Effects of forage type on diversity in bacterial pellets isolated from liquid and solid phases of the rumen content in sheep and goats.

Two sheep and two goats, fitted with a ruminal cannula, received two diets composed of 30% concentrate and 70% of either alfalfa hay (AL) or grass hay (GR) as forage in a two-period crossover design. Solid and liquid phases of the rumen were sampled from each animal immediately before feeding and 4 h post-feeding. Pellets containing solid associated bacteria (SAB) and liquid associated bacteria (LAB) were isolated from the corresponding ruminal phase and composited by time to obtain 2 pellets per animal (one SAB and one LAB) before DNA extraction. Denaturing gradient gel electrophoresis (DGGE) analysis of 16S ribosomal DNA was used to analyze bacterial diversity. A total of 78 and 77 bands were detected in the DGGE gel from sheep and goats samples, respectively. There were 18 bands only found in the pellets from sheep fed AL-fed sheep and 7 found exclusively in samples from sheep fed the GR diet. In goats, 21 bands were found only in animals fed the AL diet and 17 were found exclusively in GR-fed ones. In all animals, feeding AL diet tended (P < 0.10) to promote greater NB and SI in LAB and SAB pellets compared with the GR diet. The dendrogram generated by the cluster analysis showed that in both animal species all samples can be included in two major clusters. The four SAB pellets within each animal species clustered together and the four LAB pellets grouped in a different cluster. Moreover, SAB and LAB clusters contained two clear subclusters according to forage type. Results show that in all animals bacterial diversity was more markedly affected by the ruminal phase (solid vs. liquid) than by the type of forage in the diet.

The influence of diet on the effectiveness of garlic oil and cinnamaldehyde to manipulate in vitro ruminal fermentation and methane production.

The objective of this study was to evaluate the effects of increasing doses [0 (control: CON), 20, 60, 180 and 540 mg/L incubation medium] of garlic oil (GO) and cinnamaldehyde (CIN) on in vitro ruminal fermentation of two diets. Batch cultures of mixed ruminal microorganisms were inoculated with ruminal fluid from four sheep fed a medium-concentrate diet (MC; 50 : 50 alfalfa hay : concentrate) or four sheep fed a high-concentrate diet (HC; 15 : 85 barley straw : concentrate). Diets MC and HC were representative of those fed to dairy and fattening ruminants, respectively. Samples of each diet were used as incubation substrates for the corresponding inoculum, and the incubation was repeated on 4 different days (four replicates per experimental treatment). There were GO × diet-type and CIN × diet-type interactions (P < 0.001–0.05) for many of the parameters determined, indicating different effects of both oils depending on the diet type. In general, effects of GO were more pronounced for MC compared with HC diet. Supplementation of GO did not affect (P > 0.05) total volatile fatty acid (VFA) production at any dose. For MC diet, GO at 60, 180 and 540 mg/L decreased (P < 0.05) molar proportion of acetate (608, 569 and 547 mmol/mol total VFA, respectively), and increased (P < 0.05) propionate proportion (233, 256 and 268 mmol/mol total VFA, respectively), compared with CON values (629 and 215 mmol/mol total VFA for acetate and propionate, respectively). A minimum dose of 180 mg of GO/L was required to produce similar modifications in acetate and propionate proportions with HC diet, but no effects (P > 0.05) on butyrate proportion were detected. Methane/VFA ratio was reduced (P < 0.05) by GO at 60, 180 and 540 mg/L for MC diet (0.23, 0.16 and 0.10 mol/mol, respectively), and by GO at 20, 60, 180 and 540 mg/L for HC diet (0.19, 0.19, 0.16 and 0.08 mol/mol, respectively), compared with CON (0.26 and 0.21 mol/mol for MC and HC diets, respectively). No effects (P = 0.16–0.85) of GO on final pH and concentrations of NH3-N and lactate were detected. For both diet types, the highest CIN dose decreased (P < 0.05) production of total VFA, gas and methane, which would indicate an inhibition of fermentation. Compared with CON, CIN at 180 mg/L increased (P < 0.05) acetate proportion for the MC (629 and 644 mmol/mol total VFA for CON and CIN, respectively) and HC (525 and 540 mmol/mol total VFA, respectively) diets, without affecting the proportions of any other VFA or total VFA production. Whereas for MC diet CIN at 60 and 180 mg/L decreased (P < 0.05) NH3-N concentrations compared with CON, only a trend (P < 0.10) was observed for CIN at 180 mg/L with the HC diet. Supplementation of CIN up to 180 mg/L did not affect (P = 0.18–0.99) lactate concentrations and production of gas and methane for any diet. The results show that effectiveness of GO and CIN to modify ruminal fermentation may depend on diet type, which would have practical implications if they are confirmed in vivo.

Aislamiento y caracterización de bacterias endosimbióticas procedentes de suelos contaminados por cobre en Chile

El uso intensivo de compuestos de cobre como herbicidas y fungicidas provoca la contaminación de suelos de uso agrícola debido a la acumulación de este metal en las capas más superficiales del suelo. Se sabe que la presencia de cobre y otros metales pesados afecta negativamente a las interacciones simbióticas que se establecen entre bacterias diazotróficas de los géneros Rhizobium, Sinorhizobium y Bradyrhizobium y leguminosas de interés agrícola (Laguerre et al., 2006). El objetivo de este trabajo es estudiar la diversidad de cepas endosimbióticas de leguminosas en suelos agrícolas chilenos que presentan un elevado contenido en cobre como resultado de la contaminación con residuos de extracciones mineras. Además, se pretende caracterizar el nivel de resistencia a cobre en las cepas aisladas con objeto de identificar aquellas altamente eficientes que puedan ser utilizadas como inoculantes microbianos. Para ello, se han prospectado 9 suelos agrícolas de las regiones III, V y VI de Chile con contenidos muy variables de metales. Utilizando estos suelos como inóculos de plantas trampa de leguminosas se ha obtenido una colección de 362 cepas aisladas de nódulos de guisante (Pisum sativum), judía (Phaseolus vulgaris) y alfalfa (Medicago sativa). Los análisis filogenéticos y los ensayos de resistencia a cobre realizados han permitido caracterizar y seleccionar aquellas cepas con mayores niveles de resistencia a este metal. Los resultados demuestran que los suelos altamente contaminados por cobre poseen una menor diversidad de bacterias endosimbióticas; las cepas más resistentes han sido aisladas de los suelos con niveles de contaminación intermedia. Los análisis fenotípicos y moleculares realizados sobre las cepas más resistentes han demostrado la existencia de sistemas de resistencia a cobre inducibles por este metal y potencialmente implicados en su homeostasis.

