 gaseous emissions, which might be explained by the less homogeneous distribution with a wider range of data.

Validation and aplication of Bioelectrical Impedance (BIA) method to determine body and carcass composition and nutrient retention of growing rabbits = Validación y aplicación de la técnica de la Impedancia Bioeléctrica (BIA) para determinar la composición corporal, de la canal y la retención de nutrientes de conejos en crecimiento

Los objetivos de esta tesis fueron 1) obtener y validar ecuaciones de predicción para determinar in vivo la composición corporal y de la canal de conejos en crecimiento de 25 a 77 días de vida utilizando la técnica de la Impedancia Bioeléctrica (BIA), y 2) evaluar su aplicación para determinar diferencias en la composición corporal y de la canal, así como la retención de nutrientes de animales alimentados con diferentes fuentes y niveles de grasa. El primer estudio se realizó para determinar y después validar, usando datos independientes, las ecuaciones de predicción obtenidas para determinar in vivo la composición corporal de los conejos en crecimiento. Se utilizaron 150 conejos a 5 edades distintas (25, 35, 49, 63 y 77 días de vida), con un rango de pesos entre 231 y 3138 g. Para determinar los valores de resistencia (Rs,) and reactancia (Xc,) se usó un terminal (Model BIA-101, RJL Systems, Detroit, MI USA) con cuatro electrodos. Igualmente se registró la distancia entre electrodos internos (D), la longitud corporal (L) y el peso vivo (PV) de cada animal. En cada edad, los animales fueron molidos y congelados (-20 ºC) para su posterior análisis químico (MS, grasa, proteína, cenizas y EB). El contenido en grasa y energía de los animales se incrementó, mientras que los contenidos en proteína, cenizas y agua de los animales disminuyeron con la edad. Los valores medios de Rs, Xc, impedancia (Z), L y D fueron 83.5 ± 23.1 , 18.2 ± 3.8 , 85.6 ± 22.9 , 30.6 ± 6.9 cm y 10.8 ± 3.1 cm. Se realizó un análisis de regresión lineal múltiple para determinar las ecuaciones de predicción, utilizando los valores de PV, L and Z como variables independientes. Las ecuaciones obtenidas para estimar los contenidos en agua (g), PB (g), grasa (g), cenizas (g) and EB (MJ) tuvieron un coeficiente de determinación de (R2) de 0.99, 0.99, 0.97, 0.98 y 0.99, y los errores medios de predicción relativos (EMPR) fueron: 2.79, 6.15, 24.3, 15.2 y 10.6%, respectivamente. Cuando el contenido en agua se expresó como porcentaje, los valores de R2 y EMPR fueron 0.85 and 2.30%, respectivamente. Al predecir los contenidos en proteína (%MS), grasa (%MS), cenizas (%MS) y energía (kJ/100 g MS), se obtuvieron valores de 0.79, 0.83, 0.71 y 0.86 para R2, y 5.04, 18.9, 12.0 y 3.19% para EMPR. La reactancia estuvo negativamente correlacionada con el contenido en agua, cenizas y PB (r = -0.32, P < 0.0001; r = -0.20, P < 0.05; r = -0.26, P < 0.01) y positivamente correlacionada con la grasa y la energía (r = 0.23 y r = 0.24; P < 0.01). Sin embargo, Rs estuvo positivamente correlacionada con el agua, las cenizas y la PB (r = 0.31, P < 0.001; r = 0.28, P < 0.001; r = 0.37, P < 0.0001) y negativamente con la grasa y la energía (r = -0.36 y r = -0.35; P < 0.0001). Igualmente la edad estuvo negativamente correlacionada con el contenido en agua, cenizas y proteína (r = -0.79; r = -0.68 y r = -0.80; P < 0.0001) y positivamente con la grasa y la energía (r = 0.78 y r = 0.81; P < 0.0001). Se puede concluir que el método BIA es una técnica buena y no invasiva para estimar in vivo la composición corporal de conejos en crecimiento de 25 a 77 días de vida. El objetivo del segundo estudio fue determinar y validar con datos independientes las ecuaciones de predicción obtenidas para estimar in vivo la composición de la canal eviscerada mediante el uso de BIA en un grupo de conejos de 25 a 77 días, así como testar su aplicación para predecir la retención de nutrientes y calcular las eficacias de retención de la energía y del nitrógeno. Se utilizaron 75 conejos agrupados en 5 edades (25, 35, 49, 63 y 77 días de vida) con unos pesos que variaron entre 196 y 3260 g. Para determinar los valores de resistencia (Rs, ) y reactancia (Xc, ) se usó un terminal (Model BIA-101, RJL Systems, Detroit, MI USA) con cuatro electrodos. Igualmente se registró la distancia entre electrodos internos (D), la longitud corporal (L) y el peso vivo (PV) del cada animal. En cada edad, los animales fueron aturdidos y desangrados. Su piel, vísceras y contenido digestivo fueron retirados, y la canal oreada fue pesada y molida para posteriores análisis (MS, grasa, PB, cenizas y EB). Los contenidos en energía y grasa aumentaron mientras que los de agua, cenizas y proteína disminuyeron con la edad. Los valores medios de Rs, Xc, impedancia (Z), L y D fueron 95.9±23.9 , 19.5±4.7 , 98.0±23.8 , 20.6±6.3 cm y 13.7±3.1 cm. Se realizó un análisis de regresión linear múltiple para determinar las ecuaciones de predicción, utilizando los valores de PV, L and Z como variables independientes. Los coeficientes de determinación (R2) de las ecuaciones obtenidas para estimar los contenidos en agua (g), PB (g), grasa (g), cenizas (g) and EB (MJ) fueron: 0.99, 0.99, 0.95, 0.96 y 0.98, mientras que los errores medios de predicción relativos (EMPR) fueron: 4.20, 5.48, 21.9, 9.10 y 6.77%, respectivamente. Cuando el contenido en agua se expresó como porcentaje, los valores de R2 y EMPR fueron 0.79 y 1.62%, respectivamente. Cuando se realizó la predicción de los contenidos en proteína (%MS), grasa (%MS), cenizas (%MS) y energía (kJ/100 g MS), los valores de R2 fueron 0.68, 0.76, 0.66 and 0.82, y los de RMPE: 3.22, 10.5, 5.82 and 2.54%, respectivamente. La reactancia estuvo directamente correlacionada con el contenido en grasa (r = 0.24, P < 0.05), mientras que la resistencia guardó una correlación positiva con los contenidos en agua, cenizas y proteína (r = 0.55, P < 0.001; r = 0.54, P < 0.001; r = 0.40, P < 0.005) y negativa con la grasa y la energía (r = -0.44 y r = -0.55; P < 0.001). Igualmente la edad estuvo negativamente correlacionada con los contenidos en agua, cenizas y PB (r = -0.94; r = -0.85 y r = -0.75; P < 0.0001) y positivamente con la grasa y la energía (r = 0.89 y r = 0.90; P < 0.0001). Se estudió la eficacia global de retención de la energía (ERE) y del nitrógeno (ERN) durante todo el periodo de cebo (35-63 d), Los valores de ERE fueron 20.4±7.29%, 21.0±4.18% and 20.8±2.79% en los periodos 35 a 49, 49 a 63 y 35 a 63 d, respectivamente. ERN fue 46.9±11.7%, 34.5±7.32% y 39.1±3.23% para los mismos periodos. La energía fue retenida en los tejidos para crecimiento con una eficiencia del 52.5% y la eficiencia de retención de la energía como proteína y grasa fue de 33.3 y 69.9% respectivamente. La eficiencia de utilización del nitrógeno para crecimiento fue cercana al 77%. Este trabajo muestra como el método BIA es técnica buena y no invasiva para determinar