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.

Productive performance of brown-egg laying pullets from hatching to 5 weeks of age as affected by fiber inclusion, feed form, and energy concentration of the diet

The effects of fiber inclusion, feed form, and energy concentration of the diet on the growth performance of pullets from hatching to 5 wk age were studied in 2 experiments. In Experiment 1, there was a control diet based on cereals and soybean meal, and 6 extra diets that included 2 or 4% of cereal straw, sugar beet pulp (SBP), or sunflower hulls (SFHs) at the expense (wt/wt) of the whole control diet. From hatching to 5 wk age fiber inclusion increased (P < 0.05) ADG and ADFI, and improved (P < 0.05) energy efficiency (EnE; kcal AMEn/g ADG), but body weight (BW) uniformity was not affected. Pullets fed SFH tended to have higher ADG than pullets fed SBP (P = 0.072) with pullets fed straw being intermediate. The feed conversion ratio (FCR) was better (P < 0.05) with 2% than with 4% fiber inclusion. In Experiment 2, 10 diets were arranged as a 2×5 factorial with 2 feed forms (mash vs. crumbles) and 5 levels of AMEn (2,850, 2,900, 2,950, 3,000, and 3,050 kcal/kg). Pullets fed crumbles were heavier and had better FCR than pullets fed mash (P < 0.001). An increase in the energy content of the crumble diets reduced ADFI and improved FCR linearly, but no effects were detected with the mash diets (P < 0.01 and P < 0.05 for the interactions). Feeding crumbles tended to improve BW uniformity at 5 wk age (P = 0.077) but no effects were detected with increases in energy concentration of the diet. In summary, the inclusion of moderate amounts of fiber in the diet improves pullet performance from hatching to 5 wk age. The response of pullets to increases in energy content of the diet depends on feed form with a decrease in feed intake when fed crumbles but no changes when fed mash. Feeding crumbles might be preferred to feeding mash in pullets from hatching to 5 wk age.

Feed form and energy concentration of the diet affect growth performance and digestive tract traits of brown-egg laying pullets from hatching to 17 weeks of age

The influence of feed form and energy concentration of the diet on growth performance and the development of the gastrointestinal tract (GIT) was studied in brown-egg laying pullets. Diets formed a 2 x 5 factorial with 2 feed forms (mash vs. crumbles) and 5 levels of energy differing in 50 kcal AMEn/kg. For the entire study (0 to 17 wk of age) feeding crumbles increased ADFI (52.9 vs. 49.7 g; P < 0.001) and ADG (12.7 vs. 11.6 g; P < 0.001) and improved feed conversion ratio (FCR; 4.18 vs. 4.27; P < 0.001). An increase in the energy content of the diet decreased ADFI linearly (P < 0.001) and improved FCR quadratically (P < 0.01) but energy intake (kcal AMEn/d) was not affected. BW uniformity was higher (P < 0.05) in pullets fed crumbles than in those fed mash but was not affected (P > 0.05) by energy content of the diet. At 5, 10, and 17 wk of age, the relative weight (RW, % BW) of the GIT and the gizzard, and gizzard digesta content were lower (P < 0.05 to P < 0.001) and gizzard pH was higher (P < 0.05 to P < 0.001) in pullets fed crumbles than in pullets fed mash. Energy concentration of the diet did not affect any of the GIT variables studied. In summary, feeding crumbles improved pullet performance and reduced the RW of the GIT and gizzard, and increased gizzard pH at all ages. An increase in the energy content of the diet improved FCR from 0 to 17 wk of age. The use of crumbles and the increase in the AMEn content of the diet might be used adventageously when the objetive is to increase the BW of the pullets. However, crumbles affected the development and weight of the organs of the GIT, which might have negative effects on feed intake and egg production at the beginning of the egg laying cycle.