Technical note: Comparison of automated ribosomal intergenic spacer analysis (ARISA) and denaturing gradient gel electrophoresis (DGGE) to assess bacterial diversity in the rumen of sheep

The aim of this study was to compare automated ribosomal intergenic spacer analysis (ARISA) and denaturing gradient gel electrophoresis (DGGE) techniques to assess bacterial diversity in the rumen of sheep. Sheep were fed 2 diets with 70% of either alfalfa hay or grass hay, and the solid (SOL) and liquid (LIQ) phases of the rumen were sampled immediately before feeding (0 h) and at 4 and 8 h postfeeding. Both techniques detected similar differences between forages, with alfalfa hay promoting greater (P < 0.05) bacterial diversity than grass hay. In contrast, whereas ARISA analysis showed a decrease (P < 0.05) of bacterial diversity in SOL at 4 h postfeeding compared with 0 and 8 h samplings, no variations (P > 0.05) over the postfeeding period were detected by DGGE. The ARISA technique showed lower (P < 0.05) bacterial diversity in SOL than in LIQ samples at 4 h postfeeding, but no differences (P > 0.05) in bacterial diversity between both rumen phases were detected by DGGE. Under the conditions of this study, the DGGE was not sensitive enough to detect some changes in ruminal bacterial communities, and therefore ARISA was considered more accurate for assessing bacterial diversity of ruminal samples. The results highlight the influence of the fingerprinting technique used to draw conclusions on factors affecting ruminal bacterial diversity.

Identification of the method to quantify soluble fibre and the effect of the source of fibre on the ileal and faecal digestibility of soluble and insoluble fibre in rabbits = Identificación del método para cuantificar la fibra soluble y el efecto de la fuente de fibra en la digestibilidad ileal y fecal de la fibra soluble e insoluble en conejos