in vivo la composición de la canal y la retención de nutrientes en conejos en crecimiento de 25 a 77 días de vida. En el tercer estudio, se llevaron a cabo dos experimentos con el fin de investigar los efectos del nivel de inclusión y de la fuente de grasa, sobre los rendimientos productivos, la mortalidad, la retención de nutrientes y la composición corporal total y de la canal eviscerada de conejos en crecimiento de 34 a 63 d de vida. En el Exp. 1 se formularon 3 dietas con un diseño experimental factorial 3 x 2 con el tipo de grasa utilizada: Aceite de Soja (SBO), Lecitinas de Soja (SLO) y Manteca (L) y el nivel de inclusión (1.5 y 4%) como factores principales. El Exp. 2 también fue diseñado con una estructura factorial 3 x 2, pero usando SBO, Aceite de Pescado (FO) y Aceite de Palmiste como fuentes de grasa, incluidas a los mismos niveles que en el Exp. 1. En ambos experimentos 180 animales fueron alojados en jaulas individuales (n=30) y 600 en jaulas colectivas en grupos de 5 animales (n=20). Los animales alimentados con un 4% de grasa añadida tuvieron unos consumos diarios y unos índices de conversión más bajos que aquellos alimentados con las dietas con un 1.5% de grasa. En los animales alojados en colectivo del Exp. 1, el consumo fue un 4.8% más alto en los que consumieron las dietas que contenían manteca que en los animales alimentados con las dietas SBO (P = 0.036). La inclusión de manteca tendió a reducir la mortalidad (P = 0.067) en torno al 60% y al 25% con respecto a las dietas con SBO y SLO, respectivamente. La mortalidad aumentó con el nivel máximo de inclusión de SLO (14% vs. 1%, P < 0.01), sin observarse un efecto negativo sobre la mortalidad con el nivel más alto de inclusión de las demás fuentes de grasa utilizadas. En los animales alojados colectivo del Exp. 2 se encontró una disminución del consumo (11%), peso vivo a 63 d (4.8%) y de la ganancia diaria de peso (7.8%) con la inclusión de aceite de pescado con respecto a otras dietas (P < 0.01). Los dos últimos parámetros se vieron especialmente más reducidos cuando en las dietas se incluyó el nivel más alto de FO (5.6 y 9.5%, respectivamente, (P < 0.01)). Los animales alojados individualmente mostraron unos resultados productivos muy similares. La inclusión de aceite pescado tendió (P = 0.078) a aumentar la mortalidad (13.2%) con respecto al aceite de palmiste (6.45%), siendo intermedia para las dietas que contenían SBO (8.10%). La fuente o el nivel de grasa no afectaron la composición corporal total o de la canal eviscerada de los animales. Un incremento en el nivel de grasa dio lugar a una disminución de la ingesta de nitrógeno digestible (DNi) (1.83 vs. 1.92 g/d; P = 0.068 en Exp. 1 y 1.79 vs. 1.95 g/d; P = 0.014 en Exp. 2). Debido a que el nitrógeno retenido (NR) en la canal fue similar para ambos niveles (0.68 g/d (Exp. 1) y 0.71 g/d (Exp. 2)), la eficacia total de retención del nitrógeno (ERN) aumentó con el nivel máximo de inclusión de grasa, pero de forma significativa únicamente en el Exp. 1 (34.9 vs. 37.8%; P < 0.0001), mientras que en el Exp. 2 se encontró una tendencia (36.2 vs. 38.0% en Exp. 2; P < 0.064). Como consecuencia, la excreción de nitrógeno en heces fue menor en los animales alimentados con el nivel más alto de grasa (0.782 vs. 0.868 g/d; P = 0.0001 en Exp. 1, y 0.745 vs. 0.865 g/d; P < 0.0001 en Exp.2) al igual que el nitrógeno excretado en orina (0.702 vs. 0.822 g/d; P < 0.0001 en Exp. 1 y 0.694 vs. 0.7999 g/d; P = 0.014 en Exp.2). Aunque no hubo diferencias en la eficacia total de retención de la energía (ERE), la energía excretada en heces disminuyó al aumentar el nivel de inclusión de grasa (142 vs. 156 Kcal/d; P = 0.0004 en Exp. 1 y 144 vs. 154 g/d; P = 0.050 en Exp. 2). Sin embargo, la energía excretada como orina y en forma de calor fue mayor en el los animales del Exp. 1 alimentados con el nivel más alto de grasa (216 vs. 204 Kcal/d; P < 0.017). Se puede concluir que la manteca y el aceite de palmiste pueden ser considerados como fuentes alternativas al aceite de soja debido a la reducción de la mortalidad, sin efectos negativos sobre los rendimientos productivos o la retención de nutrientes. La inclusión de aceite de pescado empeoró los rendimientos productivos y la mortalidad durante el periodo de crecimiento. Un aumento en el nivel de grasa mejoró el índice de conversión y la eficacia total de retención de nitrógeno. ABSTRACT The aim of this Thesis is: 1) to obtain and validate prediction equations to determine in vivo whole body and carcass composition using the Bioelectrical Impedance (BIA) method in growing rabbits from 25 to 77 days of age, and 2) to study its application to determine differences on whole body and carcass chemical composition, and nutrient retention of animals fed different fat levels and sources. The first study was conducted to determine and later validate, by using independent data, the prediction equations obtained to assess in vivo the whole body composition of growing rabbits. One hundred and fifty rabbits grouped at 5 different ages (25, 35, 49, 63 and 77 days) and weighing from 231 to 3138 g were used. A four terminal body composition analyser was used to obtain resistance (Rs, ) and reactance (Xc, ) values (Model BIA-101, RJL Systems, Detroit, MI USA). The distance between internal electrodes (D, cm), body length (L, cm) and live BW of each animal were also registered. At each selected age, animals were slaughtered, ground and frozen (-20 ºC) for later chemical analyses (DM, fat, CP, ash and GE). Fat and energy body content increased with the age, while protein, ash, and water decreased. Mean values of Rs, Xc, impedance (Z), L and D were 83.5 ± 23.1 , 18.2 ± 3.8 , 85.6 ± 22.9 , 30.6 ± 6.9 cm and 10.8 ± 3.1 cm. A multiple linear regression analysis was used to determine the prediction equations, using BW, L and Z data as independent variables. Equations obtained to estimate water (g), CP (g), fat (g), ash (g) and GE (MJ) content had, respectively, coefficient of determination (R2) values of 0.99, 0.99, 0.97, 0.98 and 0.99, and the relative mean prediction error (RMPE) was: 2.79, 6.15, 24.3, 15.2 and 10.6%, respectively. When water was expressed as percentage, the R2 and RMPE were 0.85 and 2.30%, respectively. When prediction of the content of protein (%DM), fat (%DM), ash (%DM) and energy (kJ/100 g DM) was done, values of 0.79, 0.83, 0.71 and 0.86 for R2, and 5.04, 18.9, 12.0 and 3.19% for RMPE, respectively, were obtained. Reactance was negatively correlated with water, ash and CP content (r = -0.32, P < 0.0001; r = -0.20, P < 0.05; r = -0.26, P < 0.01) and positively correlated with fat and GE (r = 0.23 and r = 0.24; P < 0.01). Otherwise, resistance was positively correlated with water, ash and CP (r = 0.31, P < 0.001; r = 0.28, P < 0.001; r = 0.37, P < 0.0001) and negatively correlated with fat and energy (r = -0.36 and r = -0.35; P < 