Influence of the main cereal and feed form of the rearing phase diets on performance, digestive tract, and body traits of brown-egg laying pullets from hatch to 17 weeks of age

We hypothesize that pullets could respond similarly, independent of feed form, to the feeding of diets based on corn or wheat supplemented with adequate NSP enzymes. Also, pullets would quickly adapt their gastrointestinal tract and modify productive performance accordingly, when switched from crumbles to mash feeds. The aim of this research was to evaluate the effects of feeding crumbles for different periods of time, followed by feeding mash to 17 wk of age, on performance, gastrointestinal tract development, and body measurements of brown-egg laying pullets fed diets based on corn or wheat.

Productive performance of brown-egg laying pullets from hatching to 5 weeks of age as affected by fiber inclusion, feed form, and energy concentration of the diet1

The effects of fiber inclusion, feed form, and energy concentration of the diet on the growth performance of pullets from hatching to 5 wk age were studied in 2 experiments. In Experiment 1, there was a control diet based on cereals and soybean meal, and 6 extra diets that included 2 or 4% of cereal straw, sugar beet pulp (SBP), or sunflower hulls (SFHs) at the expense (wt/wt) of the whole control diet. From hatching to 5 wk age fiber inclusion increased (P < 0.05) ADG and ADFI, and improved (P < 0.05) energy efficiency (EnE; kcal AMEn/g ADG), but body weight (BW) uniformity was not affected. Pullets fed SFH tended to have higher ADG than pullets fed SBP (P = 0.072) with pullets fed straw being intermediate. The feed conversion ratio (FCR) was better (P < 0.05) with 2% than with 4% fiber inclusion. In Experiment 2, 10 diets were arranged as a 2×5 factorial with 2 feed forms (mash vs. crumbles) and 5 levels of AMEn (2,850, 2,900, 2,950, 3,000, and 3,050 kcal/kg). Pullets fed crumbles were heavier and had better FCR than pullets fed mash (P < 0.001). An increase in the energy content of the crumble diets reduced ADFI and improved FCR linearly, but no effects were detected with the mash diets (P < 0.01 and P < 0.05 for the interactions). Feeding crumbles tended to improve BW uniformity at 5 wk age (P = 0.077) but no effects were detected with increases in energy concentration of the diet. In summary, the inclusion of moderate amounts of fiber in the diet improves pullet performance from hatching to 5 wk age. The response of pullets to increases in energy content of the diet depends on feed form with a decrease in feed intake when fed crumbles but no changes when fed mash. Feeding crumbles might be preferred to feeding mash in pullets from hatching to 5 wk age.

Identification of lumichrome as a Sinorhizobium enhancer of alfalfa root respiration and shoot growth

Sinorhizobium meliloti bacteria produce a signal molecule that enhances root respiration in alfalfa (Medicago sativa L.) and also triggers a compensatory increase in whole-plant net carbon assimilation. Nuclear magnetic resonance, mass spectrometry, and ultraviolet–visible absorption identify the enhancer as lumichrome, a common breakdown product of riboflavin. Treating alfalfa roots with 3 nM lumichrome increased root respiration 21% (P < 0.05) within 48 h. A closely linked increase in net carbon assimilation by the shoot compensated for the enhanced root respiration. For example, applying 5 nM lumichrome to young alfalfa roots increased plant growth by 8% (P < 0.05) after 12 days. Soaking alfalfa seeds in 5 nM lumichrome before germination increased growth by 18% (P < 0.01) over the same period. In both cases, significant growth enhancement (P < 0.05) was evident only in the shoot. S. meliloti requires exogenous CO2 for growth and may benefit directly from the enhanced root respiration that is triggered by lumichrome. Thus Sinorhizobium–alfalfa associations, which ultimately form symbiotic N2-reducing root nodules, may be favored at an early developmental stage by lumichrome, a previously unrecognized mutualistic signal. The rapid degradation of riboflavin to lumichrome under many physiological conditions and the prevalence of riboflavin release by rhizosphere bacteria suggest that events demonstrated here in the S. meliloti–alfalfa association may be widely important across many plant–microbe interactions.