La presente tesis constituye un avance en el estudio de los métodos para cuantificar la fibra soluble y los efectos de las fracciones de fibra y las fuentes de fibra sobre la digestión de las diferentes fracciones de fibra (soluble e insoluble) en el conejo. Hay un efecto positivo de la fibra soluble sobre la salud intestinal de los conejos y, por ende, una reducción de la mortalidad en animales destetados. Pese a esto, no está claro si estos efectos se deben específicamente a la fracción soluble. Por lo que los objetivos generales de esta tesis fueron: 1) comparar diferentes metodologías químicas e in vitro para cuantificar la fibra soluble y estudiar las posibles interferencias en la cuantificación de la fibra soluble por las mucinas, y viceversa, 2) determinar los efectos de la fibra, el lugar de fermentación, el método para valorar la fibra soluble e insoluble, y la corrección de la fibra soluble por el contenido intestinal de mucinas sobre la digestibilidad de las distintas fracciones de la fibra y 3) evaluar los efectos individuales de las fracciones soluble e insoluble de la fibra de pulpa de remolacha y de manzana, sobre la digestibilidad de la fibra soluble e insoluble y los parámetros digestivos. Para ello se llevaron a cabo 4 estudios. En el primer estudio se compararon diferentes metodologías químicas e in vitro para valorar la fibra soluble de diferentes alimentos y se estudió la posible interferencia en la determinación de la fibra soluble y mucinas. Para ello se utilizaron seis ingredientes (pulpa de remolacha, pectinas de pulpa de remolacha, pulpa de remolacha lavada, paja de cereal, cascarilla de girasol y lignocelulosa) y siete piensos de conejos con diferentes niveles de fibra soluble. En un primer experimento se analizó la fibra dietética total (FDT), la fibra dietética insoluble (FDI), la fibra dietética soluble (FDS), la fibra neutro detergente corregida por cenizas y proteínas (aFNDmo-pb), y la digestibilidad in vitro 2 pasos pepsina/pancreatina (residuo corregido por cenizas y proteína, ivMSi2) de los ingredientes y piensos. Además la fibra soluble se calculó mediante la diferencia entre FDT-FDI (FDSFDI), FDT- ivMSi2 (FDSivMSi2), y FDT - aFNDmo-pb (FDSaFNDmo-pb). Cuando la fibra soluble se determinó directamente como FDS o se calculó como FDT-FDI no se observaron diferencias (109 g/kg MS, en promedio). Sin embargo, cuando la fibra soluble se calculó como FDT - aFNDmo-pb su valor fue un 40% menor (153 g/kg MS. P < 0,05), mientras que la FDSFDI (124 g/kg MS) no fue diferente a ninguna de las otras metodologías. La correlación entre los tres métodos fue elevada (r > 0,96. P < 0,001. n = 13), pero disminuyó o incluso desapareció cuando la pulpa o las pectinas de la remolacha fueron excluidas del análisis. En un segundo experimento, se comparó el método ivDMi2 usando crisoles (método de referencia) con una modificación del mismo usando bolsas ANKOM digeridas individualmente o en colectivo para simplificar la determinación de la FDSivMSi2. La FDSivMSi2 no difirió entre los métodos comparados. En un tercer experimento, se analizó la posible interferencia entre la determinación de la fibra soluble y las mucinas intestinales. Se observó un contenido de FDT y de mucinas elevado en las muestras de pectinas de remolacha (994 y 709 g/kg MS), así como en el moco intestinal de conejo (571 y 739 g/kg MS) cuando se aplicó el método de mucinas por precipitación con etanol. Sin embargo, después de aplicar una pectinasa en el material precipitado, la cantidad de mucinas recuperadas en las muestras de pectinas de remolacha fue cercana a cero, mientras que en el moco intestinal fue similar a los resultados previos al uso de la enzima. Con los resultados de este ensayo se estimaron los carbohidratos de mucinas retenidos en los contenidos digestivos y se propuso una corrección para la determinación de la digestibilidad de la FDT y fibra soluble. En conclusión, la contaminación de las mucinas de la digesta con fibra soluble se soluciona usando pectinasas. El segundo estudio se centró en estudiar: 1) el efecto del tipo de fibra, 2) el sitio de fermentación, 3) el método para cuantificar fibra y 4) la corrección por mucinas sobre la digestibilidad de la fibra. Para ello se formularon tres piensos con diferentes niveles de fibra soluble (FDT-aFNDmo-pb). Un pienso bajo en fibra soluble (LSF. 85 g/kg DM), un pienso medio en fibra soluble (MSF. 102 g/kg DM), y un pienso alto en fibra soluble (HSF. 145 g/kg DM). Estos piensos se obtuvieron reemplazando un 50% del heno del alfalfa en el pienso MSF por una mezcla de pulpa de manzana y remolacha (HSF) o por una mezcla de cascarilla de avena y proteína de soja (LSF). Se utilizaron 30 conejas canuladas para determinar la digestibilidad ileal y fecal. La digestibilidad cecal se calculó mediante diferencia entre la digestibilidad fecal e ileal. La fibra insoluble se determinó como aFNDmo-pb, IDF, e ivMSi2, mientras que la fibra soluble se calculó como FDSFDI, FDSaFNDmo-pb, y FDSivMSi2. La digestibilidad de la FDT y la fibra soluble se corrigieron por las mucinas. La concentración de mucinas en la digesta ileal y fecal, aumento desde el grupo LSF hasta el grupo con el pienso HSF (P < 0,01). La corrección por mucinas aumentó las digestibilidades de la FDT y la fibra soluble a nivel ileal, mientras que a nivel cecal las redujo. (P < 0.01). El coeficiente de digestibilidad ileal de FDT aumentó desde el grupo LSF al grupo HSF (0,12 vs. 0,281. P < 0,01), sin diferencias en el coeficiente de digestibilidad cecal (0,264), por lo que la tendencia a nivel fecal entre los grupos se mantuvo. El coeficiente de digestibilidad ileal de la fibra insoluble aumento desde el grupo con el pienso LSF al grupo con el pienso HSF (0,113 vs. 0,210. P < 0,01), sin diferencias a nivel cecal (0,139) y sin efecto del método usado, resultando en una digestibilidad elevada a nivel fecal, con tendencias similares a las observadas a nivel ileal. El coeficiente de digestibilidad de la FND fue elevada en comparación con la FDI o la ivMSi2 (P > 0.01). El coeficiente de la digestibilidad ileal de la fibra soluble fue mayor en el grupo LSF respecto al grupo LSF (0,436 vs. 0,145. P < 0,01) y el método no afectó a esta determinación. El coeficiente de la digestibilidad cecal de la fibra soluble se redujo desde el grupo LSF hasta el grupo HSF (0,721 vs. 0,492. P < 0,05). El valor más bajo de digestibilidad cecal y fecal de fibra soluble fue medido con el método FDSaFNDmo-pb (P < 0,01). Se observó una alta correlación entre las digestibilidades de la fibra soluble determinada como FDSFDI, FDSaFNDmo-pb, y FDSivMSi2, por lo tanto la información proporcionada por una u otra metodología fueron similares. Sin embargo, cuando se compararon con efectos fisiológicos (producción de mucinas y peso del ciego y pH del ciego de un trabajo previo), la FDSaFNDmo-pb globalmente mostró estar mejor correlacionado con estos parámetros fisiológicos. En conclusión, la corrección por mucinas es necesaria para determinar la digestibilidad ileal de la FDT y fibra soluble, mientras que la elección de uno u otro método es menos relevante. La inclusión de pulpa de manzana y remolacha incrementa la cantidad de FDT que desaparece antes de llegar al ciego. En el tercer estudio se estudió el efecto de la fracción fibrosa soluble e insoluble de la pulpa de remolacha y el método de cuantificación de la fibra soluble e insoluble sobre la digestibilidad de la fibra y algunos parámetros digestivos. Para ello se formularon cuatro piensos con niveles similares de fibra insoluble (315g aFNDmo-pb/kg MS) y proteína (167 g/kg MS). El pienso control contuvo el nivel más bajo de fibra soluble (30,3 g/kg, con cascarilla de girasol y paja como fuente de fibra). Un segundo pienso se obtuvo mediante la sustitución de 60 g de almidón/kg del pienso control por pectinas de remolacha (82,9 g fibra soluble/kg MS). Los otras dos piensos resultaron de la sustitución parcial de las fuentes de fibra del pienso control por la fracción insoluble de la pulpa de remolacha y la pulpa de remolacha entera (42.2 y 82.3 g fibra soluble/kg MS, respectivamente). Cincuenta y seis conejos en cebo (14/pienso), de 2,4  0.21 kg de peso, fueron usados para determinar la digestibilidad ileal y fecal de la FDT, FDI, aFNDmo-pb, FDSFDI, y FDSaFNDmo-pb. La concentración de mucinas en el íleon y heces se utilizaron para corregir la digestibilidad de la FDT y fibra soluble. También se midió el peso de diferentes segmentos del tracto digestivo y el pH del contenido digestivo. Los conejos alimentados con el pienso de fibra insoluble de pulpa de remolacha mostraron los consumos más bajos con respecto a los demás grupos (124 vs. 139 g/d, respectivamente. P < 0,05). El flujo de mucinas ileales fue más alto (P < 0.05) en el grupo alimentado con el pienso de pectinas de remolacha (9,0 g/d en promedio) que los del grupo control (4,79 g/d), mostrando los otros dos grupos valores intermedios, sin detectarse diferencias a nivel fecal. La digestibilidad ileal de la FDT (corregida por mucinas) y la fibra insoluble no se vieron afectadas por el tipo de pienso. El método usado para determinar la fibra insoluble afectó su digestibilidad ileal (0,123 para FDI vs. 0,108 para aFNDmo-pb. P < 0.01). De todas formas, los métodos no afectaron al cálculo de la fibra fermentada antes del ciego (4,9 g/d en promedio). Los conejos alimentados con el pienso de pulpa de remolacha y con el pienso con la fracción insoluble de la pulpa de remolacha mostraron las digestibilidades fecales más altas de la fibra insoluble (0,266 en promedio vs. 0,106 del grupo control), mientras que en los animales