0.0001). Moreover, age was negatively correlated with water, ash and CP content (r = -0.79; r = -0.68 and r = -0.80; P < 0.0001) and positively correlated with fat and energy (r = 0.78 and r = 0.81; P < 0.0001). It could be concluded that BIA is a non-invasive good method to estimate in vivo whole body composition of growing rabbits from 25 to 77 days of age. The aim of the second study was to determine and validate with independent data, the prediction equations obtained to estimate in vivo carcass composition of growing rabbits by using the results of carcass chemical composition and BIA values in a group of rabbits from 25 to 77 days. Also its potential application to predict nutrient retention and overall energy and nitrogen retention efficiencies was analysed. Seventy five rabbits grouped at 5 different ages (25, 35, 49, 63 and 77 days) with weights ranging from 196 to 3260 g were used. A four terminal body composition analyser (Model BIA-101, RJL Systems, Detroit, MI USA) was used to obtain resistance (Rs, ) and reactance (Xc, ) values. The distance between internal electrodes (D, cm), body length (L, cm) and live weight (BW, g) were also registered. At each selected age, all the animals were stunned and bled. The skin, organs and digestive content were removed, and the chilled carcass were weighed and processed for chemical analyses (DM, fat, CP, ash and GE). Energy and fat increased with the age, while CP, ash, and water decreased. Mean values of Rs, Xc, impedance (Z), L and D were 95.9±23.9 , 19.5±4.7 , 98.0±23.8 , 20.6±6.3 cm y 13.7±3.1 cm. A multiple linear regression analysis was done to determine the equations, using BW, L and Z data as parameters. Coefficient of determination (R2) of the equations obtained to estimate water (g), CP (g), fat (g), ash (g) and GE (MJ) content were: 0.99, 0.99, 0.95, 0.96 and 0.98, and relative mean prediction error (RMPE) were: 4.20, 5.48, 21.9, 9.10 and 6.77%, respectively. When water content was expressed as percentage, the R2 and RMPE were 0.79 and 1.62%, respectively. When prediction of protein (%DM), fat (%DM), ash (%DM) and energy (kJ/100 g DM) content was done, R2 values were 0.68, 0.76, 0.66 and 0.82, and RMPE: 3.22, 10.5, 5.82 and 2.54%, respectively. Reactance was positively correlated with fat content (r = 0.24, P < 0.05) while resistance was positively correlated with water, ash and protein carcass content (r = 0.55, P < 0.001; r = 0.54, P < 0.001; r = 0.40, P < 0.005) and negatively correlated with fat and energy (r = -0.44 and r = -0.55; P < 0.001). Moreover, age was negatively correlated with water, ash and CP content (r = -0.97, r = -0.95 and r = -0.89, P < 0.0001) and positively correlated with fat and GE (r = 0.95 and r = 0.97; P < 0.0001). In the whole growing period (35-63 d), overall energy retention efficiency (ERE) and nitrogen retention efficiency (NRE) were studied. The ERE values were 20.4±7.29%, 21.0±4.18% and 20.8±2.79%, from 35 to 49, 49 to 63 and from 35 to 63 d, respectively. NRE was 46.9±11.7%, 34.5±7.32% and 39.1±3.23% for the same periods. Energy was retained in body tissues for growth with an efficiency of approximately 52.5% and efficiency of the energy for protein and fat retention was 33.3 and 69.9%, respectively. Efficiency of utilization of nitrogen for growth was near to 77%. This work shows that BIA it’s a non-invasive and good method to estimate in vivo carcass composition and nutrient retention of growing rabbits from 25 to 77 days of age. In the third study, two experiments were conducted to investigate the effect of the fat addition and source, on performance, mortality, nutrient retention, and the whole body and carcass chemical composition of growing rabbits from 34 to 63 d. In Exp. 1 three diets were arranged in a 3 x 2 factorial structure with the source of fat: Soybean oil (SBO), Soya Lecithin Oil (SLO) and Lard (L) and the dietary fat inclusion level (1.5 and 4%) as the main factors. Exp. 2 had also arranged as a 3 x 2 factorial design, but using SBO, Fish Oil (FO) and Palmkernel Oil (PKO) as fat sources, and included at the same levels than in Exp. 1. In both experiments 180 animals were allocated in individual cages (n=30) and 600 in collectives cages, in groups of 5 animals (n=20). Animals fed with 4% dietary fat level showed lower DFI and FCR than those fed diets with 1.5%. In collective housing of Exp. 1, DFI was a 4.8% higher in animals fed with diets containing lard than SBO (P = 0.036), being intermediate for diet with SLO. Inclusion of lard also tended to reduce mortality (P = 0.067) around 60% and 25% with respect SBO and SLO diets, respectively. Mortality increased with the greatest level of soya lecithin (14% vs. 1%, P < 0.01). In Exp. 2 a decrease of DFI (11%), BW at 63 d (4.8%) and DWG (7.8%) were observed with the inclusion of fish oil with respect the other two diets (P < 0.01). These last two traits impaired with the highest level of fish oil (5.6 and 9.5%, respectively, (P < 0.01)). Animals housed individually showed similar performance results. The inclusion of fish oil also tended to increase (P = 0.078) mortality (13.2%) with respect palmkernel oil (6.45%), being mortality of SBO intermediate (8.10%). Fat source and level did not affect the whole body or carcass chemical composition. An increase of the fat sources addition led to a decrease of the digestible nitrogen intake (DNi) (1.83 vs. 1.92 g/d; P = 0.068 in Exp. 1 and 1.79 vs. 1.95 g/d; P = 0.014 in Exp. 2). As the nitrogen retained (NR) in the carcass was similar for both fat levels (0.68 g/d (Exp. 1) and 0.71 g/d (Exp. 2)), the overall efficiency of N retention (NRE) increased with the highest level of fat, but only reached significant level in Exp. 1 (34.9 vs. 37.8%; P < 0.0001), while in Exp. 2 a tendency was found (36.2 vs. 38.0% in Exp. 2; P < 0.064). Consequently, nitrogen excretion in faeces was lower in animals fed with the highest level of fat (0.782 vs. 0.868 g/d; P = 0.0001 in Exp. 1, and 0.745 vs. 0.865 g/d; P < 0.0001 in Exp.2). The same effect was observed with the nitrogen excreted as urine (0.702 vs. 0.822 g/d; P < 0.0001 in Exp. 1 and 0.694 vs. 0.7999 g/d; P = 0.014 in Exp.2). Although there were not differences in ERE, the energy excreted in faeces decreased as fat level increased (142 vs. 156 Kcal/d; P = 0.0004 in Exp. 1 and 144 vs. 154 g/d; P = 0.050 in Exp. 2). In Exp. 1 the energy excreted as urine and heat production was significantly higher when animals were fed with the highest level of dietary fat (216 vs. 204 Kcal/d; P < 0.017). It can be concluded that lard and palmkernel oil can be considered as alternative sources to soybean oil due to the reduction of the mortality, without negative effects on performances or nutrient retention. Inclusion of fish impaired animals´ productivity and mortality. An increase of the dietary fat level improved FCR and overall protein efficiency retention.