Expression of early nodulin genes in alfalfa mycorrhizae indicates that signal transduction pathways used in forming arbuscular mycorrhizae and Rhizobium-induced nodules may be conserved

Transcripts for two genes expressed early in alfalfa nodule development (MsENOD40 and MsENOD2) are found in mycorrhizal roots, but not in noncolonized roots or in roots infected with the fungal pathogen Rhizoctonia solani. These same two early nodulin genes are expressed in uninoculated roots upon application of the cytokinin 6-benzylaminopurine. Correlated with the expression of the two early nodulin genes, we found that mycorrhizal roots contain higher levels of trans-zeatin riboside than nonmycorrhizal roots. These data suggest that there may be conservation of signal transduction pathways between the two symbioses—nitrogen-fixing nodules and phosphate-acquiring mycorrhizae.

A synthetic cryIC gene, encoding a Bacillus thuringiensis δ-endotoxin, confers Spodoptera resistance in alfalfa and tobacco

Spodoptera species, representing widespread polyphagous insect pests, are resistant to Bacillus thuringiensis δ-endotoxins used thus far as insecticides in transgenic plants. Here we describe the chemical synthesis of a cryIC gene by a novel template directed ligation–PCR method. This simple and economical method to construct large synthetic genes can be used when routine resynthesis of genes is required. Chemically phosphorylated adjacent oligonucleotides of the gene to be synthesized are assembled and ligated on a single-stranded, partially homologous template derived from a wild-type gene (cryIC in our case) by a thermostable Pfu DNA ligase using repeated cycles of melting, annealing, and ligation. The resulting synthetic DNA strands are selectively amplified by PCR with short specific flanking primers that are complementary only to the new synthetic DNA. Optimized expression of the synthetic cryIC gene in alfalfa and tobacco results in the production of 0.01–0.2% of total soluble proteins as CryIC toxin and provides protection against the Egyptian cotton leafworm (Spodoptera littoralis) and the beet armyworm (Spodoptera exigua). To facilitate selection and breeding of Spodoptera-resistant plants, the cryIC gene was linked to a pat gene, conferring resistance to the herbicide BASTA.

Expression of alfalfa mosaic virus coat protein in tobacco mosaic virus (TMV) deficient in the production of its native coat protein supports long-distance movement of a chimeric TMV

Alfalfa mosaic virus (AlMV) coat protein is involved in systemic infection of host plants, and a specific mutation in this gene prevents the virus from moving into the upper uninoculated leaves. The coat protein also is required for different viral functions during early and late infection. To study the role of the coat protein in long-distance movement of AlMV independent of other vital functions during virus infection, we cloned the gene encoding the coat protein of AlMV into a tobacco mosaic virus (TMV)-based vector Av. This vector is deficient in long-distance movement and is limited to locally inoculated leaves because of the lack of native TMV coat protein. Expression of AlMV coat protein, directed by the subgenomic promoter of TMV coat protein in Av, supported systemic infection with the chimeric virus in Nicotiana benthamiana, Nicotiana tabacum MD609, and Spinacia oleracea. The host range of TMV was extended to include spinach as a permissive host. Here we report the alteration of a host range by incorporating genetic determinants from another virus.

NADH-Glutamate Synthase in Alfalfa Root Nodules. Genetic Regulation and Cellular Expression1

