EFFECT OF TYPE OF DIETARY CARBOHYDRATES ON DIGESTION...

UNIVERSIDAD POLITÉCNICA DE MADRID

2013

ESCUELA TÉCNICA SUPERIOR DE INGENIEROS AGRÓNOMOS

EFFECT OF TYPE OF DIETARY

CARBOHYDRATES ON DIGESTION,

INTESTINAL BARRIER AND GROWTH

PERFORMANCE IN 25-D WEANED

RABBITS

Efecto del tipo de carbohidratos de la dieta sobre la

digestión, la barrera intestinal y los rendimientos

productivos en gazapos destetados con 25-d de edad

TESIS DOCTORAL

UNIVERSIDAD POLITÉCNICA DE MADRID

2013

Mª Soledad Gómez Conde

Ingeniero Agrónomo

2013

DEPARTAMENTO DE PRODUCCIÓN ANIMAL

ESCUELA TÉCNICA SUPERIOR DE INGENIEROS AGRÓNOMOS

EFFECT OF TYPE OF DIETARY

CARBOHYDRATES ON DIGESTION,

INTESTINAL BARRIER AND GROWTH

PERFORMANCE IN 25-D WEANED

RABBITS

Mº Soledad Gómez Conde

Ingeniero Agrónomo

DIRECTORES DE TESIS

Rosa Mª Carabaño Luengo Javier García Alonso

Director de tesis Director de tesis

“Educar la mente sin educar al corazón,

no es educar en absoluto”.

―Aristóteles‖

AGRADECIMENTOS

Siendo sincera, he de reconocer que este último capítulo de la tesis me ha costado empezar-

lo y lo he demorado incluso hasta el último momento. En parte quizás por el miedo a que,

después de tanto tiempo para presentarla, olvidase con las prisas a los muchos que de ma-

nera totalmente desinteresada y a lo mejor incluso sin saberlo, habéis contribuído a que

esto haya llegado a su fin. No puedo mencionar a todas las personas que os habéis cruzado

por más o menos tiempo en estos años conmigo, pero sabed que habéis formado parte de un

camino, más largo de lo que en un principio imaginé, que me ha conducido hasta aquí y que

pese a los buenos y malos momentos que han ido surgiendo, he de confesar me ha hecho

muy feliz… por todo ello y para todos vosotros GRACIAS mil.

A Rosa y Javier, por escogerme dentro de aquel magnífico grupo de compañeros de Pro-

ducción Animal y darme la oportunidad de acceder a una formación excepcional. Gracias

por desarrollar un proyecto pluridisciplinar e innovador que despertó mis inquietudes, que

estructuró mi cabeza, que me hizo crecer como persona y que me acompaña a diario en mi

andadura profesional. Gracias a los dos por vuestra confianza, por no darme por perdida y

por tener paciencia con cada una de mis idas y venidas.

A todos los profesores del Departamento por su apoyo, formación y grandes consejos, que

nunca he dejado de recibir incluso después de mi paso por la Escuela. Espero que seáis

conscientes de lo mucho que aprecio la gran labor que hacéis, más incluso con el paso del

tiempo.

A Juan Lora, por la gran ayuda con los animales y enseñarme “pilares fundamentales de la

nutrición Animal”. A Luis Pérez Alba por el esmero que puso en la fabricación de las die-

tas. A Pablo Eiras por su ayuda con el espectofotómetro. A “Pasturas” porque siempre es-

tuvo ante cualquier imprevisto. A Ignacio Badiola y Ana Rozas por todo el soporte en la

parte de mirobiología. A Miguel Ángel Ibañez por su contribución en los análisis estadísti-

cos.

A todos mis amigos “becarios” de aquella época a los que debo muchas horas de esfuerzo y

cariño… Ana Espinosa y mi rubia Mª Ángeles, Susana, Nuno, Filipe, Encarna, Diego, Juan

Manuel, Beatriz, Juanra, Javi, Oscar, Gloria (y tantos más)… y no becarios Pilar, Mari-

gel…Pese a que hubo momentos en los que nos quejábamos, ¡qué de buenos ratos y risas

hemos pasamos juntos!. Esperemos que nunca hablen las paredes de ese laboratorio. El

tiempo y la distancia han hecho que a muchos de vosotros ya no os vea tan amenudo y “os

echo mucho de menos”.

A Mariano Gorrachategui por su ayuda con la parte del almidón de esta tesis y por los mo-

mentos que compartimos en un año muy difícil para los dos y que me hizo descubrir muchas

buenas personas.

A los más recientes: Naty por ser un ejemplo a diario de que la profesionalidad, el compa-

ñerismo y la amistad pueden ir de la mano. A Santi, Antonio … que me han dado ánimos en

esta última parte para que no decayera en mi empeño. A Silvia, por esos buenos ratos que

hemos ido descubriendo juntas “corriendo”.

A Joan, porque es sin saberlo una de las personas que me hizó lanzarme a este último y de-

finitivo esfuerzo. Gracias porque en un momento en el que lo necesitaba, me brindaste una

oportunidad dentro de tu equipo, cerraste una etapa e hiciste que resurgiera reforzado mi

“espíritu inquieto”. Espero no defraudar la confianza que depositaste en mí y que me sigas

haciendo partícipe de muchos otros proyectos.

Pero esta tesis quiero dedicársela y agradecérsela de manera especial a toda mi familia.

Nos ha constado mucho, pero prometí que la acabaría y entre todos “lo hemos consegui-

do”.

Gracias a todos “los abus” y “tíos” por entretener, cuidar y querer de la manera tan es-

pecial en que lo hacéis a nuestros hijos, tenéis la gran recompensa de saber que ellos os

adoran.

A Vicente y Maricarmen, por no desalentarse en estos años intentando conocerme, respetar

en silencio las ausencias y aprender a quererme.

A mis padres por su amor incondicional, por apoyar y respetar todas mis decisiones, por

enseñarme a luchar y no poner freno a todas mis inquietudes. Porque tanto en los momentos

felices como las adversidades, han sido un ejemplo de respeto y superación y el espejo en el

que cada día me gustaría reflejarme. Soy consciente que esta tesis ha sido larga, que pude

no seguir el orden correcto y que habréis sufrido más de una vez en silencio…gracias por-

que nunca me lo hicisteis sentir.

A Daniel, porque en mi vida hay un antes y un después a ti. Gracias por tu entrega, por tu

amor, por desvivirte por que se cumplan cada uno de mis sueños, por darme la mejor parte

de mi vida, por desear formar una familia junto a mí, por conocer lo bueno y malo de mí y

pese a todo quererme. Este último esfuerzo ha sido especialmente duro para los dos, gracias

por creer, por ceder, por comprender, por respetar y sentirte orgulloso de mí, por ser un

compañero fiel con el que espero emprender si el tiempo nos respeta, muchos otros proyec-

tos.

Finalmente, a Guille, María y Álvaro, nuestras tres pequeñas fierecillas que han ido apare-

ciendo y llenando totalmente nuestras vidas. Gracias por recordarnos que todo puede ser

sencillo, por llenar la casa de risas y conventirla en nuestro hogar. Confío en que guardéis

parte de vuestra infancia como un tesoro, que seáis prudentes pero inquietos, que no acep-

téis que nada es imposible, que seais capaces de reconocer que os equivocáis, aprendáis lo

grande que os hará el pedir perdón, que sintáis la inmensidad de compartir lo que tenéis y

que nada os desvíe de la misión más grande que tenéis en la vida que es la de intentar ser

felices.

Con mi mas absoluto agradecimiento, respeto y admiración a todos vosotros.

Marisol

Madrid, 1 de Septiembre de 2013.

Index

I

INDEX OF CONTENTS

ABBREVIATION LIST

RESUMEN

SUMMARY

CHAPTER 1: LITERATURE REVIEW AND OBJECTIVES

1 INTRODUCTION AND OBJECTIVES ........................................................................ 15

2 CHARACTERIZATION AND QUANTIFICATION OF FIBRE AND STARCH ...... 22

2.1 Fibre .............................................................................................................................. 22

2.2 Starch ............................................................................................................................ 26

2.3 The rabbit digestive tract ......................................................................................... 30

2.3.1. Effect of age and weaning on digestion................................................................ 30

2.3.2. Utilisation of carbohydrates: fibre and starch ...................................................... 33

2.3.2.1.-Fibre .................................................................................................................. 33

2.3.2.2.-Starch ................................................................................................................. 36

2.4 Criteria for evaluating intestinal health ........................................................................ 40

2.4.1. Gut barrier function .............................................................................................. 40

2.4.1.1.-The GI epithelium ............................................................................................. 41

2.4.1.2.-The gut associated lymphoid tissue (GAL T) ................................................... 44

2.4.1.3.-The intestinal microbiota ................................................................................... 47

3 REFERENCES ............................................................................................................... 51

CHAPTER 2: NEUTRAL DETERGENT SOLUBLE FIBER IMPROVES GUT BARRIER

FUNCTION IN TWENTY -FIVE-DAY-OLD RABBITS

1. ABSTRACT ................................................................................................................... 71

2. INTRODUCTION .......................................................................................................... 72

3. MATERIALS AND METHODS ................................................................................... 72

3.1 Animals ......................................................................................................................... 72

Index

II

3.2 Diets .............................................................................................................................. 73

Experiment 1: Determination of Ileal Digestibility, Mucosa Morphology and Sucrase

Activity ........................................................................................................................... 74

Experiment 2: Characterization of Lamina Propria Lymphocytes ................................. 75

Experiment 3: Characterization of Intestinal Microbiota and Determination of Mortality

Rate ................................................................................................................................. 76

3.3 Chemical Analysis ........................................................................................................ 76

3.4 Statistical Analysis ....................................................................................................... 78

4 RESULTS ....................................................................................................................... 78


Activity ........................................................................................................................... 78

Experiment 2: Characterization of Lamina Propria Lymphocytes ................................. 79

Experiment 3: Characterization of Intestinal Microbiota and Determination of Mortality

........................................................................................................................................ 79

5. DISCUSSION ................................................................................................................. 80

6. LITERATURE CITED ................................................................................................... 84

CHAPTER 3: EFFECT OF NEUTRAL DETERGENT SOLUBLE FIBRE ON

DIGESTION, INTESTINAL MICROBIOTA AND PERFORMANCE IN TWENTY-FIVE-

DAY-OLD WEANED RABBITS

1 ABSTRACT. .................................................................................................................. 99

2. INTRODUCTION ........................................................................................................ 101

3. MATERIAL AND METHODS.................................................................................... 102

3.1 Diets ............................................................................................................................ 102

3.2 Animals and housing .................................................................................................. 103

3.3 Digestibility trial ......................................................................................................... 103

3.4 Stomach, caecal fermentation and similarity rate of intestinal microbiota trial ......... 104

3.5 Growth trial ................................................................................................................ 105

3.6 Analytical methods ..................................................................................................... 106

3.7 Statistical analysis ...................................................................................................... 106

4. RESULTS ..................................................................................................................... 107

4.1 Faecal digestibility trial .............................................................................................. 107

4.2. Digestion traits ........................................................................................................... 107

Index

III

4.3 Growth trial ................................................................................................................ 108

5. DISCUSSION ............................................................................................................... 109

6. CONCLUSIONS .......................................................................................................... 111

7. ACKNOWLEDGEMENT ............................................................................................ 111

8. REFERENCES ............................................................................................................. 112

CHAPTER 4: EFFECT OF SOURCE OF STARCH ON DIGESTION, INTESTINAL

MICROBIOTA AND PERFORMANCE IN TWENTY-FIVE-DAY-OLD WEANED

RABBITS

1 ABSTRACT ................................................................................................................. 125

2. INTRODUCTION ........................................................................................................ 127


3.1 Animals and housing .................................................................................................. 128

3.2 Experimental diets ...................................................................................................... 128

3.3 Growth trial ................................................................................................................ 129

3.4 Digestion trial ............................................................................................................. 130

3.5 Microbiota trial ........................................................................................................... 132

3.6 Chemical analysis ....................................................................................................... 132

3.7 Statistical analysis ...................................................................................................... 133

4. RESULTS ..................................................................................................................... 134

4.1. Growth trial ............................................................................................................... 134

4.2 . Digestion trial ........................................................................................................... 134

4.3. Microbiota trial .......................................................................................................... 136

5. DISCUSSION ............................................................................................................... 136

5.1 Conclusions ................................................................................................................ 138

6. REFERENCES ............................................................................................................. 139

CHAPTER 5: DETERMINATION OF FACECAL DRY MATTER DIGESTIBILITY

1 ABSTRACT ................................................................................................................. 155

2. INTRODUCTION ........................................................................................................ 156


3.1. Experimental procedure ............................................................................................. 156

Index

IV

3.2 .Statistical analysis ..................................................................................................... 157