del pienso con pectinas esta digestibilidad fue un 47% mayor respecto al pienso control (P < 0,001). La digestibilidad fecal de la fibra insoluble fue un 20% más alta cuando se usó la FND en lugar de FDI para determinarla (P < 0.001). Esto hizo variar la cantidad de fibra insoluble fermentada a lo largo del tracto digestivo (9,5 ó 7,5 g/d cuando fue calculada como FDI o aFNDmo-pb, respectivamente. P < 0,001). Las digestibilidades ileales de la fibra soluble fueron positivas cuando los análisis de fibra soluble de los contenidos ileales fueron corregidos por mucinas, (P < 0,001) excepto para la digestibilidad ileal de la FDSIDF del grupo control. Una vez corregidas por mucinas, los conejos alimentados con los piensos que contuvieron la fracción soluble de la pulpa de remolacha (pienso de pectina y pulpa de remolacha) mostraron una mayor digestibilidad ileal de la fibra soluble, respecto al grupo control (0,483 vs. -0,010. P = 0.002), mientras que el grupo del pienso de fibra insoluble de pulpa de remolacha mostró un valor intermedio (0,274). La digestibilidad total de la fibra soluble fue similar entre todos los grupos (0.93). Los conejos alimentados con pulpa de remolacha y su fracción insoluble mostraron los pesos relativos más altos del estómago respecto a los del pienso control y de pectinas (11 y 56 % respectivamente; P < 0,05). Por otra parte, el peso relativo del ciego aumentó en los animales que consumieron tanto la fracción soluble como insoluble de la pulpa de remolacha, siendo un 16% más pesados (P < 0,001) que el grupo control. El pH del contenido cecal fue más bajo en los animales del grupo de pulpa de remolacha que en los del grupo control (5,64 vs. 6,03; P < 0,001), mientras que los del grupo de pectinas y de fibra insoluble de pulpa de remolacha mostraron valores intermedios. En conclusión, el efecto positivo de la pulpa de remolacha en el flujo de mucinas a nivel ileal se debe a la fracción soluble e insoluble de la pulpa de remolacha. La mitad de la fibra soluble de la pulpa de remolacha desaparece antes de llegar al ciego, independientemente si esta proviene de pectinas puras o de la pulpa de remolacha. El pH cecal esta mejor correlacionado con la cantidad de FDT que desaparece antes del ciego más que con la que se degrada en el ciego. En el último estudio se estudiaron los efectos de la fibra soluble e insoluble de la pulpa de manzana sobre la digestibilidad de la fibra y algunos parámetros digestivos. Cuatro dietas fueron formuladas con niveles similares de fibra insoluble (aFNDmo-pb 32,4%) y proteína (18,6% ambos en base seca). El pienso control contuvo el nivel más bajo de fibra soluble (46 g de fibra soluble/kg, con cascarilla de girasol y paja de cereales como la fuentes de fibra). Un segundo pienso fue obtenido mediante la sustitución de 60 g de almidón/kg del pienso control por pectinas de manzana (105 g fibra soluble/kg). Los otros dos piensos se obtuvieron por la substitución de parte de las fuentes de fibra del pienso control por pulpa de manzana o pulpa de manzana despectinizada (93 y 71 g de fibra soluble/kg, respectivamente). La digestibilidad fecal fue determinada en 23 conejos/pienso con 1.68 ± 0.23 kg de peso vivo, los cuales fueron sacrificados a los 60 d edad para recolectar su contenido digestivo para determinar digestibilidad ileal y otros parámetros digestivos. La fibra soluble de manzana (pectinas y pulpa entera) estimuló el flujo ileal de mucinas (P = 0,002), pero no asi la pulpa despectinizada. La corrección por mucinas incrementó la digestibilidad de la FDT y la fibra soluble a nivel fecal, y especialmente a nivel ileal. Cerca de la mitad de la fibra soluble proveniente de los piensos con cualquiera de las fracciones de la pulpa de manzana fue degradada a nivel ileal, sin mostrar diferencias entre los grupos (46 y 86% en promedio a nivel ileal y fecal respectivamente). La inclusión de pulpa despectinizada de manzana mejoró la digestibilidad de la FND a nivel fecal (P < 0,05) pero no a nivel ileal. El contenido cecal de los conejos alimentados con la pulpa de manzana tuvieron el pH cecal más ácido que los del pienso control (5,55 vs. 5,95. P < 0,001), mientras que los animales con el pienso de pectinas de manzana y de pulpa de manzana despectinizada mostraron valores intermedios. En conclusión los efectos positivo de la pulpa de manzana en el flujo de mucinas se debió principalmente a la fracción soluble de la pulpa de manzana. La mitad de la fibra soluble fue degradada antes del ciego independientemente de si esta provino de las pectinas o de la pulpa de manzana. El pH cecal estuvo mejor correlacionado con la cantidad de FDT fermentada en todo el tracto digestivo y antes de llegar al ciego que con la que se degradó en el ciego. Al integrar los resultados de los estudio 2, 3 y 4 se concluyó que la corrección de mucinas de los contenidos digestivos al determinar FDT y fibra soluble es necesaria para ajustar los cálculos de su digestibilidad. Esta corrección es mucho más importante a nivel ileal y en dietas bajas en fibra soluble. Por otra parte, la FDT desapareció en proporciones importantes antes de llegar al ciego, especialmente en piensos que contienen pulpa de remolacha o de manzana o alguna fracción soluble o insoluble de las mismas y estas diferencias observadas entre los piensos a nivel ileal se correlacionaron mejor con el pH cecal, lo que indicaría que la FDT se solubilizó antes de llegar al ciego y una vez en esté fermentó. Estos resultados implican que determinar la fibra soluble como FDSaFNDmo-pb es la mejor opción y que en la determinación de la digestibilidad de la FDT y fibra soluble se debe considerar la corrección por mucinas especialmente a nivel ileal y en piensos bajos en fibra soluble. ABSTRACT The present thesis constitutes a step forward in advancing the knowledge of the methods to quantify soluble fibre and the effects of the fibre fractions and source of fibre on the site the digestion of different fractions of fibre (soluble and insoluble) in the rabbit. There is a positive effect of soluble fibre on rabbit digestive health and therefore on the reduction of mortality in weaning rabbits. Nevertheless, it is no so clear that the effects of soluble fibre on rabbits are due particularly to this fraction. This thesis aims: 1) to compare the quantification of soluble fibre in feeds using different chemical and in vitro approaches, and to study the potential interference between soluble fibre and mucin determinations, 2) to identify the effects of type of fibre, site of fermentation, method to quantify insoluble and soluble fibre, and correction of the intestinal soluble fibre content for intestinal mucin on the digestibility of fibre fractions and 3) to evaluate the individual effect of soluble and insoluble fibre from sugar beet pulp and apple pulp on ileal and faecal soluble and insoluble digestibility and digestive traits. These objectives were developed in four studies: The first study compared the quantification of soluble fibre in feeds using different chemical and in vitro approaches, and studied the potential interference between soluble fibre and mucin determinations. Six ingredients, sugar beet pulp (SBP), SBP pectins, insoluble SBP, wheat straw, sunflower hulls and lignocellulose, and seven rabbit diets, differing in soluble fibre content, were evaluated. In experiment 1, ingredients and diets were analysed for total dietary fibre (TDF), insoluble dietary fibre (IDF), soluble dietary fibre (SDF), aNDFom (corrected for protein, aNDFom-cp) and 2-step pepsin/pancreatin in vitro DM indigestibility (corrected for ash and protein, ivDMi2). Soluble fibre was estimated by difference using three procedures: TDF - IDF (SDFIDF), TDF - ivDMi2 (SDFivDMi2), and TDF - aNDFom-cp (SDFaNDFom-cp). Soluble fibre determined directly (SDF) or by difference, as SDFivDMi2 were not different (109 g/kg DM, on average). However, when it was calculated as SDFaNDFom-cp the value was 40% higher (153 g/kg DM, P < 0.05), whereas SDFIDF (124 g/kg DM) did not differ from any of the other methods. The correlation between the four methods was high (r ≥ 0.96. P ≤ 0.001. n = 13), but it decreased or even disappeared when SBP pectins and SBP were excluded and a lower and more narrow range of variation of soluble fibre was used. In experiment 2, the ivDMi2 using crucibles (reference method) were compared to those made using individual or collective ankom bags in order to simplify the determination of SDFivDMi2. The ivDMi2 was not different when using crucibles or individual or collective ankom bags. In experiment 3, the potential interference between soluble fibre and intestinal mucin determinations was studied using rabbit intestinal raw mucus, digesta and SBP pectins, lignocelluloses and a rabbit diet. An interference was observed between the determinations of soluble fibre and crude mucin, as the content of TDF and apparent crude mucin were high in SBP pectins (994 and 709 g/kg DM) and rabbit intestinal raw mucus (571 and 739 g/kg DM). After a pectinase treatment, the coefficient of apparent mucin recovery of SBP pectins was close to zero, whereas that of rabbit mucus was not modified. An estimation of the crude mucin carbohydrates retained in digesta TDF is proposed to correct TDF and soluble fibre digestibility. In conclusion, the values of soluble fibre depend on the methodology used. The contamination of crude mucin with soluble fibre is avoided using pectinase. The second study focused on the effect of type of fibre, site of fermentation, method for quantifying insoluble and soluble dietary fibre, and their correction for intestinal