NADH-dependent glutamate synthase (NADH-GOGAT; EC 1.4.1.14) is a key enzyme in primary nitrogen assimilation in alfalfa (Medicago sativa L.) root nodules. Here we report that in alfalfa, a single gene, probably with multiple alleles, encodes for NADH-GOGAT. In situ hybridizations were performed to assess the location of NADH-GOGAT transcript in alfalfa root nodules. In wild-type cv Saranac nodules the NADH-GOGAT gene is predominantly expressed in infected cells. Nodules devoid of bacteroids (empty) induced by Sinorhizobium meliloti 7154 had no NADH-GOGAT transcript detectable by in situ hybridization, suggesting that the presence of the bacteroid may be important for NADH-GOGAT expression. The pattern of expression of NADH-GOGAT shifted during root nodule development. Until d 9 after planting, all infected cells appeared to express NADH-GOGAT. By d 19, a gradient of expression from high in the early symbiotic zone to low in the late symbiotic zone was observed. In 33-d-old nodules expression was seen in only a few cell layers in the early symbiotic zone. This pattern of expression was also observed for the nifH transcript but not for leghemoglobin. The promoter of NADH-GOGAT was evaluated in transgenic alfalfa plants carrying chimeric β-glucuronidase promoter fusions. The results suggest that there are at least four regulatory elements. The region responsible for expression in the infected cell zone contains an 88-bp direct repeat.

NADH-Glutamate Synthase in Alfalfa Root Nodules. Immunocytochemical Localization1

In root nodules of alfalfa (Medicago sativa L.), N2 is reduced to NH4+ in the bacteroid by the nitrogenase enzyme and then released into the plant cytosol. The NH4+ is then assimilated by the combined action of glutamine synthetase (EC 6.3.1.2) and NADH-dependent Glu synthase (NADH-GOGAT; EC 1.4.1.14) into glutamine and Glu. The alfalfa nodule NADH-GOGAT protein has a 101-amino acid presequence, but the subcellular location of the protein is unknown. Using immunocytochemical localization, we determined first that the NADH-GOGAT protein is found throughout the infected cell region of both 19- and 33-d-old nodules. Second, in alfalfa root nodules NADH-GOGAT is localized predominantly to the amyloplast of infected cells. This finding, together with earlier localization and fractionation studies, indicates that in alfalfa the infected cells are the main location for the initial assimilation of fixed N2.

Winter Survival of Transgenic Alfalfa Overexpressing Superoxide Dismutase1

To test the hypothesis that enhanced tolerance of oxidative stress would improve winter survival, two clones of alfalfa (Medicago sativa) were transformed with a Mn-superoxide dismutase (Mn-SOD) targeted to the mitochondria or to the chloroplast. Although Mn-SOD activity increased in most primary transgenic plants, both cytosolic and chloroplastic forms of Cu/Zn-SOD had lower activity in the chloroplast SOD transgenic plants than in the nontransgenic plants. In a field trial at Elora, Ontario, Canada, the survival and yield of 33 primary transgenic and control plants were compared. After one winter most transgenic plants had higher survival rates than control plants, with some at 100%. Similarly, some independent transgenic plants had twice the herbage yield of the control plants. Prescreening the transgenic plants for SOD activity, vigor, or freezing tolerance in the greenhouse was not effective in identifying individual transgenic plants with improved field performance. Freezing injury to leaf blades and fibrous roots, measured by electrolyte leakage from greenhouse-grown acclimated plants, indicated that the most tolerant were only 1°C more freezing-tolerant than alfalfa clone N4. There were no differences among transgenic and control plants for tetrazolium staining of field-grown plants at any freezing temperature. Therefore, although many of the transgenic plants had higher winter survival rates and herbage yield, there was no apparent difference in primary freezing injury, and therefore, the trait is not associated with a change in the primary site of freezing injury.

Glycolytic Flux Is Adjusted to Nitrogenase Activity in Nodules of Detopped and Argon-Treated Alfalfa Plants1