4. RESULTS AND DISCUSSION ................................................................................... 157

5. REFERENCES ............................................................................................................. 161

CHAPTER 6: GENERAL CONCLUSIONS AND IMPLICATIONS

1. Conclusions: ................................................................................................................. 171

2. Implications: ................................................................................................................. 172

Abbreviation Keys

Abbreviation keys

VII

ABBREVIATION KEYS / ABREVIATURAS

Abbrevia-

tion/Abreviatura

ENGLISH CASTELLANO

ºC Degree Celsius Grado centigrade

% Percentage Porcentaje

µl Microlitre Microlitro

µg Microgram Microgramo

µm Micometre Micrómetro

µmol Micromole Micromol

AIs Autoinducers Autoinductores

ADF Acid detergent fibre Fibra ácido detergente

ADL Acid-detergent lignin Lignina ácido detergente

ADFI Average daily feed intake Consumo medio diario

AH Alfalfa hay Heno de alfalfa

AID Apparent ileal digestibility Digestibilidad ileal aparente

AOAC Association of Official Analytical Chemist

B. Bacteroides

B-AP Beet-Apple pulp Pulpa de remolacha-manzana

BOE Boletín Oficial del Estado

BW Body weight Peso vivo

CF Crude fibre Fibra bruta

Cm Centimitre Centímetro

CP Crude Protein Proteína bruta

D Day Día

DA Dehydrated alfalfa Alfalfa deshidratada

DIA Apparent ileal digestibility Digestibilidad ileal aparente

DM Dry matter Materia seca

Dry matterid Ileal digestibility of dry matter Digestibilidad ileal de la mate-

rial seca

DMd Dry matter digestibility Digestibilidad de la materia

seca

DF Dietary fibre Fibra dietética

Abbreviation keys

VIII

DP/DE ratio Digestible protein/digestible energy Proteína digestible/energía

digestible

E Escherichia

ERE Epizootic Rabbit Enteropathy Enteropatía epizoótica del

conejo

F Fibrobacter

FD Frequency of detection Frecuencia de detección

g Gram Gramos

g/d Gram per day Gramo por día

g/kg DM Gram per kilogram of dry matter Gramo por kilogramo de mate-

rial seca

GALT Gut associated lymphoid tissue Tejido linfoide asociado a intes-

tino

GI Gastrointestinal Gastrointestinal

h Hour Hora

IELs Intraepitelial lymphocytes Linfocitos intraepiteliares

IgA Immunoglobulin A Inmunoglobulina A

IgE Immunoglobulim E Inmunoglobulina E

IgM Immunoglobulina M Inmunoglobulina M

IL2 Interleukin 2 Interleulina 2

Kcal Kilocalorie Kilocaloría

kg Kilogram Kilogramo

LP Lamina propria

mg Miligram Miligramo

MHCI Immune histocompatibility complex Complejo immune de histo-

compatibilidad

min Minute Minuto

MJ Megajoule Megajulio

mm Milimitre Milímetro

mol Mole Mol

MPO Myeloperoxidase Mieloperoxidasa

mRNA Messenger RNA ARN mensajero

N Number Número

NDF Neutral Detergent fiber Fibra neutro detergente

Abbreviation keys

IX

NDSF Neutral Detergent soluble fibre Fibra neutro detergente soluble

NO Nitric Oxide Óxido nítrico

NSP Non-starch polysaccharides Polisacáridos no amiláceos

OH Oat Hull Cáscara de avena

P Probability Probabilidad

Plinear Linear Probability Probabilidad del efecto lineal

Ppm Parts-per-million Partes por millón

PQuadratic Quadratic Probability Probabilidad del efecto

cuadrátic

Proteinid Ileal digestibility of crude protein Digestibilidad ileal de la proteí-

na bruta

R. Ruminococcus

RDS Rapidly digestible starch Almidón rápidamente digesti-

ble

rRNA Ribosomal RNA ARN Ribosomal

RS Resistant starch Almidón resistente

SAS Statistical Analysis Systems

SE Standard error Error estándar

SIgA Secretory IgA Inmonuglobulina A secretora

SDS Slowly digestible starch Almidón lentamente digestible

Spp. Species

Starchid Ileal digestibilidad of starch Digestibilidad ileal del almidón

TDF Total dietary fibre Fibra dietética total

Th T-Helpers T-cooperadores

UK United Kingdom Reino Unido

USA United sStates of America Estados Unidos

VFAs Volatile fatty acids Ácidos grasos volatiles

vs. Versus

Yb Ytterbium Iterbio

Yb2O3 Ytterbium oxide Óxido de iterbio

Resumen

Resumen

3

RESUMEN

Los objetivos globales de esta tesis han sido estudiar el efecto que los carbohidratos

de la dieta ejercen sobre los rendimientos productivos, la barrera intestinal, y la digestión de

animales destetados a 25 días de edad. Además se ha estudiado cuál es el mejor periodo para

determinar la digestibilidad fecal tras el destete a esta edad.

En el primer experimento se estudió el efecto de la fibra neutro detergente soluble

(FNDS) sobre la barrera intestinal, digestión, microbiota intestinal y rendimientos producti-

vos de gazapos en gazapos en la fase post-destete.

Se diseñaron tres piensos isonutritivos en los que la única fuente de variación fueron

los niveles de fibra soluble. A partir de una dieta control (AH) con 103 g/kg de materia seca

de FNDS y alfalfa como fuente principal de fibra, se sustituyó la mitad de esta alfalfa por

una mezcla de pulpa de remolacha y pulpa de manzana (75:25) en el pienso B-AP y por una

mezcla de cascarilla y concentrado de proteína de soja (88:12) en el pienso OH, obteniéndo-

se 131 y 79 g/kg de FNDS sobre materia seca, respectivamente. Los conejos se destetaron a

25 días y fueron alimentados con los piensos experimentales hasta los 35 días de edad, mo-

mento en el que se sacrificaron para la determinación de la digestibilidad ileal aparente

(DIA) de la materia seca (MS), proteína bruta (PB) y almidón, la morfología de la mucosa, y

actividad enzimática en el yeyuno, el tejido linfoide asociado a la mucosa, así como la mi-

crobiota intestinal. Para la determinación de la morfología de la mucosa se utilizaron adicio-

nalmente 19 animales lactantes de 35 días de edad. Para el estudio de la tasa de mortalidad,

se utilizaron 118 animales más por tratamiento que recibieron los piensos experimentales

durante las dos semanas post-destete y posteriormente un pienso comercial hasta los 60 días

de edad. Los animales recibieron durante todo el experimento medicación en el agua de be-

bida (100 ppm de apramicina sulfato y 120 ppm de tilosina tartrato).

El nivel de fibra soluble mejoró los parámetros que se utilizaron para la caracteriza-

ción del estado de la barrera intestinal. Los conejos alimentados con el mayor nivel de

FNDS en el pienso presentaron una mayor longitud de los villi (P=0.001), un mayor ratio

longitud villi/profundidad de las criptas (8.14; P=0.001), una mayor actividad disacaridásica

(8671 µmol de glucosa/g de proteína; P=0.019), así como una mayor digestibilidad ileal

Resumen

4

(96.8%; P=0.002), observándose una reducción en el flujo ileal de almidón a medida que se

incrementó el nivel de fibra soluble en el pienso (1,2 vs 0,5 g/d; P=0.001). Los animales

lactantes a 35 días de edad presentaron un ratio longitud de villi/profundidad de las criptas

menor que el observado en aquéllos alimentados con el pienso B-AP (6.70), pero superior al

de los piensos AH y OH. Niveles inferiores de NDFS tendieron (P=0.074) a incrementar la

respuesta inmune de tipo celular (linfocitos CD8+). El pienso también afectó a la producción

de IL2 (CD25+; P=0.029; CD5+CD25+; P=0.057), pero sin llegar a establecerse una clara

relación con el nivel de fibra soluble. La diversidad de la microbiota intestinal no se vio

afectada por el pienso (P ≥ 0.38). Los animales alimentados con las piensos B-AP y AH

presentaron una reducción en la frecuencia de detección de Clostridium perfringens tanto en

íleon (P=0.062) como en ciego (4.3 vs. 17.6%, P =0.047), comparado con el pienso OH.

Además la tasa de mortalidad (118 gazapos/pienso) disminuyó de 14.4% en el pienso OH a

5.1% en el pienso B-AP.

Entre los 32 y los 35 días de edad se determinó la digestibilidad fecal aparente

(14/pienso) de la materia seca (MS), energía bruta (EB), proteína bruta (PB), fibra neutro

detergente (FND), fibra ácido detergente (FAD) y almidón. Este grupo, junto con otros nue-

ve animales por tratamiento se utilizaron para determinar el peso del estómago y el ciego, la

concentración cecal de ácidos grasos volátiles (AGV) y amoniaco (NH3), así como las tasas

de similitud de la microbiota intestinal. Además se estudiaron los rendimientos productivos

(35 animales/tratamiento) de los gazapos durante todo el período de cebo, consumiendo los

piensos experimentales desde el destete hasta los 35 días y posteriormente un pienso comer-

cial hasta los 60 días de edad. Niveles crecientes de FNDS mejoraron la digestibilidad fecal

de la materia seca (MS) y energía (P<0.001). La inclusión FNDS aumentó de manera lineal

el peso del contenido cecal (P=0.001) y el peso del aparato digestivo completo (P=0.008), y

en los días previos al sacrificio disminuyó de manera lineal el consumo medio diario

(P=0.040). Se observó además, una disminución lineal (P≤0.041) del pH del estómago. No

se encontró relación entre el pH, la concentración y proporciones molares de AGV y el nivel

de FNDS. El pienso pareció tener un efecto, incluso superior al de la madre, sobre la tasa de

similitud de la microbiota, y los efectos fueron mayores a nivel cecal que ileal. La eficacia

alimenticia aumentó de manera lineal en un 12% entre piensos extremos tras el destete (25-

39d) y en un 3% en el período global de cebo con niveles mayores de NDFS. El consumo

medio diario durante la fase post-destete y durante todo el período de cebo, tendió a aumen-

Resumen

5

tar (P≤0.079) con niveles mayores de FNDS, sin embargo no se apreció efecto sobre la ga-

nancia media diaria (P≥0.15).

En conclusión, el incremento del nivel de fibra soluble en el pienso parece resultar

beneficioso para la salud del animal ya que mejora la integridad de la mucosa, y reduce la

frecuencia de detección de potenciales patógenos como C. perfringens y Campylobacter

spp. Conforme a estos resultados, debería tenerse en cuenta el contenido en fibra soluble en

la formulación de piensos de conejos en la fase post-destete.

El objetivo del segundo experimento fue determinar el efecto de la fuente de almidón

sobre la digestión, la microbiota intestinal y los rendimientos productivos en conejos deste-

tados con 25 días de edad. Se formularon tres piensos isonutritivos en los que se modifica-

ron las principales fuentes de almidón: trigo crudo, trigo cocido y una combinación de trigo

y arroz cocido. Dos grupos de 99 y 193 animales se destetaron con 25 días de edad. El pri-

mero de ellos se utilizó para la determinación de los parámetros productivos conforme al

mismo protocolo seguido en el experimento anterior. El segundo de los grupos se utilizó

para la determinación de la digestibilidad fecal de 32 a 35 d, la digestibilidad ileal aparente

(DIA) a 35 d, la morfología de la mucosa intestinal, los parámetros de fermentación cecal;

así como, la caracterización de la microbiota intestinal. Se utilizaron además dos grupos

adicionales de animales 384 (medicados) y 177 (no medicados) para estudiar el efecto de la

suplementación con antibióticos en el agua de bebida sobre la mortalidad. El procesado tér-

mico del trigo mejoró ligeramente la digestibilidad ileal del almidón (P=0.020) pero no mo-

dificó el flujo final de almidón que alcanzó el ciego, observándose una mayor frecuencia de

detección de Campylobacter spp. y Ruminococcus spp. en ciego (P≤0.023), pero sin cambios

a nivel ileal. El procesado térmico del trigo no afectó tampoco a los parámetros productivos,

la mortalidad, la digestibilidad ileal y fecal o la morfología de la mucosa. La sustitución par-

cial del trigo cocido por arroz cocido, penalizó la digestibilidad ileal del almidón (P=0.020)

e incrementó el flujo ileal de este nutriente al ciego (P=0.007). Sin embargo no afectó a la

mortalidad, pese a que se detectaron cambios en la microbiota tanto a nivel ileal como cecal,

disminuyendo la frecuencia de detección de Campylobacter spp. (en íleon y ciego), Helico-

bacter spp. (en íleon) y Ruminococcus spp (en ciego) e incrementando Bacteroides spp. (en

ciego) (P≤0.046). El empleo de arroz cocido en las piensos post-destete no tuvieron efectos

sobre los parámetros productivos, la mortalidad, la digestibilidad ileal y fecal a excepción

del almidón, o la morfología de la mucosa. La suplementación con antibiótico redujo la fre-

Resumen

6

cuencia de detección de la mayoría de las bacterias estudiadas (P≤0.048), sobre todo para

Campylobacter spp., Clostridium perfringens y Propionibacterium spp. (P≤0.048), obser-

vándose un efecto mayor a nivel ileal que cecal, lo que se asoció a la bajada significativa

(P<0.001) de la mortalidad.