In vitro properties of the first ORF protein from mouse LINE-1 support its role in ribonucleoprotein particle formation during retrotransposition

LINEs are transposable elements, widely distributed among eukaryotes, that move via reverse transcription of an RNA intermediate. Mammalian LINEs have two ORFs (ORF1 and ORF2). The proteins encoded by these ORFs play important roles in the retrotransposition process. Although the predicted amino acid sequence of ORF1 is not closely related to any known proteins, it is highly basic; thus, it has long been hypothesized that ORF1 protein functions to bind LINE-1 (L1) RNA during retrotransposition. Cofractionation of ORF1 protein and L1 RNA in extracts from both mouse and human embryonal carcinoma cells indicated that ORF1 protein binds L1 RNA, forming a ribonucleoprotein particle. Based on UV crosslinking and electrophoretic mobility-shift assays using purified components, we demonstrate here that the ORF1 protein encoded by mouse L1 binds nucleic acids with a strong preference for RNA and other single-stranded nucleic acids. Furthermore, multiple copies of ORF1 protein appear to bind single-stranded nucleic acid in a manner suggesting positive cooperativity; such binding characteristics are likely to be facilitated by the protein–protein interactions detected among molecules of ORF1 polypeptide by coimmunoprecipitation. These observations are consistent with the formation of ribonucleoprotein particles containing L1 RNA and ORF1 protein and provide additional evidence for the role of ORF1 protein during retrotransposition of L1.

Homospermidine synthase, the first pathway-specific enzyme of pyrrolizidine alkaloid biosynthesis, evolved from deoxyhypusine synthase

Pyrrolizidine alkaloids are preformed plant defense compounds with sporadic phylogenetic distribution. They are thought to have evolved in response to the selective pressure of herbivory. The first pathway-specific intermediate of these alkaloids is the rare polyamine homospermidine, which is synthesized by homospermidine synthase (HSS). The HSS gene from Senecio vernalis was cloned and shown to be derived from the deoxyhypusine synthase (DHS) gene, which is highly conserved among all eukaryotes and archaebacteria. DHS catalyzes the first step in the activation of translation initiation factor 5A (eIF5A), which is essential for eukaryotic cell proliferation and which acts as a cofactor of the HIV-1 Rev regulatory protein. Sequence comparison provides direct evidence for the evolutionary recruitment of an essential gene of primary metabolism (DHS) for the origin of the committing step (HSS) in the biosynthesis of pyrrolizidine alkaloids.

Detyrosination of Tubulin Regulates the Interaction of Intermediate Filaments with Microtubules In Vivo via a Kinesin-dependent Mechanism

Posttranslationally modified forms of tubulin accumulate in the subset of stabilized microtubules (MTs) in cells but are not themselves involved in generating MT stability. We showed previously that stabilized, detyrosinated (Glu) MTs function to localize vimentin intermediate filaments (IFs) in fibroblasts. To determine whether tubulin detyrosination or MT stability is the critical element in the preferential association of IFs with Glu MTs, we microinjected nonpolymerizable Glu tubulin into cells. If detyrosination is critical, then soluble Glu tubulin should be a competitive inhibitor of the IF–MT interaction. Before microinjection, Glu tubulin was rendered nonpolymerizable and nontyrosinatable by treatment with iodoacetamide (IAA). Microinjected IAA-Glu tubulin disrupted the interaction of IFs with MTs, as assayed by the collapse of IFs to a perinuclear location, and had no detectable effect on the array of Glu or tyrosinated MTs in cells. Conversely, neither IAA-tyrosinated tubulin nor untreated Glu tubulin, which assembled into MTs, caused collapse of IFs when microinjected. The epitope on Glu tubulin responsible for interfering with the Glu MT–IF interaction was mapped by microinjecting tubulin fragments of α-tubulin. The 14-kDa C-terminal fragment of Glu tubulin (α-C Glu) induced IF collapse, whereas the 36-kDa N-terminal fragment of α-tubulin did not alter the IF array. The epitope required more than the detyrosination site at the C terminus, because a short peptide (a 7-mer) mimicking the C terminus of Glu tubulin did not disrupt the IF distribution. We previously showed that kinesin may mediate the interaction of Glu MTs and IFs. In this study we found that kinesin binding to MTs in vitro was inhibited by the same reagents (i.e., IAA-Glu tubulin and α-C Glu) that disrupted the IF–Glu MT interaction in vivo. These results demonstrate for the first time that tubulin detyrosination functions as a signal for the recruitment of IFs to MTs via a mechanism that is likely to involve kinesin.