To investigate the short-term (30–240 min) interactions among nitrogenase activity, NH4+ assimilation, and plant glycolysis, we measured the concentrations of selected C and N metabolites in alfalfa (Medicago sativa L.) root nodules after detopping and during continuous exposure of the nodulated roots to Ar:O2 (80:20, v/v). Both treatments caused an increase in the ratios of glucose-6-phosphate to fructose-1,6-bisphosphate, fructose-6-phosphate to fructose-1,6-bisphosphate, phosphoenolpyruvate (PEP) to pyruvate, and PEP to malate. This suggested that glycolytic flux was inhibited at the steps catalyzed by phosphofructokinase, pyruvate kinase, and PEP carboxylase. In the Ar:O2-treated plants the apparent inhibition of glycolytic flux was reversible, whereas in the detopped plants it was not. In both groups of plants the apparent inhibition of glycolytic flux was delayed relative to the decline in nitrogenase activity. The decline in nitrogenase activity was followed by a dramatic increase in the nodular glutamate to glutamine ratio. In the detopped plants this was coincident with the apparent inhibition of glycolytic flux, whereas in the Ar:O2-treated plants it preceded the apparent inhibition of glycolytic flux. We propose that the increase in the nodular glutamate to glutamine ratio, which occurs as a result of the decline in nitrogenase activity, may act as a signal to decrease plant glycolytic flux in legume root nodules.

Expression of β-Amylase from Alfalfa Taproots1

Alfalfa (Medicago sativa L.) roots contain large quantities of β-amylase, but little is known about its role in vivo. We studied this by isolating a β-amylase cDNA and by examining signals that affect its expression. The β-amylase cDNA encoded a 55.95-kD polypeptide with a deduced amino acid sequence showing high similarity to other plant β-amylases. Starch concentrations, β-amylase activities, and β-amylase mRNA levels were measured in roots of alfalfa after defoliation, in suspension-cultured cells incubated in sucrose-rich or -deprived media, and in roots of cold-acclimated germ plasms. Starch levels, β-amylase activities, and β-amylase transcripts were reduced significantly in roots of defoliated plants and in sucrose-deprived cell cultures. β-Amylase transcript was high in roots of intact plants but could not be detected 2 to 8 d after defoliation. β-Amylase transcript levels increased in roots between September and October and then declined 10-fold in November and December after shoots were killed by frost. Alfalfa roots contain greater β-amylase transcript levels compared with roots of sweetclover (Melilotus officinalis L.), red clover (Trifolium pratense L.), and birdsfoot trefoil (Lotus corniculatus L.). Southern analysis indicated that β-amylase is present as a multigene family in alfalfa. Our results show no clear association between β-amylase activity or transcript abundance and starch hydrolysis in alfalfa roots. The great abundance of β-amylase and its unexpected patterns of gene expression and protein accumulation support our current belief that this protein serves a storage function in roots of this perennial species.

Developmental Expression and Substrate Specificities of Alfalfa Caffeic Acid 3-O-Methyltransferase and Caffeoyl Coenzyme A 3-O-Methyltransferase in Relation to Lignification1

The biosynthesis of monolignols can potentially occur via two parallel pathways involving free acids or their coenzyme A (CoA) esters. Caffeic acid 3-O-methyltransferase (COMT) and caffeoyl CoA 3-O-methyltransferase (CCOMT) catalyze functionally identical reactions in these two pathways, resulting in the formation of mono- or dimethoxylated lignin precursors. The activities of the two enzymes increase from the first to the sixth internode in stems of alfalfa (Medicago sativa L.), preceding the deposition of lignin. Alfalfa CCOMT is highly similar at the amino acid sequence level to the CCOMT from parsley, although it contains a six-amino acid insertion near the N terminus. Transcripts encoding both COMT and CCOMT are primarily localized to vascular tissue in alfalfa stems. Alfalfa CCOMT expressed in Escherichia coli catalyzes O-methylation of caffeoyl and 5-hydroxyferuloyl CoA, with preference for caffeoyl CoA. It has low activity against the free acids. COMT expressed in E. coli is active against both caffeic and 5-hydroxyferulic acids, with preference for the latter compound. Surprisingly, very little extractable O-methyltransferase activity versus 5-hydroxyferuloyl CoA is present in alfalfa stem internodes, in which relative O-methyltransferase activity against 5-hy-droxyferulic acid increases with increasing maturity, correlating with increased lignin methoxyl content.