En conclusión, los resultados de este experimento indican que la fuente de almidón

afecta a la microbiota intestinal pero no influiye sobre la salud del animal. En relación al

procesado, el uso de trigo cocido junto con arroz cocido no mejora los resultados obtenidos

con trigo duro, si bienserían necesarios más experimentos que confirmaran este punto.

El último de los experimentos se centró en un aspecto metodológico. Dado que, los

conejos destetados presentan un patrón digestivo diferente al de un animal adulto resultado

de su inmadurez digestiva, el objetivo buscado era tratar de determinar el mejor procedi-

miento para la determinación de la digestibilidad fecal en los gazapos en la fase post-destete.

Para tal fin se utilizaron 15 animales/tratamiento de tres camadas diferentes que se desteta-

ron con 25 días, suministrándoles un pienso comercial de crecimiento-cebo. Se registró el

consumo medio diario y la excreción diaria de heces desde el día 25 hasta el día 40 de edad

para la determinación de la digestibilidad de la MS. La camada afectó al consumo medio

diario y la excreción de heces (P=0.013 y 0.014, respectivamente), observándose una ten-

dencia (P=0.061) en la digestibilidad. La edad afectó (P<0.001) a todos estos factores, in-

crementándose de manera más evidente la excreción que la ingestión de materia seca en la

primera semana de vida, para aumentar de forma paralela a partir de la segunda. La correla-

ción entre el consumo medio diario fue mayor con la excreción de heces del mismo día que

con la del día siguiente, por lo que se utilizó el primero para la determinación de la digestibi-

lidad de la MS (MSd). La MSd disminuyó de manera lineal hasta los 32 días de edad

(2.17±0.25 unidades porcentuales por día), mientras que permaneció constante desde los 32

a los 40 días (69.4±0.47%). Por otro lado, la desviación estándar de la MSd se redujo cuando

se incrementó el período de recogida de 2 a 6 días en un 54%.

Conforme a los resultados obtenidos, se puede concluir que no es aconsejable co-

menzar las pruebas de digestibilidad antes de los 32 días de edad y que el número de anima-

les necesario para detectar diferencias significativas entre tratamientos dependerá del perío-

do de recogida de heces.

Summary

Summary

9

SUMMARY

The global aim of this thesis has been to study the effect of dietary carbohydrates on

growth, performance, digestion and intestinal barrier in 25-d weaned rabbits. In addition

there has also been studied which is the best period to determine the fecal digestibility after

weaning.

The first experiment focused on the effect of Neutral Detergent Soluble Fibre

(NDSF) on gut barrier function, digestion, intestinal microbiota and growth performance n

rabbits in the post-weaning period.

Three isonutritive diets which only varied in the levels of soluble fiber were formu-

lated such as it described as follows: a control diet (AH) containing 103 g of neutral deter-

gent soluble fiber, including alfalfa as main source of fiber, was replaced by a mixture of

beet and apple pulp (75-25) in the B-AP diet and, by a mix of oat hulls and soybean protein

concentrate (88:12) in the OH diet, resulting 131 and 79 g of NDFS/kg of dry matter, re-

spectively. Rabbits, weaned at 25 days of age, were fed the experimental diets up to 35 days

of age, moment in which they were slaughtered for apparent ileal digestibility (AID) of dry

matter (DM), crude protein (CP) and starch, mucosa morphology, sucrose activity, charac-

terization of lamina propria lymphocytes and intestinal microbiota. To assess mucosal mor-

phology, 19 suckling 35-d-old rabbits were also used. For mortality study, besides these an-

imals, 118 additional rabbits per treatment were fed the experimental diets for two weeks

period and thereafter received a commercial diet until 60 days of age. Rabbits were water

medicated during the whole experimental period (100 ppm de apramicine sulphate and 120

ppm of tylosine tartrate). Level of soluble fiber improved all the parameters used for the

characterization of the intestinal barrier condition. Villous height of the jejunal mucosa in-

creased with dietary soluble fiber (P=0.001). Villous height of jejunal mucosa increased

with dietary soluble fiber (P = 0.001). Rabbits fed the highest level of soluble fiber (BA-P

diet) showed the highest villous height/crypth depth ratio (8.14; P = 0.001), sucrase specific

activity (8671 µmol glucose/ g protein; P = 0.019), and the greatest ileal starch digestibility

(96.8%; P = 0.002). The opposite effects were observed in rabbits fed decreased levels of

soluble fiber (AH and OH diets; 4.70, 5,848 µmol of glucose/g of protein, as average, re-

spectively). The lowest ileal starch digestibility was detected for animal fed OH diet

Summary

10

(93.2%). Suckling rabbits of the same age showed a lower villous height/crypt depth ratio

(6.70) compared with the B-AP diet group, but this ration was higher that the AH or OH diet

groups. Lower levels of soluble fiber tended (P = 0.074) to increase the cellular immune

response (CD8+ lymphocytes). Diet affected IL-2 production (CD25+, P = 0.029;

CD5+CD25+, P = 0.057), with no clear relationship between soluble fiber and IL-2. The

intestinal microbiota biodiversity was not affected by diets (P ≥ 0.38). Animals fed B-AP

and AH diets had a reduced cecal frequency of detection compatible with Campylobacter

spp. (20.3 vs. 37.8, P = 0.074), and Clostridium perfringens (4.3 vs. 17.6%, P = 0.047),

compared with the OH diet group. Moreover, the mortality rates decreased from 14.4 (OH

diet) to 5.1% (B-AP diet) with the increased presence of soluble fiber in the diet.

Between 32 and 35 days of age, faecal apparent digestibility of dry matter (DM),

gross energy (GE), crude protein (CP), neutral detergent fiber (NDF), acid detergent fiber

(ADF) and starch was determined (14/diet). This group, plus another nine rabbits/diet were

used to determine weight of stomach and caecum and their contents, cecal fermentation

traits and similarity rate (SR) of intestinal microbiota. Besides, growth performance parame-

ters (35 rabbits/diet) were studied during the whole fattening period, in animals consuming

the experimental feed after the weaning up to 35 days of age and later on a commercial diet

up animals reached 60 days of age. Increasing levels of Neutral Detergent Soluble Fiber

improved faecal dry matter and energy digestibility (P<0.001). NDSF inclusion improved

linearly weight of the caecal content (P=0.001) and the total gastrointestinal tract (P=0.008),

and in the previous days to slaughter a linear decrease of daily feed intake in diet with high-

est level of soluble fiber was also observed. Stomach pH decreased linearly with increasing

levels of NDFS (P≤0.041). No relation between NDSF level on pH, concentration and molar

proportion of VFA was found. Treatments appeared to influence the similarity rate of mi-

crobiota, even higher to mother effect. These effects were higher in ileum than in caecum. A

linear positive effect of feed efficiency was observed, which increased around 12% in the

two weeks post-weaning (25-39d) and 3% in the whole fattening period between extreme

diets with highest levels of soluble fiber. Average daily feed intake during the two weeks

after weaning and in the whole fattening period, tended (P≤0.079) to increase with highest

levels of NDSF; although there were no effect on daily weight gain (≥0.15).

In conclusion, an increase of soluble fiber in the feed seems to be beneficial for ani-

mal health, due to improve mucose integrity and reduce detection frequency of those poten-

Summary

11

tial pathogens like C. perfringens and Campylobacter spp. According to these results, level

of soluble fiber should be taking care in feed rabbit formulation in the post-weaning period.

The objective of the second experiment was to determine the effect of source of

starch on digestion, intestinal microbiota and growth performance in twenty-five-day old

weaned rabbits. To accomplish with this aim three iso-nutritive diets were formulated with

different source of starch: raw wheat, boiled wheat and a combination of boiled wheat and

boiled rice. Two groups of 99 and 193 rabbits were weaned at 25 days of age. The first

group was used for growth performance determination following the same protocol than in

previous experiment. The second group was used to determine faecal digestibility from 32 to

35 d, apparent ileal digestibility (AID) at 35 d, jejunal mucosa morphology, caecal fermenta-

tion traits and characterization of intestinal microbiota. For mortality, two additional groups

of 384 (medicated) and 177 (not medicated) were used in order to study the effect of antibi-

otic water supply supplementation. Heat processing of starch slightly improved ileal digesti-

bility of starch (P=0.020) but did not modify the flow of starch to the caecum. An increase in

frequency of detection of Campylobacter spp. y Ruminococcus spp. was observed in the

caecum (P≤0.023), with no changes at ileal level. Heat processing of wheat did not modify

growth performance, mortality, ileal or faecal digestibility and mucosa morphology. Partial

substitution of boiled wheat for boiled rice in the diet impaired ileal starch digestibility

(P=0.020) and increased the ileal flow of this nutrient to the caecum (P=0.007). However, it

did not affect mortality rate, although changes in the ileal and caecal intestinal microbiota

were detected, decreasing the frequency of detection of Campylobacter spp. (both ileum and

caecum), Helicobacter spp. (at ileum) and Ruminococcus spp (at caecum) and increasing the

Bacteroides spp. (at caecum) (P≤0.046). The effect of boiled rice supplementation did not

alter growth performance, mortality, ileal or faecal digestibility of other nutrients than

starch, and mucosa morphology. Medication of rabbits reduced the ileal frequency of detec-

tion of most bacteria studied (P≤0.048), especially for Campylobacter spp., Clostridium

perfringens y Propionibacterium spp. (P≤0.048), resulting the effect higher at ileal than cae-

cal level and relating it with a strong reduction of mortality rate (P<0.001).

In conclusion, the results of this experiment make think that the source of starch af-

fects the intestinal microbiota but they do not seem to influence animal health. In relation to

the effect of heat processed the use of cooked wheat or cooked rice it does not seem to im-

Summary

12

prove the results obtained with hard wheat, but there would be necessary more experiments

that were confirming this point.

The last experiment focused on a methodological aspect. Considering that, weaned

rabbits have a different digestive pattern than older animals due to their digestive immaturi-

ty; the fixed objective was to determine the best procedure for faecal digestibility determina-

tion in young rabbits in the post-weaning period. Fifteen rabbits from 5 different litters were

weaned at 25 days of age and fed with a commercial feed. Feed intake and faeces excretion

were recorded daily from 25 to 40 days of age for dry matter digestibility (DMd) determina-

tion. Litter affected daily DM intake and excretion (P=0.013 y 0.014, respectively) and tend-

ed to affect DMd (P=0.061). Age affected all these factors (P<0.001), but ingestion in-

creased slowly than dry matter excretion during the first week buth they evolved similarly in

the second week. The correlation between daily feed intakes was higher with the faeces ex-

cretion of the day than with faeces excretion of the next day, and the first values were used

to determine daily DMd. The DMd decreased linearly from weaning to 32 d of age

(2.17±0.25 percentage units per day), whereas from 32 to 40 d remained constant

(69.4±0.47%). On the other hand, average standard deviation of DMd decreased by 54%

when the length of collection period increased from 2 to 6d.

Consequently to the obtained results, it could be concluded that it would not be ad-

visable to start digestibility trials before the 32 days of age and that the number of animals

required to detect a significant difference among means would depend on the collection pe-

riod.

Chapter 1

Literature review and objectives

Chapter 1: Literature Review and Objectives

15

1 INTRODUCTION AND OBJECTIVES

The infectious diseases of the digestive system represent 71% of the total rabbit dis-

eases. This percentage traditionally high, get even worse since the Epizootic Rabbit Enter-

opathy (ERE) turned up. The digestive pathology is responsible of the 60% of the rabbit

mortality in the fattening period and of important reductions in feed efficiency and animal

growth, causing a delay in the total fattening period within one or two weeks. Utilization of

Growth promoters has been the most habitual way of controlling mortality, which has led

that the cost of the veterinary treatments has been multiplied for 2,5 and thus, decreasing the

net profit of the farms (Rosell et al., 2009) . Together with ERE, there continue existing oth-

er digestive diseases produced in most of the cases by different strains of E. coli.