mucin on fibre digestibility. Three diets differing in soluble fibre were formulated (85 g/kg DM soluble fibre, in the low soluble fibre [LSF] diet; 102 g/kg DM in the medium soluble fibre [MSF] diet; and 145 g/kg DM in the high soluble fibre [HSF] diet). They were obtained by replacing half of the dehydrated alfalfa in the MSF diet with a mixture of beet and apple pulp (HSF diet) or with a mix of oat hulls and soybean protein (LSF diet). Thirty rabbits with ileal T-cannulas were used to determine total tract apparent digestibility (CTTAD) and ileal apparent digestibility (CIAD). Caecal digestibility was determined by difference between CTTAD and CIAD. Insoluble fibre was measured as aNDFom-cp, IDF, and ivDMi2, whereas soluble fibre was calculated as SDFaNDFom-cp, SDFIDF, SDFivDMi2. The intestinal mucin content was used to correct the TDF and soluble fibre digestibility. Ileal and faecal concentration of mucin increased from the LSF to the HSF diet group (P < 0.01). Once corrected for intestinal mucin, The CTTAD and CIAD of TDF and soluble fibre increased whereas caecal digestibility decreased (P < 0.01). The CIAD of TDF increased from the LSF to the HSF diet group (0.12 vs. 0.281. P < 0.01), with no difference in the caecal digestibility (0.264), resulting in a higher CTTAD from the LSF to the HSF diet group (P < 0.01). The CIAD of insoluble fibre increased from the LSF to the HSF diet group (0.113 vs. 0.21. P < 0.01), with no difference in the caecal digestibility (0.139) and no effect of fibre method, resulting in a higher CTTAD for rabbits fed the HSF diet compared with the MSF and LSF diets groups (P < 0.01). The CTTAD of aNDFom-cp was higher compared with IDF or ivDMi2 (P < 0.01). The CIAD of soluble fibre was higher for the HSF than for the LSF diet group (0.436 vs. 0.145. P < 0.01) and fibre method did not affect it. Caecal soluble fibre digestibility decreased from the LSF to the HSF diet group (0.721 vs. 0.492. P < 0.05). The lowest caecal and faecal soluble fibre digestibility was measured using SDFaNDFom-cp (P < 0.01). There was a high correlation among the digestibilities of soluble fibre measured as SDFaNDFom-cp, SDFIDF, and SDFivDMi2. Therefore, these methodologies provide similar information. However, the method that seems to be globally better related to the physiological traits (ileal flow of mucins, and relative weight of the caecum and caecal pH from previous work) was the SDFaNDFom-cp. In conclusion, a correction for intestinal mucin is necessary for ileal TDF and soluble fibre digestibility whereas the selection of the fibre method has a minor relevance. The inclusion of sugar beet and apple pulp increased the amount of TDF fermented in the small intestine. The third study examined the effect of fibre fractions of sugar beet pulp (SBP) and the method for quantifying soluble and insoluble fibre on soluble and insoluble fibre digestibility and digestive traits. Four diets were formulated with similar level of insoluble fibre (aNDFom-cp: 315 g/kg DM) and protein (167 g/kg DM). Control diet contained the lowest level of soluble fibre (30.3 g/kg DM, including sunflower hulls and straw as sole sources of fibre). A second diet was obtained by replacing 60 g starch/kg of control diet with SBP pectins (82.9 g soluble fibre/kg DM). Two more diets were obtained by replacing part of the fibrous sources of the control diet with either insoluble SBP fibre or SBP (42.2 and 82.3 g soluble fibre/kg DM, respectively). Fifty six (14/diet) rabbits weighing 2.40  0.213 kg were used to determine faecal and ileal digestibility of total dietary fibre (TDF), insoluble dietary fibre (IDF), neutral detergent fibre corrected for ash and CP (aNDFom-cp) and soluble fibre estimated as SDFaNDFom-cp and SDFIDF. Faecal and ileal mucin content was used to correct TDF and soluble fibre digestibility. It was also recorded weight of digestive segments and digesta pH. Rabbits fed insoluble SBP showed the lowest feed intake with respect to the other 3 diets (124 vs. 139 g/d, respectively. P < 0.05). Ileal mucin flow was higher (P < 0.05) in animals fed pectin and SBP diets (9.0 g/d, as average) than those fed control diet (4.79 g/d), showing InsSBP group an intermediate value. No differences on mucin content were detected at faecal level. There was no diet effect on the CIAD of TDF (corrected for mucin) and insoluble fibre. Fibre methodology influenced the CIAD of insoluble fibre (0.123 for IDF vs. 0.108 for aNDFom-cp. P < 0.01). Anyway, the amount of insoluble fibre fermented before the caecum did not differ between both methods (4.9 g/d, on average). Rabbits fed insoluble SBP and SBP diets showed the highest CTTAD of insoluble fibre (0.266 on average vs. 0.106 for control group), whereas those fed pectin diet had an intermediate value (0.106. P < 0.001). The CTTAD of insoluble fibre measured with IDF was higher than that measured with aNDFom-cp (by 20%. P < 0.001). It led that the amount of insoluble fibre fermented along the digestive tract were different (9.5 or 7.5 g/d when calculated as IDF or aNDFom-cp, respectively; P < 0.001). When the CIAD of soluble fibre was corrected for mucin they became positive (P < 0.001) except for control group measured as SDFIDF. Once corrected for mucin content, rabbits fed soluble fibre from SBP (pectin and SBP groups) showed higher CIAD of soluble fibre than control group (0.483 vs. -0.019. respectively), whereas the value for insoluble SBP group was intermediate 0.274. The CTTAD of soluble fibre (mucin corrected) was similar among diets 0.93. Rabbits fed with SBP and insoluble SBP diets showed higher total digestive tract and stomach relative weight than those fed pectin and control diets (by 11 and 56 %. respectively, P < 0.05). The caecal relative weight did not differ in rabbits fed pectin, insoluble SBP, and SBP diets (62 g/kg BW, as average) and they were on average 16% higher (P < 0.001) than in control group. Caecal content of rabbits fed SBP diet was more acid than those fed control diet (5.64 vs. 6.03. P < 0.001), whereas those from pectin and insoluble SBP diets showed intermediate values. In conclusion, the positive effect of SBP fibre on ileal mucin flow was due to both its soluble and insoluble fibre fraction. Half of the soluble SBP fibre was degraded before the caecum independently it came from pectin or SBP. The caecal pH correlated better with the ileal amount of fermented TDF in the digestive tract rather than with that fermented in the caecum. The last study examined the effect of soluble and insoluble fibre of apple pulp on fibre digestibility and digestive traits. Four diets were formulated with similar level of insoluble fibre (aNDFom-cp: 324 g/kg DM) and protein (18.6 g/kg DM). Control diet contained the lowest level of soluble fibre (46 g soluble fibre/kg DM, including oat hulls and straw as sole sources of fibre). A second diet was obtained by replacing 60 g starch/kg of control diet with apple pectins (105 g soluble fibre/kg DM). Two more diets were obtained by substituting part of the fibrous sources of the control diet by either apple pulp or depectinized apple pulp (93 and 71 g soluble fibre/kg, respectively). The CTTAD was determined in 23 rabbits/diet weighing 1.68  0.23 kg BW, and 23 rabbits/diet were slaughtered at 60 d of age to collect ileal digesta to determine CIAD and record other digestive traits. Soluble fibre from apple stimulated ileal flow of mucin (P = 0.002), but depectinized apple pulp did not. The correction for mucin increased the digestibility of crude protein, total dietary fibre, and soluble fibre at faecal, but especially at ileal level, depending in this case on the diet. Around half of the soluble fibre in diets containing any fibre fraction from apple was degraded at ileal level, with no differences among these diets (0.46 vs. 0.066 for control group, P=0.046). Faecal soluble fibre digestibility was 0.86 on average for all groups). Inclusion of the apple insoluble fibre improved NDF digestibility at faecal (0.222 vs. 0.069. P < 0.05) but not at ileal level. Caecal content of rabbits fed apple pulp diet was more acid than those fed control diet (5.55 vs. 5.95. P < 0.001), whereas those from pectin and depectinised apple pulp diets showed intermediate values. In conclusion, the positive effect of apple fibre on ileal mucin flow was mainly due to its soluble fibre fraction. Half of the soluble apple fibre was degraded before the caecum independently it came from pectin or apple pulp. The caecal pH correlated better with the total and ileal amount of fermented TDF in the digestive tract rather than with that fermented in the caecum. The results obtained in the studies 2, 3 and 4 were considered together. These results showed that the mucin correction is necessary when the TDF and soluble fibre digestibility is determined, and it correction is more important at ileal level and in diets with low level of soluble fibre. On another hand, incrementing the soluble fibre using sugar beet and apple pulp increased the amount of TDF disappear before the caecum. Moreover, the caecal pH correlated better with the ileal amount of fermented TDF in the digestive tract rather than with that fermented in the caecum. This suggests that an ileal fibre solubilisation may occur rather than ileal fermentation. Therefore the implications of this work were that: the estimation of soluble fibre as SDFaNDFom-cp is an adequate method considering its correlation with the physiological effects; and the TDF and soluble fibre digestibility must be corrected with intestinal mucins, especially when the ileal digestibility is determined.