The Mr 140,000 Intermediate Chain of Chlamydomonas Flagellar Inner Arm Dynein Is a WD-Repeat Protein Implicated in Dynein Arm Anchoring

Previous structural and biochemical studies have revealed that the inner arm dynein I1 is targeted and anchored to a unique site located proximal to the first radial spoke in each 96-nm axoneme repeat on flagellar doublet microtubules. To determine whether intermediate chains mediate the positioning and docking of dynein complexes, we cloned and characterized the 140-kDa intermediate chain (IC140) of the I1 complex. Sequence and secondary structural analysis, with particular emphasis on β-sheet organization, predicted that IC140 contains seven WD repeats. Reexamination of other members of the dynein intermediate chain family of WD proteins indicated that these polypeptides also bear seven WD/β-sheet repeats arranged in the same pattern along each intermediate chain protein. A polyclonal antibody was raised against a 53-kDa fusion protein derived from the C-terminal third of IC140. The antibody is highly specific for IC140 and does not bind to other dynein intermediate chains or proteins in Chlamydomonas flagella. Immunofluorescent microscopy of Chlamydomonas cells confirmed that IC140 is distributed along the length of both flagellar axonemes. In vitro reconstitution experiments demonstrated that the 53-kDa C-terminal fusion protein binds specifically to axonemes lacking the I1 complex. Chemical cross-linking indicated that IC140 is closely associated with a second intermediate chain in the I1 complex. These data suggest that IC140 contains domains responsible for the assembly and docking of the I1 complex to the doublet microtubule cargo.

The Chlamydomonas IDA7 Locus Encodes a 140-kDa Dynein Intermediate Chain Required to Assemble the I1 Inner Arm Complex

To identify new loci that are involved in the assembly and targeting of dynein complexes, we have screened a collection of motility mutants that were generated by insertional mutagenesis. One such mutant, 5B10, lacks the inner arm isoform known as the I1 complex. This isoform is located proximal to the first radial spoke in each 96-nm axoneme repeat and is an important target for the regulation of flagellar motility. Complementation tests reveal that 5B10 represents a new I1 locus, IDA7. Biochemical analyses confirm that ida7 axonemes lack at least five I1 complex subunits. Southern blots probed with a clone containing the gene encoding the 140-kDa intermediate chain (IC) indicate that the ida7 mutation is the result of plasmid insertion into the IC140 gene. Transformation with a wild-type copy of the IC140 gene completely rescues the mutant defects. Surprisingly, transformation with a construct of the IC140 gene lacking the first four exons of the coding sequence also rescues the mutant phenotype. These studies indicate that IC140 is essential for assembly of the I1 complex, but unlike other dynein ICs, the N-terminal region is not critical for its activity.

Identification and Stereospecificity of the First Three Enzymes of 3-Dimethylsulfoniopropionate Biosynthesis in a Chlorophyte Alga1

Many marine algae produce 3-dimethylsulfoniopropionate (DMSP), a potent osmoprotective compound whose degradation product dimethylsulfide plays a central role in the biogeochemical S cycle. Algae are known to synthesize DMSP via the four-step pathway, l-Met → 4-methylthio-2-oxobutyrate → 4-methylthio-2-hydroxybutyrate → 4-dimethylsulfonio-2-hydroxy-butyrate (DMSHB) → DMSP. Substrate-specific enzymes catalyzing the first three steps in this pathway were detected and partially characterized in cell-free extracts of the chlorophyte alga Enteromorpha intestinalis. The first is a 2-oxoglutarate-dependent aminotransferase, the second an NADPH-linked reductase, and the third an S-adenosylmethionine-dependent methyltransferase. Sensitive radiometric assays were developed for these enzymes, and used to show that their activities are high enough to account for the estimated in vivo flux from Met to DMSP. The activities of these enzymes in other DMSP-rich chlorophyte algae were at least as high as those in E. intestinalis, but were ≥20-fold lower in algae without DMSP. The reductase and methyltransferase were specific for the d-enantiomer of 4-methylthio-2-hydroxybutyrate in vitro, and both the methyltransferase step and the step(s) converting DMSHB to DMSP were shown to prefer d-enantiomers in vivo. The intermediate DMSHB was shown to act as an osmoprotectant, which indicates that the first three steps of the DMSP synthesis pathway may be sufficient to confer osmotolerance.

Intermediate hosts of Toxoplasma gondii and food safety:epidemiology, genetics and parasite survival in meat-producing animals in Italy