Mortality in the fattening period usually turns up in the two following weeks after

weaning. Changes in housing conditions (type of cage, temperature, etc…) and in the animal

nutrition, together with an incomplete development of the defensive mechanism in the ani-

mal, might favor the outbreak of the most frequent bacterial dysbiosis (colibacilosis and

clostridiosis) described in rabbits around weaning. Pathogen presence: either identified or a

multi factorial origin (bacteria, virus and coccidian) with many factors acting in cascade

(Peeters et al., 2000), are the principal factors which cost enteric diseases. However, there

are some factors as the intestinal saprophyte microbiota and the animal’s own defense

mechanism which tends to impede or reduce the growth of pathogens and the development

of diseases. This explains why some animals survive to controlled infections caused by

pathogenic strains, even though they present clinical symptoms (morbid animals). The

mechanisms by which a non-pathogenic species predominates over a pathogenic one, the so-

called ―competitive exclusion‖, are complex. According to Hampson et al. (2001), there are

several routes by which that competition can be produced, including differences in growth

on a specific substrate, efficiency of mucosa colonization and production of inhibitory sub-

stances of pathogen development (short chain fatty acids, de-conjugated bile salts and bacte-

ricides).

There is the general agreement that nutrition can concern the incidence of enteric

pathologic processes. The supply of unbalanced diets has been related with the appearance

of digestive disorders by means of two mechanisms:


16

- Promoting a higher retention time of the digesta in the digestive tract or

- Causing a higher flow or easily available substrates into the fermentative area.

In both situations, the alteration of the intestinal microbiota has been postulated as

the possible primary cause of these pathologies. Considering the particular case of rabbit

nutrition, level of starch and fibre (usually inversely correlated), type of fibre and level and

type of protein are the factors of the diet more related with the appearance of digestive trou-

bles (De Blas et al., 1981; de Blas et al., 1999; Gidenne, 2003; Gidenne and García, 2006).

However, the relationship between these nutrients and the intestinal microbiota (pathogenic

or commensal bacteria) has not been well established yet. The present thesis will focus on

fibre and starch, that accounts for around 60% of chemical composition of rabbit diets.

Fibre is the major fraction in rabbit diets and accounts for 35-50% of them. The im-

portance of fibre in rabbit is due to its influence on rate of passage, and its function as sub-

strate for microbiota, both related to rabbit health and performance. The concept of fibre, its

quantification and characterization either of the total fraction or of its different constituents

are still under revision and discussion (DeVries, 2010). The difficulty to find an agreement

on the dietary fibre concept lay down on its complex physical structure and chemical com-

position of cell wall, and by the wide diversity of cells types, and accordingly of cell walls,

that constitute the different plant tissues, and on the wide and different physiological effects

of the different constituents. It implies that the quantitative analysis of the whole compo-

nents of this fraction cannot be obtained by any analytical method or combination of meth-

ods.

Usually in animals and especially in rabbit nutrition, the insoluble fraction of fibre

has been considered more important, and this is reflected by the fact that nutritional recom-

mendations include only this fraction. Insoluble fibre accounts for around 77% of the total

fibre in rabbit diets, it can be quantified by using relatively easy methodology, and it is the

best dietary fraction to predict the dietary digestible energy value. Neutral detergent fibre

(NDF) is the most usual method to quantify insoluble fibre for herbivores (Mertens, 2003).

Many studies have demonstrated that rabbits require at least around 30 and 33% of NDF,

depending on the age. Lower NDF level delay the rate of passage, reduce performance and

increase the risk of suffering digestive pathologies (Gidenne and García, 2007; De Blas and

Mateos, 2010). However, not only the level of insoluble fibre is important for the animal,


17

but also its chemical (degree of lignification) and physical characteristics (particle size) as

they influence rate of passage and its fermentability (Nicodemus et al., 1999 and 2006).

Other fibre characteristics as water holding and buffer capacity also influence rabbit diges-

tive physiology (García et al., 2000).

On the opposite, there are few studies dealing with the effect exerted by soluble fibre

on rabbit traits, despite of it might have a major influence on intestinal microbiota than in-

soluble fibre, due to its higher fementability (Trocino et al., 2012). The reason is that soluble

fibre is a minority fraction inside total fibre (around 23% of total fibre in rabbit diets), as

well to its heterogeneous composition and the methodology troubles to quantify and charac-

terize it. One of the most important properties of soluble fibre might be its capacity of gel

forming and increase viscosity in the digestive tract, but is has been scarcely investigated in

rabbits.

Both, soluble and insoluble fibres are components of digestible fibre which is an im-

portant substrate for microbial growth. Faecal digestibility of insoluble fibre (NDF) is

around 0.3 (García et al., 2002) whereas that of soluble fibre might be around 0.75, and 0.3

of this value might be digested before reaching the caecum (Gidenne, 1992; Carabaño et al.,

2001). However, it is not possible to separate the effects of digestible fibre coming from

soluble or insoluble fibre with the available information, as the feedstuffs rich in soluble

fibre are also usually rich in digestible insoluble fibre (.e.g. sugar beet pulp). Besides, the

nutritional and physiological importance of the site of fibre fermentation (that might depend

on the proportions of soluble and insoluble digestible fibre) is still not clarified.

Most of the effects exerted by fibre on the rabbit depend on its effect on intestinal

microbiota. However, as it has already been mentioned, there was an important difficulty to

study the influence of any dietary component on microbiota, as the traditional cultivate tech-

niques only allowed to work with around one fourth of the intestinal microbiota. Alternative

techniques for solve this problem were use as the volatile fatty acid concentration, fibrolytic

activity or the microbial nitrogen synthesized. But in many circumstances they do not seem

to reflect adequately the changes produced in the microbiota population (Abecia et al.,

2007). Recently, the development of new molecular tools to characterize intestinal microbio-

ta allow to study more properly what happen with microbiota, and consequently identify

new effects of the different dietary fractions (Carabaño et al., 2006).


18

Starch accounts for less than 18% in rabbit diets and is almost completely digested in

the digestive tract of rabbits. In fact, faecal digestibility of starch during week 4 of life, in

still-suckling young rabbits beginning to consume feed, has been observed to be almost total

(0.98-0.99) practically independently of the source of starch (Blas, 1986; Gidenne et al.,

2007). However, higher faecal losses of starch have been reported for different sources of

maize in youngest rabbits (Gidenne and Pérez, 1993; Maertens and Luzi, 1995).

Starch digestion mainly takes place in the small intestine. But, it starts in the stomach

and can also be extended to the large intestine. Ileal digestibility of the starch not always

reaches a 100% value, and depends of his botanical origin and of the feed manufacturing

processes, as well as of the maturity of the digestive system of the animal. For this reason,

Asp et al. (1992) introduced in humans the concept of resistant starch, which is defined as

the fraction of starch and intermediate metabolites of its degradation which escape to the

enzymatic digestion and enter in the large intestine where they could be fermented. In this

sense, different studies have demonstrated the presence of amylase activity in the caeco-

colic segment (Yoshida et al., 1968; Blas, 1986; Makkar and Singh, 1987; Marounek et al.,

1995) and it has also been reported the presence of stable high counts of amylolytic bacteria

in the caecal contents of 2- to 7-week old rabbits (Padilha et al., 1995). The amount of starch

fermented in the caeco-colic segment of 4- to 6-week old rabbits can range from 0.03 (Gal-

lois et al., 2008) in a diet containing 113 and 147 g/kg DM of starch (wheat and wheat bran)

and cellulose respectively, to 0.11 (Gutiérrez et al., 2002) in a diet containing 226 (wheat)

and 141 g/kg DM of starch and cellulose. This result suggests that the amount of starch

reaching the caecum increase at early ages when the rabbit is fed with high-starch diets, and

that an interaction with the source of starch cannot be excluded. As it was observed by

Gutiérrez et al. (2002b) who reported higher starch concentration in the terminal ileum of

early-weaned 35-day old rabbit when using pea instead of wheat (60 and 36 g/kg DM, re-

spectively).

As faecal digestibility of starch is almost complete (0.99), either extrusion or cooking

or enzyme supplementation cannot increase it. But they can reduce the amount of starch

escaping from the small intestine as suggest the significant decreases in ileal starch concen-

tration in 35-days-old rabbits reported when cooking pea or wheat and add enzymes (Gutiér-

rez et al., 2002) or extruding maize in 29- or 50-days-old rabbits (Gidenne et al., 2005).


19

Evaluation of this resistant starch could be of particular interest in order to study the

possible consequences on caeco-colic fermentative activity, as well as on the stability of the

microbial ecosystem and the digestive health. Besides, the compounds resulting from bacte-

rial fermentation (mainly lactate and volatile fatty acids VFAs) can be used for the animal as

energetic substratum but with a lower energetic efficiency, which in humans has been esti-

mated as 45% of the enzymatic hydrolysis (Livesey, 1990). However, as it also occurs with

fibre, there are few studies which deal the effect of starch flux on microbiota population.

Additionaly, many experiments have been performed to elicit the respective effects

of fibre and starch on the incidence of diarrhea in the growing rabbit, particularly just after

the weaning (Colin et al., 1976; De Blas et al., 1986; Blas et al., 1994; Bennegardi et al.,

2001). This period is particularly critical since there is a large incidence of digestive trou-

bles, and also due to the active digestion maturation process which occurs by the time that

feed intake is increasing sharply. An increase in the dietary starch/fibre ratio (<30% NDF,

<15% ADF, >20% starch) could lead to both a lower ileal flow of dry matter and bacterial

biomass production in the caecum of the young rabbit, which is often associated with a low-

er fermentative activity (higher pH), modified fermentation pattern (higher butyrate propor-

tion) and a lower fibrolytic activity of bacteria (Bellier and Gidenne, 1996; Gidenne et al.,

2000, 2002, 2004a; Nicodemus et al., 2003a, 2004).

However, it still remain difficult to explain how these changes in caecal digestive

processes determine the greater incidence of digestive troubles (diarrhea mainly) observed

with low fibre diets. Probably the microbial ecosystem is largely affected, such the archaeal

community who was twofold higher with a standard diet than with a fibre-deficient (Benne-

gadi et al., 2003). Furthermore, when dietary NDF was reduced from 30 to 25%, microbiota

diversity increased at ileum but was reduced at the caecum (Nicodemus et al., 2004). Be-

sides, the negative effect of a deficient fibre level on rabbit digestive health was also shown

using experimental infection model reproducing a colibacillosis (Gidenne and Licois, 2005).

The main limitation of all these experiments were that compared diets where the lev-

el of fibre was inversely related to the level of starch. Consequently, when a study reported a

positive effect of an increase of dietary fibre content on digestive health, it was in fact diffi-

cult to exclude if there was also and effect of the reduced starch level. This question has

tried to be elicited in some experiments. De Blas et al (1986) and Carabaño et al. (1988),

using eight diets with a wide ranges of fibre (from 9.8 to 32% ADF on DM) and starch


20

(from 12 to 30% on DM), showed that fibre more than starch explain the changes observed

in the level of fermentation and in the protein content in the caecum (mainly from microbial

protein). Diets with similar level of fibre and differing in starch content did not produce

changes in the cecal fermentation, suggesting that starch was digested at ileum. Furthermore,

mortality was more associated to ingestion of fibre deficient diets than with variations in

starch content. Similar results was observed by a large scale study using a network of 6 ex-

perimental breeding unit (GEC French group) through a 2x2 factorial design (two level of

starch ―12 vs 19%‖ combined with two ADF levels ―15 vs 19%‖. The results obtained

showed that only fibre (not starch) played a role in digestive trouble occurrence (Gidenne et

al., 20004b). Furthermore, by comparing iso-fibre diets but with several starch sources vary-

ing in their intestinal digestion (maize, wheat and barley), Gidenne et al. (2005) observed no

effect of starch ileal flow on diarrhea incidence in the weaned rabbit. Fibre level plays thus a

major role in the determinism of the digestive trouble in the classically weaned rabbit (28-35

days old). In the particular case of early weaned rabbits (25 days of age), Gutiérrez et al.

(2002) observed that mortality remained low and similar with diets having 36 vs. 30% of

NDF, but mortality tended to increase after a feed shift (at 39 d of age, from a commercial

diet) for those previously fed with 36% NDF diet.

Traditionally, enteric pathologies were controlled by the addition of antibiotics to

feed. However, the European ban on the use of antimicrobials to prevent digestive diseases

and the narrow range of molecules allowed as veterinarian treatment in rabbit farming have

stimulated the search of alternative solutions. Between the proposed alternatives, nutrition

has been outlined as one of the most important factors to bearing in mind as a consequence

of the effect that digestion products might have on the competitive exclusion phenomena as

well as on the mechanisms of intestinal barrier.

Consequently, the new aims/targets that it tries to be approached inside the frame of

the animal nutrition are:

- Maximize the intestinal functions of digestion, absorption and intestinal barrier, fo-

cusing on one hand on avoiding all those hurts that can take place on the intestinal

mucosa, as well as on favoring the mechanisms of repair of these hurts as soon as

they have turned up.