Taxonomía y caracterización del arbusto forrajero Atriplex halimus L.

El objetivo de esta Tesis Doctoral ha sido la revisión de la taxonomía infraespecífica de la especie Atriplex halimus L., la delimitación del área geográfica que ocupan sus taxones y su caracterización morfológica. En base a las citas bibliográficas y material de herbario consultado se ha trazado un mapa de distribución de la especie, que delimita su distribución como planta espontánea a la Cuenca Mediterránea, desierto de Siria, Macizo de Hoggar y Sahara Occidental. El resto debe considerarse poblaciones naturalizadas o cultivadas. La taxonomía actual, basada en relaciones filogenéticas, nos indica que lo adecuado es separar las poblaciones de A. halimus en dos clados que se caracterizan por tener diferente nivel de ploidía. Para definir el área de distribución de los clados diploide y tetraploide se ha determinado la ploidía de 19 poblaciones en el espacio comprendido entre las dos áreas de distribución conocidas. Y como resultado el área de distribución de ambos clados puede definirse trazando una recta que une el estrecho de Gibraltar con Estambul, las poblaciones al sur de esta línea son tetraploides a las que habría que añadir las poblaciones del este y sur de la isla de Cerdeña que son también tetraploides. Se han determinado cuales son los parámetros morfológicos que permiten caracterizar ambos clados, para lo cual se han estudiado 52 poblaciones que comprenden toda el área de distribución de la especie. Se han evaluado caracteres cualitativos y cuantitativos de valvas fructíferas, plántulas cultivadas en cámara y arbustos de 2 a 4 años situados en la misma parcela. El único elemento fiable para determinar la pertenencia al clado diploide o tetraploide es la medición del nivel de ploidía. Ninguno de los caracteres morfológicos estudiados es plenamente fiable para determinar si los individuos de una población pertenecen al clado diploide o tetraploide a pesar de realizarse las mediciones sobre poblaciones cultivadas en igualdad de condiciones de suelo y clima, y en condiciones muy favorables para la expresión de caracteres cualitativos y cuantitativos. Los caracteres cualitativos observados que guardan una mayor correlación con el nivel de ploidía son: la base de las hojas de las plántulas, el porte del arbusto, la ramificación, el ángulo de inserción de las ramas y la longitud de la inflorescencia. Respecto a los datos cuantitativos los más relevantes han sido, la longitud de los cotiledones y la longitud del limbo de la 1ª, 3ª o 5ª hoja de la plántula o la razón entre este valor y la anchura del limbo. ABSTRACT The aim of this Thesis has been in the revision of the infraspecies taxa of Atriplex halimus L., the delimitation of their natural growing area and their morphological characterization. Using references to relevant literature and consultation of herbariums, it has been possible to map the distribution of the species as spontaneous plant in the Mediterranean Basin, Desert of Syria, Massif of Hoggar and Western Sahara. The remaining populations must be considered as naturalized or cultivated. The current taxonomy, based on phylogenetic relationships, indicates that A.halimus should be divided in two clades. These clades are characterized by their different ploidy level. Samples of nineteen populations collected from localizations between the defined distribution area of both clades have been characterized for their ploidy level. As a result, it can be seen that the line of clade separation can be trazed from Istambul tothe Strait of Gibraltar, where the populations north to this line are classified as diploid and those south of the line are clsassified as tetraploid. The only exception of this rule is in Sardinia, hwre populations to the West and South of the Island are also classified as tetraploid. The morphological parameters, which include the characterization of both clades, have been defined, and the study of fifty-two populations from the entire distribution area according to these parameters have been analysed. The qualitative and quantitative characteristics of the fruit valves have been evaluated, with seedlings grown in a culture chamber, and 2-4 years old shrubs grown in the same orchard. None of the morphological characters under study are fully reliable to determine whether individuals in a population belong to clade diploid or tetraploid. This is despite measurements being made on populations grown under the same conditions of soil and climate, and in very favorable conditions for the expression of qualitative and quantitative traits. The qualitative characteristics that show a higher correlation with the ploidy level are: the base of the leaves of the seedlings, the bearing bush, the branch arrangement, the angle of insertion of the branches and the inflorescence length. With regards to the quantitative data the most relevant were the length of the cotyledons and the length of the blade of the 1st, 3rd or 5th leaves of the seedling, and the ratio of any of this values and the leaf blade width. The only reliable way to determine the diploid or tetraploid clade membership is the measurement of ploidy level.