Toxoplasma gondii is a coccidian parasite with a global distribution. The definitive host is the cat (and other felids). All warm-blooded animals can act as intermediate hosts, including humans. Sexual reproduction (gametogony) takes place in the final host and oocysts are released in the environment, where they then sporulate to become infective. In intermediate hosts the cycle is extra-intestinal and results in the formation of tachyzoites and bradyzoites. Tachyzoites represent the invasive and proliferative stage and on entering a cell it multiplies asexually by endodyogeny. Bradyzoites within tissue cysts are the latent form. T. gondii is a food-borne parasite causing toxoplasmosis, which can occur in both animals and humans. Infection in humans is asymptomatic in more than 80% of cases in Europe and North-America. In the remaining cases patients present fever, cervical lymphadenopathy and other non-specific clinical signs. Nevertheless, toxoplasmosis is life threatening if it occurs in immunocompromised subjects. The main organs involved are brain (toxoplasmic encephalitis), heart (myocarditis), lungs (pulmonary toxoplasmosis), eyes, pancreas and parasite can be isolated from these tissues. Another aspect is congenital toxoplasmosis that may occur in pregnant women and the severity of the consequences depends on the stage of pregnancy when maternal infection occurs. Acute toxoplasmosis in developing foetuses may result in blindness, deformation, mental retardation or even death. The European Food Safety Authority (EFSA), in recent reports on zoonoses, highlighted that an increasing numbers of animals resulted infected with T. gondii in EU (reported by the European Member States for pigs, sheep, goats, hunted wild boar and hunted deer, in 2011 and 2012). In addition, high prevalence values have been detected in cats, cattle and dogs, as well as several other animal species, indicating the wide distribution of the parasite among different animal and wildlife species. The main route of transmission is consumption of food and water contaminated with sporulated oocysts. However, infection through the ingestion of meat contaminated with tissue cysts is frequent. Finally, although less frequent, other food products contaminated with tachyzoites such as milk, may also pose a risk. The importance of this parasite as a risk for human health was recently highlighted by EFSA’s opinion on modernization of meat inspection, where Toxoplasma gondii was identified as a relevant hazard to be addressed in revised meat inspection systems for pigs, sheep, goats, farmed wild boar and farmed deer (Call for proposals -GP/EFSA/BIOHAZ/2013/01). The risk of infection is more highly associated to animals reared outside, also in free-range or organic farms, where biohazard measure are less strict than in large scale, industrial farms. Here, animals are kept under strict biosecurity measures, including barriers, which inhibit access by cats, thus making soil contamination by oocysts nearly impossible. A growing demand by the consumer for organic products, coming from free-range livestock, in respect of animal-welfare, and the desire for the best quality of derived products, have all led to an increase in the farming of free-range animals. The risk of Toxoplasma gondii infection increases when animals have access to environment and the absence of data in Italy, together with need for in depth study of both the prevalence and genotypes of Toxoplasma gondii present in our country were the main reasons for the development of this thesis project. A total of 152 animals have been analyzed, including 21 free-range pigs (Suino Nero race), 24 transhumant Cornigliese sheep, 77 free-range chickens and 21 wild animals. Serology (on meat juice) and identification of T. gondii DNA through PCR was performed on all samples, except for wild animals (no serology). An in-vitro test was also applied with the aim to find an alternative and valid method to bioassay, actually the gold standard. Meat samples were digested and seeded onto Vero cells, checked every day and a RT-PCR protocol was used to determine an eventual increase in the amount of DNA, demonstrating the viability of the parasite. Several samples were alos genetically characterized using a PCR-RFLP protocol to define the major genotypes diffused in the geographical area studied. Within the context of a project promoted by Istituto Zooprofilattico of Pavia and Brescia (Italy), experimentally infected pigs were also analyzed. One of the aims was to verify if the production process of cured “Prosciutto di Parma” is able to kill the parasite. Our contribution included the digestion and seeding of homogenates on Vero cells and applying the Elisa test on meat juice. This thesis project has highlighted widespread diffusion of T. gondii in the geographical area taken into account. Pigs, sheep, chickens and wild animals showed high prevalence of infection. The data obtained with serology were 95.2%, 70.8%, 36.4%, respectively, indicating the spread of the parasite among numerous animal species. For wild animals, the average value of parasite infection determined through PCR was 44.8%. Meat juice serology appears to be a very useful, rapid and sensitive method for screening carcasses at slaughterhouse and for marketing “Toxo-free” meat. The results obtained on fresh pork meat (derived from experimentally infected pigs) before (on serum) and after (on meat juice) slaughter showed a good concordance. The free-range farming put in evidence a marked risk for meat-producing animals and as a consequence also for the consumer. Genotyping revealed the diffusion of Type-II and in a lower percentage of Type-III. In pigs is predominant the Type-II profile, while in wildlife is more diffused a Type-III and mixed profiles (mainly Type-II/III). The mixed genotypes (Type-II/III) could be explained by the presence of mixed infections. Free-range farming and the contact with wildlife could facilitate the spread of the parasite and the generation of new and atypical strains, with unknown consequences on human health. The curing process employed in this study appears to produce hams that do not pose a serious concern to human health and therefore could be marketed and consumed without significant health risk. Little is known about the diffusion and genotypes of T. gondii in wild animals; further studies on the way in which new and mixed genotypes may be introduced into the domestic cycle should be very interesting, also with the use of NGS techniques, more rapid and sensitive than PCR-RFLP. Furthermore wildlife can become a valuable indicator of environmental contamination with T. gondii oocysts. Other future perspectives regarding pigs include the expansion of the number of free-range animals and farms and for Cornigliese sheep the evaluation of other food products as raw milk and cheeses. It should be interesting to proceed with the validation of an ELISA test for infection in chickens, using both serum and meat juice on a larger number of animals and the same should be done also for wildlife (at the moment no ELISA tests are available and MAT is the reference method for them). Results related to Parma ham do not suggest a concerning risk for consumers. However, further studies are needed to complete the risk assessment and the analysis of other products cured using technological processes other than those investigated in the present study. For example, it could be interesting to analyze products such as salami, produced with pig meat all over the Italian country, with very different recipes, also in domestic and rural contexts, characterized by a very short period of curing (1 to 6 months). Toxoplasma gondii is one of the most diffuse food-borne parasites globally. Public health safety, improved animal production and protection of endangered livestock species are all important goals of research into reliable diagnostic tools for this infection. Future studies into the epidemiology, parasite survival and genotypes of T. gondii in meat producing animals should continue to be a research priority.

Polpa de guavira (Campomanesia cambessedeana Berg) desidratada em spray dryer: efeitos das condições de processo e composição da alimentação nas propriedades físico químicas e atividade antioxidante