21

- Control substrate that favors the growth of microbiota, on promoting competitive ex-

clusion mechanisms or on direct inhibition of pathogen growth. To reach this objec-

tive it is necessary.

- Know the flow of nutrients in the areas with the greatest microbial population (at the

end of the small intestine and caecum).

- Characterize the saprophyte or beneficial microbiota and the pathogens.

- Characterize the mechanisms of competitive exclusion among pathogenic and non-

pathogenic populations.

- Understand the role of nutrition on digestive health needs a correct characterization

of the mechanism implicated in the competitive exclusion of pathogens by the com-

mensal bacteria and of the mechanisms of pathogenicity. First nutritional experi-

ments have characterized these mechanisms by measuring the end products of the

microbial activity (VFA etc …) or the physio-chemical parameters of the ecosystem

(pH) or the enzymatic activity of intestinal microbiota. However, few studies have

been devoted studying the effect of nutrition on the colonization of both commensal

and pathogenic bacteria. The use of classical bacterial cultivation techniques implied

a large number of assays to characterize the microbiota and led to a largely incom-

plete flora pattern. Nowadays, the new molecular techniques can be a useful tool in

order to understand the complexity of this relationship (Carabaño et al., 2006), which

let in turn a greater sophistication of rabbit diets (specially of early weaning) accord-

ing to the digestive limitations of these ages and finally minimize enteric problems

and mortality.

In a general way, this PhD Thesis tried to contribute in the definition of the carbohy-

drate requirements of young rabbits. The objectives of this PhD Thesis were to study the

effect of the type of fiber (soluble vs. insoluble), the type of cereal used as source of energy

(wheat vs. rice) and the type of processing of this starch (raw vs. boiled) on growth perfor-

mance, digestion and intestinal barrier in young rabbits weaned at 25 days of age. The above

mentioned general aim is framed in turn in a General Program of Resources and food-

processing Technologies.


22

2 CHARACTERIZATION AND QUANTIFICATION OF FIBRE AND

STARCH

2.1 Fibre

The definition of fibre is essential to develop a valid methodology for its analysis.

The heterogeneous tridimensional matrix of plant cell walls make that there is no available

substances that can be used as standards to validate of the different proposed methodologies

for fibre analysis. The concept of dietary fibre used in human nutrition, and extended to all

mammals, has been periodically reviewed and clarified (Hispley, 1953; Burkitt et al., 1972;

Trowell, 1974; DeVries and Rader, 2005; DeVries, 2010), and it is defined as the feed com-

ponents resistant to mammal enzyme digestion and absorption, and that can be partially or

total fermented in the gut. More specifically, presently the Codex Committee on Nutrition

and Foods for Special Dietary Purposes of the European Union is still discussing the defini-

tion of dietary fibre (but focused only in human nutrition), as there is no official/legal defini-

tion of dietary fibre at the EU. The definition that is being discussed is: Dietary fibre (DF)

means carbohydrate polymers with ten or more monomeric units, which are not hydrolised

by the endogenous enzymes of the small intestine of humans and belong to the following

categories: i) edible carbohydrate polymers naturally occurring in the food as consumed, ii)

carbohydrate polymers, which have been obtained from food raw material by physical, en-

zymatic or chemical means and which have been shown to have a physiological effect of

benefit to health as demonstrated by generally accepted scientific evidence to competent

authorities, and iii) synthetic carbohydrate polymers which have been shown to have a phys-

iological effect of benefit to health as demonstrated by generally accepted scientific evidence

to competent authorities. When derived from a plant origin, DF may include also fractions

of lignin and/or other associated compounds (mainly lignin, proteic fractions, phenolic

compounds, waxes, saponins, phytates, cutin, phytosterols, etc). Besides, the inclusion of

carbohydrates of 3 to 9 monomeric units, mainly oligosaccharides, is left up to each nation-

al authority. This definition do not mention any specific physiological benefits of DF to hu-

man health, as they cannot be only restricted to the colon. It also implies that dietary fibre

can only be truly measured by the digestive process of the animal.

The indirect estimation of dietary fibre can be performed by using different method-

ologies (reviewed by Bach Knudsen, 2001, and Mertens, 2003), where the no fibrous com-

ponents are extracted by solubilization, by enzymatic hydrolysis or by combining both pro-


23

cedures. Once isolated, fibre residue can be quantified gravimetrically (weighing the resi-

due) or chemically (hydrolyzing the residue and determining its single constituents: sugars

and lignin). According to these procedures there are three types of methodologies: chemical-

gravimetric, enzymatic-gravimetric and enzymatic-chemical. By this way total dietary fibre

can be quantified (non-starch polysaccharides and lignin) and separated into insoluble and

soluble fibre (in aqueous solution), and obtain its monosaccharide composition. The combi-

nation of the monosaccharide composition of fibre with additional chemical information

may allow describing better fibre structure that influence its physic-chemical properties, and

accordingly, the effect exerted in the animal on the digestive physiology and digestibility.

However, these methodologies are complex, expensive, with a relatively low reproducibility

(especially for monomers determination) and difficult to implement as routine analysis.

There are alternative definitions that try to approach to the animal physiology.

Mertens (2003) propose to define insoluble dietary insoluble fibre for herbivores, consider-

ing their special digestive features, as it affects their digestibility and rate of passage. Insolu-

ble dietary fibre is defined as the indigestible (lignin) or slowly digesting organic matter of

feeds (mostly hemicelluloses and cellulose) that occupies space in the gastrointestinal tract.

This definition excludes soluble and rapidly fermenting polysaccharides of plant cell walls

(fructans, gums and pectins) that do not occupy space in a liquid environment, and show

similar digestibility than starch or protein contained in the cellular cytoplasm (and their ef-

fects on health and performance are rarely studied). The quantification of insoluble dietary

fibre is performed by the NDF procedure of Van Soest (Mertens, 2002), that is a method

more simple, rapid, economical and reproducible than the methods derived from the dietary

fibre definition, and show very close results compared to the later (Table 1). In spite of these

advantages, NDF procedure is criticized due to its variability among laboratories, especially

when it is compared with the results obtained in the analysis of other feed constituents (Xic-

cato et al., 1996). This is partially explained to the different procedures to perform this anal-

yses (Van Soest and Wine, 1967; Robertson y Van Soest, 1980; Mertens, 2002) and the dif-

ferent adaptations of these methods used in each laboratory. For this reason, Uden et al.

(2005) ask for a better description of the methodology used to determine NDF (Table 2) and

recommend to follow the procedure described by Mertens (2002) (where NDF is expressed

ash free, and it is used amylase and sodium sulfite –that improve filtration and protein ex-

traction, but also may extract lignin and phenolic complexes-). The European Group on


24

Rabbit Nutrition has also proposed some recommendations for determining feed chemical

composition to improve the reproducibility among laboratories (Gidenne et al., 2001).

Acid detergent fibre (ADF) and crude fibre (CF) are alternative methods to quantify

insoluble fibre (both determined by AOAC procedures), but neither of them fit with total or

insoluble dietary fibre definitions mentioned. ADF do not quantify all the insoluble fibre as

it dissolves hemicelluloses. Besides its residue contains pectins, except if it has been previ-

ously extracted with neutral detergent solution. The main drawback of CF lies in the high

variability in the chemical composition of its residue, as depending on the feed, it can dis-

solve up to 60% cellulose, 80% hemicelluloses and 95% lignin. As a consequence, CF di-

gestibility was higher than that of nitrogen free extract in 25% of the feeds studied by Morri-

son (1956). For these reasons, these determinations are not very useful to explain the effects

exerted by fibre on the animal, but both have demonstrated to be very useful to predict die-

tary energy value (Wiseman et al., 1992), and also show a similar or even higher reproduci-

bility than NDF.

Table 1. Comparison of insoluble and soluble fibre obtained by different methods

(adapted from Bach Knudsen, 1997; Hall, 1997 and Mertens, 2003).

Insoluble Soluble

Dietary fibre

NDF NSP1 NDSF

2

Maize 9,9 9,5 0,9 —

Soybean meal 17,0 14,9 6,3 —

Wheat bran 43,4 42,5 1,5 —

Sugar beet pulp 40,7 45,8 40,7 37,3

Grass hay 52,7 57,7 3,1 3,4

Alfalfa hay 38,0 41,6 7,7 17,7

1 Non-starch polysaccharides.

2 Neutral detergent soluble fibre.

The most important characteristics of insoluble fibre are its degree of lignification

and particle size. Lignin content is determined by using the acid detergent method –using

sulfuric acid- (ADL) described by Robertson and Van Soest (1981). This method requires

the previous extraction with acid detergent solution, and it is more laborious and variable

than NDF (Xiccato et al., 1996). Another important feature of insoluble fibre is particle size,

especially the proportion of particles larger than 0.3 mm. Its quantification is not easy, due


25

to the difficulty to separate fibre particles from other particles. It must be considered that the

big particles rich in starch or protein will be extensively digested at the small intestine, and

accordingly do not exert the same function on rate of passage than fibre particles. The only

method used to determine particle size is by wet sieving as described by García et al. (1999)

and Lebas and Lamboley (1999). Later the residue can be ground and NDF analyzed as per-

formed by Nicodemus et al. (2009).

It is more complex to asses and characterize soluble than insoluble fibre. First, the

term soluble raise the question: soluble in what solution and in what conditions? Many of

the methods mentioned allow quantifying soluble fibre dissolving it with an aqueous phos-

phate buffered solution, precipitated with diluted ethylic alcohol, weighted the residue and

corrected by its protein and ash content. However, Theander et al. (1994) demonstrated that

this method not always recovered all insoluble fibre. Furthermore, the complexity method-

ology of these methods difficult their routine uses. An alternative is to assess the fibre dis-

solved by neutral detergent extraction, the neutral detergent soluble fibre (NDSF), proposed

by Hall (1997). This is determined by difference among total dietary fibre and NDF and

mainly includes fructans, galactans, β-glucans, and pectic substances. Recently, Martínez-

Vallespín et al. (2011) have proposed some modifications in NDSF procedure in order to

improve its accuracy. However, these methods have the disadvantage of using solvents quite

different to those found in the animal gut. Accordingly, it is not sure if the values obtained

will reflect the real ones and help to explain the physiological effects observed in the ani-

mals. Finally, another possibility is to calculate the soluble fiber content by difference: or-

ganic matter – (protein + fat + sugars + starch + NDF).

Figure 1 shows a diagram including the different fibre components and the way to

asses them derived from Van Soest procedure that is the most feasible alternative in practical

conditions.


26

Figure 1. Major constituents of dietary fibre (adapted from Hall, 2003).

Dietary insoluble fibre can also be estimated by using NIR technology, that have al-

ready demonstrated its usefulness for predicting dietary dry matter, protein, fat, starch, and

even the dietary digestible energy value. However, ADF is the only fibre fraction that can be

adequately estimated by this technique, whereas both NDF and ADL are estimated with

lower precision (Xiccato et al., 2003).

2.2 Starch

Starch is a type of carbohydrate found vastly in plant tubers and seed granules.

Starch is the major reserve polysaccharide of green plants and probably the second most

abundant carbohydrate in nature after cellulose (Hizukuri, 1996). Starch occurs naturally as

discrete particles, called granules which organized in concentric alternating semi-crystalline

and amorphous layers in granules of various sizes within the endosperm (Shivus et al.,

2005). After the disintegration and dissolution of these granules, the majority of starches can

be divided in two components: amylose and amylopectin. Amylose is a linear chain of D-

glucose (around 1000) with (1-4) links and amylopectin a branched chain of D-glucose

(around 12-120) with (1-4) and (1-6) links. Numerous processes currently used in animal

feed manufacturing are able to modify the starch granule, either slightly by steaming in the

pelleting process or strongly by using temperature combined with hydration and pressure in

the extrusion process (Blas and Gidenne, 2010). Upon heating in aqueous solution, starch

swells irreversibly and its crystalline structure collapse in a phenomenon known as gelatini-

Dietary fibre

Cell content

Cell wall

Oligosaccharides

Resistant starch

Fructans

Mannans-Galactomannans

Pectic substancesGlucomannans-Galactoglucomannans

β-glucans

HemicellulosesArabinoxylans

Arabinogalactans

Xyloglucans

Cellulose

Lignin

NSP1

NDF3

NDSF2

ADF4

ADL5

1 Non-starch polysaccarides. 2 Neutral detergent soluble fibre. 3 Neutral detergent fibre.. 4 Acid detergent fibre.. 5 Acid detergent lignin.