Effect of type of fiber, site of fermentation, and method of analysis on digestibility of soluble and insoluble fiber in rabbits

The effect of type of fiber, site of fermetation, method for quantifying insoluble and soluble dietary fiber, and their correction for intestinal mucin on fiber digestibility were examined in rabbits. Three diets differing in soluble fiber were formulated (8.5% soluble fiber, on DM basis, in the low soluble fiber [LSF] diet; 10.2% in the medium soluble fiber [MSF] diet; and 14.5% in the high soluble fiber [HSF] diet). They were obtained by replacing half of the dehydrated alfalfa in the MSF diet with a mixture of beet and apple pulp (HSF diet) or with a mix of oat hulls and soybean protein (LSF diet). Thirty rabbits with ileal T-cannulas were used to determine ileal and fecal digestibility. Cecal digestibility was determined by difference between fecal and ileal digestibility. Insoluble fiber was measured as NDF, insoluble dietary fiber (IDF), and in vitro insoluble fiber, whereas soluble fiber was calculated as the difference between total dietary fiber (TDF) and NDF (TDF_NDF), IDF (TDF-IDF), and in vitro insoluble fiber (TDF-in vitro insoluble fiber). The intestinal mucin content was used to correct the TDF and soluble fiber digestibility. Ileal and fecal concentration of mucin increased from the LSF to the HSF diet group (P < 0.01). Once corrected for intestinal mucin, ileal and fecal digestibility of TDF and soluble fiber increased whereas cecal digestibility decreased (P < 0.01). Ileal digestibility of TDF increased from the LSF to the HSF diet group (12.0 vs. 28.1%; P < 0.01), with no difference in the cecum (26.4%), resulting in a higher fecal digestibility from the LSF to the HSF diet group (P < 0.01). Ileal digestibility of insoluble fiber increased from the LSF to the HSF diet group (11.3 vs. 21.0%; P < 0.01), with no difference in the cecum (13.9%) and no effect of fiber method, resulting in a higher fecal digestibility for rabbits fed the HSF diet compared with the MSF and LSF diets groups (P < 0.01).Fecal digestibility of NDF was higher compared with IDF or in vitro insoluble fiber (P < 0.01). Ileal soluble fiber digestibility was higher for the HSF than for the LSF diet group (43.6 vs. 14.5%; P < 0.01) and fiber method did not affect it. Cecal soluble fiber digestibility decreased from the LSF to the HSF diet group (72.1 vs. 49.2%; P < 0.05). The lowest cecal and fecal soluble fiber digestibility was measured using TDF-NDF (P < 0.01). In conclusion, a correction for intestinal mucin is necessary for ileal TDF and soluble fiber digestibility whereas the selection of the fiber method has a minor relevance. The inclusion of sugar beet and apple pulp increased the amount of TDF fermented in the small intestine.

Comparison of fermentation characteristics and bacterial diversity in the rumen of sheep and batch cultures of rumen microorganisms

The objective of the current study was to assess how closely batch cultures (BC) of rumen microorganisms can mimic the dietary differences in fermentation characteristics found in the rumen, and to analyse changes in bacterial diversity over the in vitro incubation period. Four ruminally and duodenally cannulated sheep were fed four diets having forage : concentrate ratios (FCR) of 70 : 30 or 30 : 70, with either alfalfa hay or grass hay as forage. Rumen fluid from each sheep was used to inoculate BC containing the same diet fed to the donor sheep, and the main rumen fermentation parameters were determined after 24 h of incubation. There were differences between BC and sheep in the magnitude of most measured parameters, but BC detected differences among diets due to forage type similar to those found in sheep. In contrast, BC did not reproduce the dietary differences due to FCR found in sheep for pH, degradability of neutral detergent fibre and total volatile fatty acid (VFA) concentrations. There were differences between systems in the magnitude of most determined parameters and BC showed higher pH values and NH3–N concentrations, but lower fibre degradability and VFA and lactate concentrations compared with sheep. There were significant relationships between in vivo and in vitro values for molar proportions of acetate, propionate and butyrate, and the acetate : propionate ratio. The automated ribosomal intergenic spacer analysis (ARISA) of 16S ribosomal deoxyribonucleic acid showed that FCR had no effect on bacterial diversity either in the sheep rumen fluid used as inoculum (IN) or in BC samples. In contrast, bacterial diversity was greater with alfalfa hay diets than those with grass hay in the IN, but was unaffected by forage type in the BC. Similarity index between the bacterial communities in the inocula and those in the BC ranged from 67·2 to 74·7%, and was unaffected by diet characteristics. Bacterial diversity was lower in BC than in the inocula with 14 peaks out of a total of 181 detected in the ARISA electropherograms never appearing in BC samples, which suggests that incubation conditions in the BC may have caused a selection of some bacterial strains. However, each BC sample showed the highest similarity index with its corresponding rumen IN, which highlights the importance of using rumen fluid from donors fed a diet similar to that being incubated in BC when conducting in vitro experiments.

Environmental niche variation and evolutionary diversification of the Brachypodium distachyon grass complex species in their native circum-Mediterranean range

PREMISE OF THE STUDY: We conducted environmental niche modeling (ENM) of the Brachypodium distachyon s.l. complex, a model group of two diploid annual grasses ( B. distachyon , B. stacei ) and their derived allotetraploid ( B. hybridum) , native to the circum-Mediterranean region. We (1) investigated the ENMs of the three species in their native range based on present and past climate data; (2) identifi ed potential overlapping niches of the diploids and their hybrid across four Quaternary windows; (3) tested whether speciation was associated with niche divergence/conservatism in the complex species; and (4) tested for the potential of the polyploid outperforming the diploids in the native range. M ETHODS: Geo-referenced data, altitude, and 19 climatic variables were used to construct the ENMs. We used paleoclimate niche models to trace the potential existence of ancestral gene fl ow among the hybridizing species of the complex. KEY RESULTS: Brachypodium distachyon grows in higher, cooler, and wetter places, B. stacei in lower, warmer, and drier places, and B. hybridum in places with intermediate climatic features. Brachypodium hybridum had the largest niche overlap with its parent niches, but a similar distribution range and niche breadth. C ONCLUSIONS: Each species had a unique environmental niche though there were multiple niche overlapping areas for the diploids across time, suggesting the potential existence of several hybrid zones during the Pleistocene and the Holocene. No evidence of niche divergence was found, suggesting that species diversifi cation was not driven by ecological speciation but by evolutionary history, though it could be associated to distinct environmental adaptations.