O objetivo desse trabalho foi obter polpa de guavira desidratada por atomização, utilizando maltodextrina ou goma arábica como agentes carreadores. Inicialmente, avaliou-se a influência das condições de processo, temperatura do ar de secagem (130, 155 e 180) °C e vazão volumétrica da mistura (20 e 40) mL/min, o tipo e concentração de agente carreador (10 e 20) % nas características físicas, físico-químicas e atividade antioxidante do produto obtido. As propriedades analisadas foram umidade, atividade de água, higroscopicidade, solubilidade, cor, distribuição e tamanho médio de partículas, morfologia, compostos fenólicos totais e atividade antioxidante. A temperatura do ar de secagem e a vazão volumétrica de alimentação influenciaram significativamente todas as propriedades da guavira em pó. A umidade e atividade de água apresentaram os menores valores na temperatura intermediária, independentemente do tipo e concentração do carreador usado. A solubilidade das amostras adicionadas de maltodextrina foram superiores às amostras com goma arábica. O aumento da concentração de agente carreador, em geral, proporcionou um aumento no parâmetro L* e diminuição dos parâmetros a* e b*, tornando as amostras mais claras e reduzindo as tonalidades vermelha e amarela. A guavira em pó apresentou coloração próxima do amarelo e marrom, com grande variação nos parâmetros de cor C* e H* em função das diferentes condições de secagem. A distribuição do tamanho de partículas não teve um padrão definido e o tamanho médio das amostras com maltodextrina foram maiores do que as com goma arábica para a temperatura do ar a 130 °C. No entanto, para as outras temperaturas (155 e 180) °C não houve um comportamento específico do tamanho das partículas em função da vazão de alimentação, tipo e ou concentração de agente carreador. A análise de microscopia eletrônica de varredura permitiu observar que as partículas obtidas tanto com maltodextrina como goma arábica apresentaram formato esférico, superfície rugosa e com adesão de partículas menores nas de maior tamanho, sendo que a superfície das partículas com goma arábica também apresentaram concavidades. A atividade antioxidante foi superior quando utilizada a temperatura de secagem intermediária. A partir das condições selecionadas na primeira etapa (temperatura do ar de 155 °C, vazão volumétrica da mistura de 40 mL/min e 10% de maltodextrina ou goma arábica) a polpa de guavira em pó foi caracterizada quanto a temperatura de transição vítrea, as isotermas de adsorção e a estabilidade à estocagem do ácido ascórbico, compostos fenólicos totais e da atividade antioxidante da polpa de guavira em pó produzida por spray drying ao longo de 120 dias. As temperaturas de transição vítrea foram de (25,2 ± 2,7 °C e 31,4 ± 0,4) °C para os pós produzidos com goma arábica e maltodextrina, respectivamente. O modelo de BET apresentou ajuste muito bom (R2>0,99) para descrever o comportamento de sorção de água das amostras nas temperaturas de (20, 30 e 40) °C. A polpa de guavira em pó produzida com goma arábica apresentou maior adsorção de água do que as amostras obtidas com maltodextrina. No estudo da estabilidade, as amostras foram acondicionadas em embalagem de polietileno laminado e armazenadas a 25 °C e umidade relativa de 75%. A embalagem de polietileno laminado foi eficiente na manutenção do teor de ácido ascórbico e atividade antioxidante da guavira em pó por um período de 120 dias, independente do carreador adicionado. O teor de compostos fenólicos para a guavira em pó com goma arábica apresentou uma redução nos primeiros 22 dias, contudo a amostra com maltodextrina manteve-se estável durante 120 dias de armazenamento.

The first extrasolar planet discovered with a new-generation high-throughput Doppler instrument

We report the detection of the first extrasolar planet, ET-1 (HD 102195b), using the Exoplanet Tracker (ET), a new-generation Doppler instrument. The planet orbits HD 102195, a young star with solar metallicity that may be part of the local association. The planet imparts radial velocity variability to the star with a semiamplitude of 63.4 ± 2.0 m s^-1 and a period of 4.11 days. The planetary minimum mass (m sin i) is 0.488MJ ± 0.015M_J. The planet was initially detected in the spring of 2005 with the Kitt Peak National Observatory (KPNO) 0.9 m coudé feed telescope. The detection was confirmed by radial velocity observations with the ET at the KPNO 2.1 m telescope and also at the 9 m Hobby-Eberly Telescope (HET) with its High Resolution Spectrograph. This planetary discovery with a 0.9 m telescope around a V = 8.05 magnitude star was made possible by the high throughput of the instrument: 49% measured from the fiber output to the detector. The ET's interferometer-based approach is an effective method for planet detection. In addition, the ET concept is adaptable to multiple-object Doppler observations or very high precision observations with a cross-dispersed echelle spectrograph to separate stellar fringes over a broad wavelength band. In addition to spectroscopic observations of HD 102195, we obtained brightness measurements with one of the automated photometric telescopes at Fairborn Observatory. Those observations reveal that HD 102195 is a spotted variable star with an amplitude of ~0.015 mag and a 12.3 ± 0.3 day period. This is consistent with spectroscopically observed Ca II H and K emission levels and line-broadening measurements but inconsistent with rotational modulation of surface activity as the cause of the radial velocity variability. Our photometric observations rule out transits of the planetary companion.

Compostos fenólicos de resíduos agroindustriais: identificação, propriedades biológicas e aplicação em matriz alimentar de base lipídica

A geração de resíduos sólidos pelas atividades agroindustriais tem criado a demanda por um reaproveitamento tecnológico desses materiais. Assim, o objetivo deste trabalho foi avaliar o potencial bioativo e tecnológico de resíduos agroindustriais, como fontes naturais de compostos fenólicos com atividade antioxidante. Foram analisados resíduos agroindustriais vinícolas, de indústrias produtoras de polpas congeladas de frutas (açaí, cajá, cupuaçu e graviola) e provenientes do beneficiamento de café e de laranja. Inicialmente, foi realizado um estudo para a determinação das condições ótimas de extração, empregando planejamento experimental multivariado com delineamento composto central rotacional, cujos resultados foram avaliados empregando a técnica de superfície de resposta. Na sequência, foram feitos a triagem dos resíduos, baseada na atividade antioxidante, e a caracterização fenólica dos extratos hidroalcoólicos obtidos dos resíduos agroindustriais. De acordo com os resultados de atividade antioxidante, engaço de uva da variedade Chenin Blanc (EC) e semente de açaí (SA) foram os resíduos selecionados, os quais seguiram para as etapas de concentração e fracionamento bioguiado de sua(s) molécula(s) bioativa(s), as quais foram posteriormente identificadas por UHPLC-ESI-LTQ-MS. Extratos brutos e concentrados foram avaliados in vitro quanto à capacidade de desativação de espécies reativas de oxigênio (radicais peroxila, ânion superóxido e ácido hipocloroso) e então, aplicados em óleo de soja, emulsão e suspensão de lipossomos, a fim de se avaliar a efetividade desses extratos como antioxidante natural em matrizes lipídicas. Concentrações intermediárias de etanol (40-60%) e alta temperatura (96°C), exceto para semente de açaí (25°C), foram as condições ótimas para a extração de antioxidantes dos resíduos agroindustriais. Epicatequina, ácido gálico, catequina e procianidina B1 foram os compostos de maior ocorrência, quando avaliados pela técnica de HPLC-DAD. O EC apresentou a maior atividade antioxidante global e SA a maior atividade entre os resíduos de polpas de frutas, laranja e café. A concentração dos extratos brutos de EC e SA, pela resina Amberlite XAD®-2, produziu aumento significativo da atividade antioxidante. Além disso, extratos brutos e concentrados apresentaram atividade antiproliferativa e anti-inflamatória. Os extratos concentrados foram fracionados por meio de Sephadex LH-20, a partir da qual foi possível identificar quatro frações de maior bioatividade para o EC e três para o SA. Procianidina B1, catequina, epicatequina e resveratrol foram identificados no extrato concentrado e frações de EC. Dezoito procianidinas poliméricas, catequina, epicatequina foram os principais compostos identificados em SA, por meio de UHPLC-ESI-LTQ-MS. Resveratrol também foi encontrado em SA pela primeira vez. Quando avaliados em óleo de soja, EC e SA demonstraram atividade pro-oxidante. Contudo, elevada atividade antioxidante foi verificada quando essas amostras foram aplicadas em sistemas lipídicos coloidais, pois retardaram o consumo de oxigênio em uma emulsão óleo/água e o período de indução na produção de dienos conjugados em uma suspensão de lipossomos. Portanto, os resíduos agroindustriais EC e SA possuem potencial tecnológico de reaproveitamento industrial podendo ser considerados possíveis matérias-primas para a obtenção de extratos ricos em antioxidantes ou pela extração de antioxidantes naturais de uso pelas indústrias farmacêutica e/ou de alimentos.