27

zation. Starch swelling is a property of amylopectin, whereas amylose has been known to

restrict it (Tester and Morrison, 1990). These gelatinization processes undergoes in two

steps. In the first step, between 60ºC and 70ºC of temperature, the granules breaking up

takes place (Colonna y Mercier, 1985). In the second step, around 90ºC, starch losses gran-

ule structure, remaining only fragments of amylopectin in an amylose solution. As the gelat-

inized starch cools in a humid environment, the amylose and amylopectin molecules associ-

ate to form a gel (Miles et al, 1985). Gelatinized starched of grains may improve enzymatic

access to glycosidic bonds and consequently, digestibility. However, if the viscous solution

is cooled or left at lower temperature for a long enough period, the linear molecules, amyl-

ose, and linear parts of amylopectin molecules retrograde and rearrange themselves to a mo-

res crystalline structure, in a process known as retrogradacion. The linear chains place them-

selves parallel and form hydrogen bridges, expelling water from the polymer network.

The treatment with -amylase of the starch submitted to the retrograded process, lib-

erates the non-retrograded starch, leaving the fraction that resist to hydrolysis that is called

resistant starch (Berry, 1986). It is particularly important due to it is not digested in the small

intestine and pass directly to caecum and colon where can be fermented by the microbial

population to volatile fatty acids (which energy value is lower than the glucose one), which

can be used as substrate for this bacterial population, producing methane and hydrogen

which reduce even more the energetic performance (Wiseman, 2006).

The molecular estructure of starch is affected by genetic, climate, agronomic condi-

tions and grain development. Morphological studies (microscopy and particle size analysis)

have reflected significant differences among starch granule shapes (polyhedral, irregular),

granular size and gelatinization temperature depending on the botanical origin (Table 3). In

general, the grains of tubers (e.g. potato) are big and spherical whereas those of the cereals

are small and polyhedric and those of the leguminous present a kidney shapes (Gallant,

1992).


28

Tabla 3. Properties of the starch granules of main raw materials uses in monogastric

nutrition (Westengen and Van del Poel, 2009)

Raw material Granule shape Granule size

(µm)

Amylose content

(%) Waxy corn Round 5-25 1 Corn Round or lenticular 5-25 28

Oat Polyhedric 5-10 23-24

Rice Polygonal 3-8 17-19

Bean Oval 30 24

Wheat Round or lenticular 2-35 23-27

Sorghum Round 15-35 23-28

Potato Oval 15-100 23

Starch properties depend on the physical and chemical characteristic such as mean

granule size, granule size distribution, amylose/amylopectin ratio, and mineral content

(Madsen and Christensen, 1996; Wani et al. 2010). The considerable differences found be-

tween grains and even within grains determine its nutritional value (Verstengen and Van der

Poel, 2009). It must be considered that digestion of starch depends on the availability of the

enzymes capable of breaking specific chemical bonds, the ability of the enzymes to come in

contact with the bonds, which will depend on the diferent proportions of the cereal grains:

Hull, pericarp, aleurone, endosperm and scutellum (see table 4), and the length of time that

the enzymes are in association with the substrates. Besides, the rate of passage of digesta

through the gut, which is affected by dietary, animal and eviromental factors, might also

alter substantially the time enzymes have to digest dietary ingredients and thus affecting

digestibility properties.

Table 4: Proportion of parts of cereal grains (%) (Kent and Evers, 1994)

Cereal Hull Pericarp&testa Aleurone Endosperm Embryo axis Scutellum

Wheat - 8,2 6,7 81,5 1,6 2,0

Barley 13 2,9 4,8 76,2 1,7 1,3

Oats 25 9,0 63,0 1,2 1,6

Rye - 10,0 86,5 1,8 1,7

Sorghum - 9,7 82,3 9,3

Maize - 6,5 2,2 79,6 1,2 10,6

Millet - 3,0 6,0 70,0 5,0


29

It is well known that grains with a high content of amylose starches are less well di-

gested than those which have starches which mainly consist of amylopectine. The rate of

digestion is very much lower in amylose compared to amylpectine (Petterson and Lindberg,

1997).

The minor components in starch which are either at the surface or inside the starch

granules are lipids and protens. The degree to which the granules are encapsulated will in-

fluence how starch will be digested in the small intestine (Verstegen and Van der Poel,

2009), due to, before the amylase can reach the starch, these proteins must be degraded first.

Cereal starches contain about 1% lipids and 0.25% proteins (Baldwin, 2001). Nonwaxy rice

starches (12.2% to 28.6% amylose) contain 0.9% to 1.3% lipids comprise of 29% to 45%

fatty acids and 48% phospholipids (Azudin and Morrison, 1986). Waxy rice starches (1.0%

to 2.3% amylose) contain negligible amountos of lipids (Azudin and Morrison, 1986).

The resistance degree to the raw starch digestion keeps relation with the degree of X-

ray diffraction, distinguishing three types of starch: A (highly digestible) of cereals, B of

tubercles as potato, more resistant to -amylase digestion and C of legumes, intermediate

between both.

Other classification could be the one proposed by Englyst et al. (1992): Rapidly di-

gestible starch (RDS), slowly digestible starch (SDS) and resistant starch (RS). In general,

starches with a high amounto of amylose are used as a source of resistant starch (RS) where-

as fully gelatinized waxy starches serve as a source of rapidly digestible starch (RDS):

Starch is usually determined in animal feeds or raw materials through the Ewers EC

(optical rotation determination) or enzymatic methods (hydrolysis followed bay glucose de-

termination). The two techniques provide, in general, very well correlated data, with slightly

higher values for the Ewers EC method (+0.5-4%). Differences for both methodologies are

greater for legumes than for cereals. And the difference lies in the fact that the Ewers EC

method may interfere with un-extracted sugars and polysaccharides.

However, during the last decade, a strong interest in quantify the different analytical

fractions of starch which resist to the small intestine enzymatic digestion has turned up. Two


30

methodologies have been developed (Champ, 1992; Englyst et al, 1992), which basically try

to reproduce which happens in vivo in the digestive tract. In both techniques, the first step is

the digestible starch extraction with an enzymatic hydrolysis: pancreatin and amylogluco-

sidase. The released glucose is measured by colorimetric, using a glucose oxidase kit. In

some cases, starch hydrolysis may be preceded by a proteolysis with trypsin and chymotryp-

sin in order to imitate the action of the stomach and the intestine. The resistant starch is

quantified directly in the residue (Champ, 1992) or by difference between the total starch

and the digestible starch that it is quantified separately (Englyst el al., 1992). The values

obtained by this methodology for RDS and SDS reflect the rate of starch digestion in vivo.

Values for RS are similar to the amounts of starch escaping digestion in the small intestine

of ileostomates, and are a guide to the amount of starch likely to enter in the colon for fer-

mentation.

2.3 The rabbit digestive tract

2.3.1. Effect of age and weaning on digestion

Animals present great changes in the gastrointestinal tract until they reach maturity.

Gastric pH tends to be acidified with the age (Brooks, 1978), at 28-35 days of age, rabbit

stomach pH varies within 3.8-4.2, which result significant different comparing with the pH

of adult animals of 2.0. Therefore, the first step of defence against pathogens is already

compromise in young animals. Besides, the gastric pH affects enzyme activities, such as

amylase and proteases, which increase their actions at lower pHs. Caecal pH also seems to

be affected by age, decreasing from 6.8 to 5.6 at 15 and 50 days of age, respectively (Padilha

et al., 1995).

Suckling rabbits consume mother’s milk up to 18 days of age, moment from which a

gradual substitution of milk for feed takes place. Maternal milk provides a diverse range of

substances that are developmentally delayed in the neonate and are thought to play a critical

role in gastrointestinal defense (Bauer et al., 2006; Maertens et al., 2006; Gallois et al.,

2007; Russ et al., 2010). Bioactive molecules such as immunoglobulin A (IgA) and lactofer-

rin are present in abundance and are potent anti-microbial agents. Maternal milk also con-

tains substances, including growth factors and cytokines, that promote the maturation of the

intestinal and the associated immune system. Interest in the immune-modulatory properties


31

is increasing due to certain constituents of maternal milk are capable of promoting immune

function in early life, which benefits may persist throughout adult life (Kelly et al., 2000).

This could explain the result observed in different works where it has been observed that

suckling rabbits excrete less pathogen coli bacilli (Gallois et al., 2005b) and contain less C.

perfringens in the caecum (Romero et al., 2007) than weaned animals at the same age and

consequently the low incidence of digestive problems in suckling rabbits consuming breed-

ing does feed.

In rabbits, the microbiota begins to develop during the lactation stage, but the fer-

mentative area only begins to grow after feed consumption. According to Lebas and Laplace

(1972), weight of caecal content (as % of total body weight) increases twofold between the

third and the fifth week of life, keeping this effect till the seventh week of life. Parallel to the

development of the fermentative area changes in the immune system are also observed. Das-

so et al. (2000) reported that the follicular area in the vermiform appendix increased between

the third and the sixth week of life and keep the same relative importance until the adult

stage. The proliferative area of the follicles also reaches a maximum at six weeks. Changes

in the immune system not only affects to the lymphocytes proliferation centers but in the

profile of lymphocytes in the lamina propria too. Campin el al. (2003) observed how the

proportion of total lymphocytes and the proportion of B cells increase in rabbits between 19

to 26 days of age as response to the antigens entering by the feed consumption and microbial

colonization. Weaning can accelerate the maturation of the immune system as suggests the

larger follicular area in the vermiform appendix and the increase of lymphocytes. Delgado et

al. (2010) also observed an increase after weaning (26 to 52 d of age) of gene expression of

several cytokines (IL-2 and IL-2 and IFN- in ileum and IFN- in appendix vermiformis)

probably related to a Th1 response. However symptoms of immaturity are still presented and

therefore weaned animals have a larger proportion of undifferentiated T cells that respond to

the double marking CD4+/CD8+ (Campin et al., 2003). Total lymphocytes recover at 32

days of age levels similar to the existing ones at 12-19 days, possibly in response to the es-

tablishment of a certain tolerance degree. All these changes may be closely related to the

evolution of intestinal microbiota after weaning (Delgado et al., 2012).

The activity of certain digestive enzymes can also be affected by the age and they

can be rapidly induced by the presentation of especific substrate (Kelly et al., 1991; Zhang et

al., 1997). Marounek et al. (1995) observed that amylase activity is two-fold higher compar-


32

ing animals of three months of ages to 1 month. Similar results have also been obtained by

Gutiérrez et al. (2002), who observed how while the lactose activity decrease from 25 to 35

days of age (4849 vs 681 U/mg of protein), saccharose and pancreatic α-amylase increased

during the same period of time, but no differences for the maltose activity were appreciated.

Factors affecting rabbit enzymes will be described more deepthly in the chapter ―Utilization

of carbohydrates: fibre and starch‖.

In a context of digestive and immune immaturity weaning takes place. Weaning in

rabbits is less stressful than in other species due to animal has already started to consume

feed. However, it still implies a situation of stress for the animal as consequence of the ma-

ternal separation, the change of housing condition and the social group. On the other hand, it

also involves a change in nutrition (from milk to feed) that supposes a decrease in the inges-

tion of immunoglobulin proceeding from the maternal milk and in the consumption of nutri-

ents (Gallois et al., 2005a), so the needs of the animal so much for the functions of growth or

for digestive system development might be not covered. It has been observed that viability

of weanling rabbits increase when weaning age is delayed (Feugier et al., 2006; Sánchez,

2006; Romero et al., 2007), effect that was even higher and more significant when compari-

son was made with weaning at very early age (as 23-d) or when sanitary conditions in the

farm were poor. However, more studies are needed to confirm this trend, as contradictory

results have been reported (Garrido et al., 2006). Early weaning also leads to a lower post-

weaning weight gain in comparison to suckling rabbits of the same age, although these dif-

ferences are compensated during the whole fattening period (Méndez et al., 1986).

At level of small intestine so much the weaning, as well as the change on feeding,

exercise an affect the morphology of the intestinal mucosa and on the enzymes associated to

it. To this respect, Gutiérrez et al. (2002) observed an intestinal atrophy in animals with 35

days weaned at 25 d in comparison to those animals that with the same age still remained

with their mothers. This was reflected by a shortening in the length of the villi and in an in-

crease in the crypts depth (585±55.9 and 69.7±6.1 µm at 35d suckling vs. 536±48.7 and

62.4±5.3 at 25d weaning). In this work, it was also seen how the histological damage on the

intestinal mucosa was accompanied by a reduction in the intestinal enzymes activities. On

this way, maltose, sucrose and lactose were reduced a 30, 40 and 72% respectively, com-

pared with sucking rabbits. This effect could be explained by the fact that these intestinal

enzymes activities are found in the apical part of the villi (Tsuboi et al., 1981, 1985).


33

However, the pancreatic -amylose was increased a 59% as a result of the weaning.

Similar effects of weaning on the intestinal enzymatic activities have also been recovered by

other authors. Corring et al. (1972) observed a positive relation between the increase of the

weight of the pancreas and the pancreatic -amylose activity in animals of 30-36d of age

weaned with 21-30d. Nevertheless, values of the -amylose pancreatic activity were in all

works, lower than those obtained in adult animals using the same methodology (García et

al., 1997).