Análisis de las poblaciones microbianas en fermentadores de flujo continuo: efecto de modificacioens técnicas para la retención de protozoos en el sistema

The objective of this work was to study the effect of two technical modifications (supplemented with sponge materials (ES) and provided with a filter system (FIL))in continuous-culture fermenters on the microbial populations and ruminal fermentation parameters over the sampling period. Six fermenters fed a 50:50 alfalfa hay: concentrate diet, inoculated with rumen liquor from sheep fed the same diet, were used in two incubation runs of 14 days each. On days 10 and 14, samples were taken for analysis of fermentation parameters (volatile fatty acids, ammonia-N and lactate) and microbial populations. None of the technical modification affected (P>0.05) concentrations of bacterial DNA and the relative abundance of fungi and archaea, but protozoal DNA concentrations were higher (P>0.05) in ES and FIL fermenters than in the control ones. However, values of protozoal DNA were about 50 times lower than in the rumen fluid used as inoculum for the ermenters. The tested technical modifications did not affect (P>0.05) any fermentation parameter, and there were no differences in fermentation parameters between days 10 and 14, with the exception of lactate production which was higher (P=0.009) on day 14 than on day 10. In conclusion, the technical modifications tested maintained protozoa in continuous culture fermenters without any effect on fermentation parameters and other microbial populations, but protozoa concentrations were still lower than those in the rumen.

Influencia del tipo de filtrado y tratamiento con Stomacher del fluido ruminal de ovejas en las poblaciones microbianas del inóculo

Four rumen-fistulated sheep fed a 66:34 alfalfa hay:concentrate diet were used as donors to investigate the effect of rumen contents’ treatment on microbial populations in the resulting fluid. Rumen contents were sampled from each individual sheep and subjected to the following treatments: SQ: squeezed through 4 layers of cheesecloth; FIL: SQ treatment and further filtration through a 100-μm nylon cloth; STO: reated with a Stomacher® for 3 min at 230 rev min-1 and followed by SQ. Microbial populations in the fluid were analysed by real-time PCR and bacterial diversity was assessed by the automated ribosomal intergenic spacer analysis (ARISA) of the 16S ribosomal DNA. Bacterial DNA concentrations and relative abundance of Ruminococcus flavefaciens, arqueal and fungal DNA did not differ (P>0.05) between treatments. In contrast, STO treatment decreased (P<0.05) protozoal DNA concentrations and increased (P<0.05) the relative abundance of Fibrobacter succinogenes compared with SQ method. There were no differences (P>0.05) between treatments either in the Shannon index or in the number of peaks in the ARISA electropherograms, indicating no effect on bacterial diversity. Studies analyzing the influence on the tested methods on fermentation characteristics of different substrates when the fluid is used as inoculum is required.

Analysis of microbial populations in Rusitec fermenters fed diets of variable composition

Fermenters are widely used to study ruminal fermentation, but information on microbial populations developing in fermenters over the incubation period is limited. Four Rusitec fermenters were fed 2 diets representative of those administered to dairy sheep(DAI; 50:50 alfalfa hay:concentrate) and fattening lambs (FAT; 15:85 barley straw:concentrate) in a crossover design with 2 14-d incubation periods to assess the evolution of the microbial populations. There were 4 fermenters per diet.

Induction of ARF tumor suppressor gene expression and cell cycle arrest by transcription factor DMP1

Expression of the DMP1 transcription factor, a cyclin D-binding Myb-like protein, induces growth arrest in mouse embryo fibroblast strains but is devoid of antiproliferative activity in primary diploid fibroblasts that lack the ARF tumor suppressor gene. DMP1 binds to a single canonical recognition site in the ARF promoter to activate gene expression, and in turn, p19ARF synthesis causes p53-dependent cell cycle arrest. Unlike genes such as Myc, adenovirus E1A, and E2F-1, which, when overexpressed, activate the ARF-p53 pathway and trigger apoptosis, DMP1, like ARF itself, does not induce programmed cell death. Therefore, apart from its recently recognized role in protecting cells from potentially oncogenic signals, ARF can be induced in response to antiproliferative stimuli that do not obligatorily lead to apoptosis.

Polyploid formation created unique avenues for response to selection in Gossypium (cotton)

A detailed restriction fragment length polymorphism map was used to determine the chromosomal locations and subgenomic distributions of quantitative trait loci (QTLs) segregating in a cross between cultivars of allotetraploid (AADD) Gossypium hirsutum (“Upland” cotton) and Gossypium barbadense (“Sea Island,” “Pima,” or “Egyptian” cotton) that differ markedly in the quality and quantity of seed epidermal fibers. Most QTLs influencing fiber quality and yield are located on the “D” subgenome, derived from an ancestor that does not produce spinnable fibers. D subgenome QTLs may partly account for the fact that domestication and breeding of tetraploid cottons has resulted in fiber yield and quality levels superior to those achieved by parallel improvement of “A” genome diploid cottons. The merger of two genomes with different evolutionary histories in a common nucleus appears to offer unique avenues for phenotypic response to selection. This may partly compensate for reduction in quantitative variation associated with polyploid formation and be one basis for the prominence of polyploids among extant angiosperms. These findings impel molecular dissection of the roles of divergent subgenomes in quantitative inheritance in many other polyploids and further exploration of both “synthetic” polyploids and exotic diploid genotypes for agriculturally useful variation.

COS1, a two-component histidine kinase that is involved in hyphal development in the opportunistic pathogen Candida albicans

Two-component histidine kinases recently have been found in eukaryotic organisms including fungi, slime molds, and plants. We describe the identification of a gene, COS1, from the opportunistic pathogen Candida albicans by using a PCR-based screening strategy. The sequence of COS1 indicates that it encodes a homolog of the histidine kinase Nik-1 from the filamentous fungus Neurospora crassa. COS1 is also identical to a gene called CaNIK1 identified in C. albicans by low stringency hybridization using CaSLN1 as a probe [Nagahashi, S., Mio, T., Yamada-Okabe, T., Arisawa, M., Bussey, H. & Yamada-Okabe, H. (1998) Microbiol. 44, 425–432]. We assess the function of COS1/CaNIK1 by constructing a diploid deletion mutant. Mutants lacking both copies of COS1 appear normal when grown as yeast cells; however, they exhibit defective hyphal formation when placed on solid agar media, either in response to nutrient deprivation or serum. In constrast to the Δnik-1 mutant, the Δcos1/Δcos1 mutant does not demonstrate deleterious effects when grown in media of high osmolarity; however both Δnik-1 and Δcos1/Δcos1 mutants show defective hyphal formation. Thus, as predicted for Nik-1, Cos1p may be involved in some aspect of hyphal morphogenesis and may play a role in virulence properties of the organism.