O uso do eucalipto em sistemas silvipastoril: acúmulo de fitomassa arbórea e de pastagem

O presente estudo foi dividido em três capitulos, todos realizados na Estação Experimental de Ciências Florestais de Anhembi/SP, entre os anos de 2014 e 2015. O primeiro estudo intitulado de \"Variação mensal da fitomassa da forragem em função do grau de cobertura do dossel em sistemas silvipastoris\", foi realizado em 3 monoculturas de 13 anos de idade, com área útil de 50 m x 30 m para coleta de pasto as mesmas efectuadas mensalmente. Os resultados apresentaram que não há relação significativa entre a cobertura do dossel e fitomassa da forragem pelo caso de que o sub-bosque estava muito sombreado. Entretanto, houve uma relação indireta entre área basal e fitomassa. Evidenciando-se que o talhão de Eucalipto urograndis apresentou as melhores condições de crescimento e disponibilidade de materia seca mensal para Bachiaria decumbens além de obter a maior porcentagem de folha entre todos os tratamentos. Ao contrario, no talhão de Pinus tecunumanii, foi encontrada a menor disponibilidade de materia seca mensal e por consequência, menor porcentagem de folha. O segundo estudo foi chamado de: \"Disponibilidade de fitomassa de B. decumbens, em um sistema silvipastoril com eucalipto: o papel da radiação\" onde o componente florestal foi o eucalyptus (COP-1377) de 2 anos de idade plantado em uma área útil de 10 ha, dividido em 3 tratamentos (onda longa-OL (39 m), onda curta-OC (21 m), e testemunha-T) e instalado em 4 blocos distintos. Foram realizadas duas coletas dutante o período de verão e de inverno, onde foi possível verificar que o tratamento OL mostrou maior disponibilidade de fitomassa a 65% de irradiância além de obter maior porcentagem da fração folha. Este foi favorecido pelo maior espaçamento entre as aléias. Contudo, houve ataque de cigarinha na pastagem, mantendo a queda da disponibilidade no período de inverno. O terceiro estudo intitulado de: \"Variaçoes arquiteturais de uma monocultura de E. urograndis em função de sua posição espacial\", foi também realizado na monocultura do primeiro estudo, numa área de 7 ha. Para este estudo, realizou-se um inventario florestal, logo após, dividiu-se as árvores por sua classe diamétrica e selecionou-se aleatoriamente 60 árvores para cubagem, e destas, escolheu-se 15 para determinação da fitomassa e respectiva densidade da madeira. Para a obtenção da fitomassa dividiu-se as árvores em três frações de análise: tronco, galhos e folha. Além disso, as 15 árvores foram divididas em: bordadura, intermediária e centro da parcela, de acordo com a sua localização. Verificou-se que a bordadura apresentou os maiores crescimentos em DAP, altura, largura de copa e, que por consequência, obteve maior volume e fitomassa em todas suas frações. Também foi possível observar que tanto a bordadura quanto o centro apresentaram maior densidade básica em função da maior copa e altura das árvores incentivando a geração de mais fitomassa foliar. Finalmente conforme os três estudos realizados neste trabalho de pesquisa, concluiu-se que a radiação solar é fator chave na produtividade da cultura forrageira, demonstrando a necessidade de mais pesquisas sobre os sistemas agroforestais e silvipastoris para o sucesso de futuros emprendimentos.

The accretion environment in Vela X-1 during a flaring period using XMM-Newton

We present analysis of 100 ks contiguous XMM-Newton data of the prototypical wind accretor Vela X-1. The observation covered eclipse egress between orbital phases 0.134 and 0.265, during which a giant flare took place, enabling us to study the spectral properties both outside and during the flare. This giant flare with a peak luminosity of 3.92+0.42-0.09 × 1037 erg s-1 allows estimates of the physical parameters of the accreted structure with a mass of ~1021 g. We have been able to model several contributions to the observed spectrum with a phenomenological model formed by three absorbed power laws plus three emission lines. After analysing the variations with orbital phase of the column density of each component, as well as those in the Fe and Ni fluorescence lines, we provide a physical interpretation for each spectral component. Meanwhile, the first two components are two aspects of the principal accretion component from the surface of the neutron star, the third component seems to be the X-ray light echo formed in the stellar wind of the companion.

Application of the Putting Women First protocol in a study on violence against immigrant women in Spain

In this paper, we describe our experience of using the Putting Women First protocol in the design and implementation of a cross-sectional study on violence against women (VAW) among 1607 immigrant women from Morocco, Ecuador and Romania living in Spain in 2011. The Putting Women First protocol is an ethical guideline for VAW research, which includes recommendations to ensure the safety of the women involved in studies on this subject. The response rate in this study was 59.3%. The prevalence of VAW cases last year was 11.7%, of which 15.6% corresponded to Ecuadorian women, 10.9% to Moroccan women and 8.6% to Romanian women. We consider that the most important goal for future research is the use of VAW scales validated in different languages, which would help to overcome the language barriers encountered in this study.