Unlike what it happens in the small intestine, the large intestine development and the

bacterial colonization is positive affected by weaning (Xicatto et al., 2001; Gutiérrez et al.,

2002; Gallois et al., 2005a; Delgado et al. 2010, 2012), which favours (as it has already been

mentioned) favours diversification of the immune system.

Alteration in the mucosa morphology and on its functional ability worsen nutrients

digestibility, which provokes that larger amount of substrate reach terminal ileum, altering

the profile of the intestinal microbiota and can facilitate the translocation of bacteria. This

situation favours the appearance of enteric diseases that cause important mortalities in two

weeks following weeks after weaning.

2.3.2. Utilisation of carbohydrates: fibre and starch

2.3.2.1.-Fibre

As happens in other species, rabbits can only use fibre from the feed through micro-

bial fermentation in the digestive tract. Besides, the efficiency of this process significantly

determines the final utilization of the feed. It must not be forgotten, than in adult rabbits,

volatile fatty acid (VFA) absorption accounts for around 30% of the basal metabolism

(Hoover and Heitman, 1972; Parker, 1976; Marty and Vernay, 1984; Gidenne, 1994).

The dietary factors affecting the variability of fibrolytic activity have been scarcely

studied, but it seems that low fibre diets might reduce it at the caecum (Gidenne et al., 2000

and 2002). Type of fibre also influence fibrolytic activity, and sugar beet pulp and wheat

bran based diets increased caecal pectinolytic, cellulolytic and xilanolytic activities, respec-


34

tively (Falcao-e-Cunha et al., 2004). This activity is evident when ileal and caecal fibre di-

gestibility is determined.

Average faecal digestibility of total dietary fibre, NDF and soluble fibre (measured

as TDF-NDF) is around 0.4, 0.3 and 0.9, respectively, but they vary mainly according to the

type of fibre (Trocino et al., 2012). Previous studies have determined that precaecal digesti-

bility of insoluble fibre is between 0 and 0.19 (crude fibre), 0.37 (non-starch polysaccarides)

or 0.43 (NDF) (Yu et al., 1987; Gidenne y Ruckebusch, 1989; Gidenne, 1992; Merino and

Carabaño, 1992) and it would be related to the pectinolytic activity in the stomach and small

intestine of the rabbit (Marounek et al., 1995). The ileal digestibility of soluble fibre is high-

er than the insoluble fraction and range from 0.2 to 0.45 (Abad et al., 2012). The monomers

best digested at ileum are uronic acids, an important constituent of pectins. In fact, around

0.75-0.80 of the fibre fermented at ileum would be pectins that are the more soluble fibre

fraction with digestibilities that range between 0.4-0.6 (Gidenne, 1992; Carabaño et al.,

2001). On the opposite site, glucose and xylose, the major monomers in most fibre sources,

showed a much lower ileal digestibility even with values close to zero (from 0.2 to 0). Ac-

cordingly raw material richest in pectins are particularly well digested in rabbits, in which

time of caecal fermentation is relatively short (10 hours) (Gidenne et al., 2010).

The result of the microbial activity is the release in the gut of volatile fatty acids

(VFA), which concentration tend to increase in the caecum with NDF level and to be re-

duced with its degree of lignification (García et al., 2002). VFAs resulting of the microbial

fermentation are important due to they can modified the environment in which microorgan-

isms are developed. Prohaszka (1980) and Wallace et al. (1989) observed how either the

acidity or the VFA concentration reduced the density of Escherichia coli in in vitro cultures,

although no clear effects have been observed in rabbits in vivo (Gidenne and Licois, 2005).

On the other hand, VFA affect directly to the cellular proliferation, gut motility and to the

absorption and secretion processes of the gastrointestinal tract. The stimulant effect on the

cellular epithelium, seems to be more important for the butyric acid that for the propionic,

and for the propionic than for the acetic acid (Kripke et al., 1989; Sakata, 1995).

Butyric acid resulting from the anaerobic fermentation in the large intestine of food

available substrates, is used in a preferential way by the enterocythes of the intestinal muco-

sal as source of energy, allowing a better regeneration of the damaged digestive mucosa and


35

a reduction of the incident of enteric pathological processes (Vernay, 1987; Chiou et al.,

1994); Which has a notorious effect on the capacity of absorption of nutrients and on the

development of the immune system associated to mucosa GALT (Lanning et al., 2000).

Within the most important factors implied in fibre digestion, the combined effect of

the particle size and the type of fiber has been pointed out. However, level of fiber seems to

have little effect on it (Gidenne et al., 2010).

Since the time of retention of the digesta in the caecum represents two thirds of the

total transit time of the digesta (Gidenne et al., 1992; Merino and Carabaño 1992, García et

al., 1999), the fermentative activity of caecum will significantly determine the final digesti-

bility of fibre. Caecal microorganisms ferment in a preferential way the fraction of the diges-

ta that is more time retained (Gidenne et al., 2010), that is, the soluble fraction and the one

with a particle size lower than 0.3 mm (Gidenne, 1993). It is due to the segregation of parti-

cles that takes place in the caecum-colon junction (Björnhag et al., 1972). In conformity

with this digestive behavior, time of cecal retention is positively correlated with the propor-

tion of short particles and negatively correlated with the proportion of large particles (García

et al., 1999).

As a result of the low retention times, caecal microorganisms will ferment in turn,

the quickest fermentable fraction of fibre together with the endogenous substances reaching

the caecum (Carabaño et al., 2009; Abad et al., 2012). Some authors have pointed out how

type of fibre affects the caecum fermentation parameters: pH and VFAs concentrations,

which represent an indirect way for measuring microbial activity. Caecal acidity and VFA

content tends to increase with the utilization of soluble fibre and low lignified insoluble fibre

like sugar beet pulp, but not with the use of high lignified long fibre such as cereal straw or

sunflower hulls (Fraga et al., 1991; Carabaño et al., 1996; García et al., 2000 and 2002a;

Falcão-e-Cunha et al., 2003). Results from different works have also showed how the VFA

profile result quite stable opposite to variations in the level and type of fiber, being obtained

as average values: 76% acetic, 6.5% propionic, 17.5% butyric (García et al., 2002a). Alt-

hough, some authors (Fraga et al., 1991; Falcão-e-Cunha et al., 2003) have observed in-

creases in the levels of acetic acid and decreases in the levels of butyric when more digesti-

ble fibre like the resulting from sugar beet pulp are included. Parallel to VFA's concentration


36

in caecum, cecal pH is reduced with the inclusion of soluble and low lignified sources of

fibre like sugar beet pulp (García et al., 2002a).

Level, type of fiber and particle size of diets, also impact on the accumulation of the

digesta in the caecum (weight of the caecal contents) through its effect on the intestinal mo-

tility that also influence feed intake and dressing out performance (García et al., 2002a).

Weight of cecal content reaches minimal values at levels of 39.3 % NDF (as % of dry mat-

ter) and, at the same content of fibre in the diet, tends to increase with higher levels of solu-

ble fiber and short particles and, and tends to reduce with lignified fibre and large particles

(Gidenne et al., 2010).

Several authors have stressed the importance of soluble fibre (usually not controlled

in formulation) on caecal fermentation (Carabaño el al., 2001; Rodríguez-Romero et al.,

2011) and on minimizing effect of ERE incidence (Fabre et al., 2006; Marguenda et al.,

2006), as well as its balance with insoluble fibre and its interaction with the sanitary condi-

tions of the farm. However, it is essential to study more more in depth the way of action of

soluble fibre, and especially its influence on intestinal microbiota, as Peters et al. (1995)

related it to enterotoxaemia due to Clostridium spiroforme.

2.3.2.2.-Starch

Starch is a quantitatively important component of rabbit feeds (contributing 100-250

g/kg). In normal circumstances its faecal digestibility is usually very high independently of

age (> 0.98, Blas and Gidenne, 2010); Thus, when is included in moderate quantities (15-

20%) turns out to be a suitable source of energy.

Starch digestion mainly takes place in the small intestine. However, starch may also

be degraded to some extent in other parts of the digestive tract, such as the stomach and

large intestine (Blas and Gidenne, 2010).

`

It cannot be realible quantified the amount of starch hydrolyzed in the stomach due in part to

the soft faeces recycling. Although, it has been observed by several authors that the starch

concentration in the gastric digesta is clearly less than in the diet (Fraga et al., 1984; Blas,

1986). Amylose from the soft faeces and saliva are the responsible of starch hidrolysis in the


37

stomach and its activity remain constant from week 4 of live independently of starch intake

(Blas, 1986), only being affected by the stomach pH. In fact, the amylase activity of the

stomach contents disappears completely if the pH is lower than 3.2 (Blas, 1986).

Although the rabbits stomach pH can reach very acid values in the antrum (Griffiths

and Davies, 1963), the buffer capacity of the diet, soft faeces and saliva prevent of acifica-

tion and maintain appreciable amylase activity (Alexander and Chowdhury, 1958; Griffiths

and Davies, 1963; Hörnicke and Mackiewicz, 1976; Blas, 1986; Vernay, 1986).

Main starch digestion takes place in the small intestine by the pancreatic amylase.

Although, other brush border enzymes of the intestinal mucose are also necessary (maltase,

amyloglucosidase). These enzymes transform dissacharides coming from the hydrolysis of

starch into monomers of glucose which absorbed in situ.

Different authors have reported that the α-amylase pancreatic activity might be insuf-

ficient at early eages, increasing rapidly between week 2 and 7 of life (Corring et al., 1972;

Marounek et al., 1995; Dojana et al., 1998; Scapinello et al., 1999) and is still increasing in

3-month-old rabbit (Marounek et al., 1995; Dojana el al., 1998). Similarly, the ontongenic

development of the amyloglucosidase activity has been studied by Ototumi et al. 2005 who

reported and increases between 37 and 60 days of age. Mores controversial results have been

obtained for intestinal maltase activity, where all experiments agree in the increase with the

age but differ in the period of this increase takes place, from 2 and 4 days of age (Toofanian,

1984 and Gallois, 2008) to 1 and 3 months (Marounek et al., 1995). Whereas Gutiérrez el al.

(2002a) and Scapinello et al. (1999) reported no changes in maltase activity between 25 and

35-day-old rabbit and 32 and 42-day-old rabbit, respectively.

The ability of the small intestine to modulate the enzyme secreation of higher starch

intake seems to be less remarkable in rabbits compared to other species. No differences ei-

ther in pancreatic amylase or in intestinal mucosal or intraluminal maltase in 42-d-old rabbit

consuming twice amount of starch than control were found by Debray et al. (2003). Fur-

thermore, Gidenne et al (2007) reported higher amylase activity in the total intestinal con-

tents in 42-day-old rabbits consuming one-third less starch than controls, while Gutiérrez et

al (2002a) reported no changes in pancreatic amylase in 35-day-old rabbits consuming twice


38

the amount of starch than controls and hinger pancreatic amylase and mucosal maltase when

consuming one-third less starch.

Weaning can also affect intestinal digestibility of starch due to it could impair the

intestinal mucosa morphology. Gutiérrez et al (2002a) observed rabbits weaned at 25 days of

age presented a significant decrease of the intestinal villous height comparing to animals

suckling at 35 days of age, and consequently a reduction of the disaccharidase activity

(maltose, lactose and sucrose). Similarly, results have also been reported by Gallois et al.

(2008) which observed a la lower amylase activity in the intestinal contents but higher malt-

ase activity in the intestinal mucose of 28-d-old rabbits that were still suckling than in those

weaned at 21-days- old, despite of there were no effect on mucosal morphology.

Starch undigested in the small intestine might be quickly hydrolysed and fermented

by the microbiota in the caeco-colic segment to lactate and volatile fatty acids (VFAs) ab-

sorbed in situ. Differences between ileal and faecal digestibility indicate certain degree of

starch fermentation, despite of the short mean retention time of digesta in the ileo-caecal

junction (6-12 h). Rabbit microflora does not remain constant throughtout the life of the rab-

bit and is stongly affected by the time of weaning. Stable high counts of amylolytic bacteria

in the caecal contents of 2- to 7-week old rabbits which remain quite stabilize from 15-day-

old have been reported (Padilha et al., 1995). Besides, Blas (1986) observe that amylase ac-

tivity in the caecal although is hardly affected with the age in 4- to 8-week-old rabbits, re-

sulted four times greater with starch rich diets compaired to low starch diets.

Ileal digestibility of starch range between 0.88 and 0.94 (Blas and Gidenne, 2010).

No clear evidence of effect of the dietary starch level of source on its ileal digestibility has

been detected. Ileal flow of starch is relatively low respect to the starch intake and has been

measured by different authors: 1.0-3.2 g/d (Gidenne, 1992), 0.3-1.3 g/d (Amber, 1997), 0.2-

2.3 g/d (Gidenne et al., 2000) and 0.5-1.1 g/d (Pinheiro, 2002), 0.6-2.3 g/d (Gutiérrez el al.

2002a), 0.8 g/d (Nicodemus et al. 2003) and 0.1 and 1.5 g/d (Soler et al., 2006) or 0.1- 0.4

g/d (Sánchez-Martínez, 2009). Thus, the amount of starch that might be fermented in the

caeco-colic segment of adult rabbits vary between 0.03 (Gallois et al., 2008) and 0.11

(Gutiérrez et al., 2002a). Results suggest that the amount of starch reaching the caecum in-

crease at early ages when rabbit is fed with hing-starch diets, and that an interaction with the

starch source cannot be excluded. As observed Pinheiro (2002), Blas et al. (1994), Gidenne


39

et al. (2005) and Gutiérrez et al. (2002), who reported higher starch concentration of starch

when include peas os maize-rich diets.

Faecal digestibility of starch in growing rabbits in almost complete (0.98-0.99), in

most cases independently of the age, starch intake or starch source (Blas y Gidenne, 2010).

Only minor but statistically significant reduction has been observed when including pea

(Blas, 1986; Gidenne et al., 2007) or maize and particularly in youngest rabbits (Michelan et

al. 2006; Cossu et al 2005). Villamide and De Blas (1991) for example, reported a faecal

digestibility of starch around 7% lower in corn starch diets compared than that of barley,

together with a lower crude protein digestibility (60% vs 67%). Nevertheless, some studies

using maize as the only or main starch source have reported faecal digestibilities of starch

similar to that from other sources 0.98 to 1.00 (Toral et al., 2002, Xiccato el al., 2002; Fur-

lan et al., 2003). This fact could be explained by the differences between varieties of particu-

lar grains or granule estructure, focusing in the ratio of amylose-amylopectin and in the

hardness of its endosperm, as a consequence of a strong link between protein matrix and

starch granules which are no broken in presence of water (Hoseney, 1991), which would be

in agreement with the lower mentionded protein digestibility (Sequeira et al., 2000).

Faecal digestibility of starch tends to decrease systematically in diets with lower

starch content in comparison with those of higher starch content, even with the same source

os starch (Blas, 1986; Blas et al., 1990, Parig-Bini et al., 1990; Gidenne, 1992, De Blas et

al., 1995; Amber, 1997; Gidenne and Pérez, 2000; de Arruda et al., 2002; Gutiérrez el al.,

2002). However, no clear conclusion should be obtained from these studies due they were

associated to a higher fibre intake that modify rate of passage and should conduce to a lower

efficiency in starch degradation.

Neither grinding affect starch digestibility, indicating no effect of particle size of

cereals on their nutritional value, unlike the particle size of fibre affects fibre digestion, rate

of passage and feed intake in rabbits (García et al. 1999). Despite everything, some authors

(Gidenne and Pérez, 1993; Carabaño, 1995) arguments that the coaser grinding of corn is

one of the reason o its relative lo digestibility. However, some authors Sequeira et al. (2000)

found greater digestibilities for wheat grinding coarsed compared to corn diets grinding fine.


40

In the last few years, alternatives as feed manufacturing processes (involving heating,

moisture and pressure) or enzyme supplementation are being studied in order not to improve

faecal digestion (already high) but reduce the amount of starch escaping from the small in-

testine. Unfortunately, there is little information available on this matter.

Nevertheless, some authors as Gutiérrez el al (2002) or Gidenne el al. (2005) have

reported significant differences in ileasl starch concentration by cooking pea or wheat and

maize, respectively, with no effect of faecal digestibility (0.99). Gutiérrez et al (2002a) also

observed a reduction of the ileal starch concentration using enzymes in diets incuding peas

and wheat.

Gutiérrez et al. (2000b) has studied the effect of substituting wheat and soyabean

meal with peas, enzyme supplementation (o vs. 1 g/kg of an enzyme complex containing β-

glucanases and α-amylases) and heat treatment of wheat and peas (raw vs. processed) in

starter isonutritive diets during the post-weaning period from 25 to 39 days of age. Ileal

starch concentration was reduced when using heat processed wheat and enzyme supplemen-

tation compared to raw peas and no enzyme supplementation. Besides, ileal starch concen-

tration was negatively correlated with weight gain and feed efficiency during the period

from 25 to 39 days and positively correlated with the mortality.

2.4 Criteria for evaluating intestinal health

2.4.1. Gut barrier function

The gastrointestinal (GI) tract is a complex ecosystem generated by the alliance of

GI epithelium, immune cells and resident microbiota. The three components of the GI

ecosystem have co-evolved such that each relies on the presence of the other two compo-

nents to achieve its normal function and activity (McCraken and Lorenz , 2001).

Intestinal barrier is not only involved in nutrients absorption. Besides, once the path-

ogen is implanted the intestinal barrier plays a fundamental role in the developments of the

protection processes, impeding the colonization of the mucosa by the opportunist pathogenic

organisms and the bacterial and toxins translocation (Kelly et al., 2000). This action in-

volves several mechanisms which are developed in cascade and turned on when required.


41

2.4.1.1.-The GI epithelium

The GI epithelium forms a barrier between the body and a luminal environment

which not only contains nutrients, but is laden with potentially hostile microorganisms and

toxins. The challenge is to allow efficient transport of nutrients across the epithelium while

rigorously excluding passage of harmful molecules and organisms into the animal.

The GI epithelium is protected by a mucosal layer of 400 µm thickness secreted by

the globet cells. Mucins are glyconconjugates made up of a protein core joined to sugar

chains of different length. The ending chains of these sugars can be sulphated or sialylated,

giving rise to acid mucins that are more resistant to microbial degradation that neutral mu-

cins that do not have those terminations (Vand Dijk et al., 2002). Longest and more

branched chains tend to present sulphated endings while the shortest and more linear chains

tend to become in sialylated (Mantle et al., 1989). Glycosiation patters are preprogrammed;

however several authors have observed how they can be affected by modifications resulting

from diets and weaning (Kelly and King, 1991; Kelly el al., 1983) and also by age (Taajtes

and Roth, 1990; Kelly and King, 1991).

Mantle and Thakore (1988) purified the small intestine and colon mucin of Rabbit.

Colon mucins are less acidic and less resistant to proteolysis. The amount and composition

of these mucins is dependent on bacterial colonization, but little known about the quantita-

tive and qualitative balance compatible with an improvement in intestinal health.

The glycosilation patterns are pre-programmed; however several authors have ob-

served how they can be modified depending on other factors like age and weaning (Kelly

and king, 1991; Kelly et al., 1993) as well as age (Taajtes and Roth, 1990; Kelly and King,

1991; King and Kelly., 1991).

The quantity and composition of the mucus layer might be altered in case of sick an-

imals (Koninks et al., 1988; Ehsanullah et al., 1982, Reid et al., 1984; Turani et al., 1986;

McGuckin et al., 2011). This situation can be even worse in the particular case of young

animals, which produce neutral and still immature mucins more capable to be degraded by

pathogens. Hezcko et al. (2000) observed an increase in the production of mucus in the first

moment of infection with E. coli 0103, suggesting that the mucosal layer is fundamental for


42

bacterial proliferation and adhesion to the mucosa and thus, for the pathogenic effects to be

shown. Controlled infection with Yersinia enterocolitica in rabbits has also shown an in-

crease the production of mucins, especially in the terminal end of the ileum and the proximal

colon where this intestinal disease is more severe (Mantle et al., 1989). Other studies (Kon-

inks et al., 1988) observed how mucin composition changed in rats infected with Nip-

postrongylus brasilensis from neutral to acidic after 10-15 days post-infection. This point

out how animals response against infections increasing the protection mechanisms in order

to facilitate the expulsion of pathogen (McGuckin et al., 2011).

Saprophyte bacteria also have the ability to regulate the synthesis, composition and

use of mucins, but the mechanisms are less studied than in the case of pathogens. Bac-

teroides, a significant genus in humans and rabbits seems to have mucinolytic properties

(Hill, 1986; Marounek et al., 2000; Sirotek et al., 2003). In humans, B. thetaiotaomicron

also has mucinolitic capacity. This would allow to saprophyte bacteria a better contact with

the mucosa, explaining the immunosuppressive effect of these bacteria (Kelly et al., 2005).

Pérez de Rozas et al. (2005) also showed that bacteria from the genera Bacteroides may act

as a probiotic in the case of ERE.

The mucus layer presents two different physical forms. The first one, very thin, vis-

cous and insoluble in water, sticks firmly to the epithelial surface. The second one, more

soluble, spreads towards the intestinal lumen and meets in contact not only with the diges-

tion product but also with the enzymes, the adhered microorganisms and the immunoglobu-

lin (Strugala et al., 2003).

The balance between degradation and secretion processes of the globet cells deter-

mines the thickness of the mucus layer. Damages on the mucus layer favour that undesirable

ligands join to the membrane receptors. Nevertheless, it is also necessary to consider that an

excessive thickness of the membrane can increase the distance for the diffusion of the prod-

ucts of the digestion, and so reduce the efficiency of absorption.

Under the mucus layer the intestinal epithelium with four different types of cells

(paneth cells, columnar epithelial cells, globet cells and enteroendocrine cells) are found.

The cellular differentiation process begins in the multipotent stem cells place in the base of

the crypts of Lieberkühn. Daughter cells then begin differentiation pathways to four differ-


43

ent cell types as they divide (Cheng and Leblond, 1974; Falk et al., 1998). Paneth cells mi-

grate to the base of the small intestinal crypt, while the remaining epithelial cell type mi-

grates for the crypt up to the small intestine villus or its analogue in the large intestine. Co-

lumnar epithelial cells also referred as to enterocytes in the small intestine or colonocytes in

the large intestine, comprise more than 80% of the epithelial cell types. Mucin-secreting

globet cells are present throughout the intestine, but increase in density along the proximal

axis. Enteroendocrine cells that integrate the intestine with the systemic nervous system are

dispersed throughout the small and large intestine (Furness et al., 1999). Intestinal epithelial

cells have a life cycle of about 2±5 days and, upon death, are exfoliated, along with any ad-

herent bacterial that may assist in the elimination of pathogens. Paneth cells have longer life

spans, surviving for up to 20 days, whereupon they are phagocytised.

Enterocytes are joined to each other through tight junctions which provide a strong

physical barrier, reducing intestinal permeability and avoiding macromolecules transport.

So, any damage on these tight junctions may favour the step of infectious and/or antigenic

agents (Fanning et al., 1999). Once mucosa continuity is lost, stem cells activate and rapidly

repair damage through the proliferation and differentiation of enterocytes by mean of pro-

cess call restitution. The rate of proliferation and differentiation of enterocytes is complicat-

ed and is affected by several factors such as: breed, age, genetic, environmental conditions

or by both resident and pathogenic microbiota (Sangild, 1997; Pluske, 2001; Kelly et al.,

2001), Without forgetting that diet can determine the intestinal microbiota and become ani-

mal more capable to suffering diseases.

So, maintenance of the structure and functionality of the intestinal mucosa does not

allow colonization by pathogenic bacteria and assures functionality of the digestive tract:

digestion, absorption, secretion and metabolism of nutrients. This explains why enteric dis-

eases are more frequent in younger animals which have an immature mucosa and during the

post-weaning periods, since there can be structural damage at the level of mucosa.

Nutritional strategies have been directed towards avoiding structural damage of the

mucosa and favouring repair mechanisms by supplying the necessary nutrients (McBurney,

1994).


44

2.4.1.2.-The gut associated lymphoid tissue (GALT)

Once the luminal barriers are overcome, bacteria and toxins get in contact with the

epithelium through membrane receptors. Then, the immune system kicks in developing sev-

eral mechanisms (specific and unspecific) which are turn on when required. Membrane re-

ceptors trigger a signal to the local immune system responsible for the protection from path-

ogens and for the regulation of the inflammatory response. The immune system is particular

complicated since is not only in charge of defending from infectious agents, but also it

should be to differentiate antigens from the diet and the intestinal saprophyte flora and to

develop an effective tolerance mechanism against them to avoid the development of aller-

gies to certain feeds and inflammation processes. Tolerance is a preferable response to de-

fence mechanism for the animal survival.

The immune innate response is considered to be the first line of defence, is unspecif-

ic and works rapidly independently that there has been previous exhibition to the antigen. In

the intestine, this type of response works through the action of the globet and paneth cells

which produce mucins and antimicrobial peptins (β-defensins) which eliminate bacteria and

avoid translocation through the intestinal barrier during infection processes (O’Neil et al.,

1999).

Additional to the innate immune response, there is an acquired immune response

half-full by the GALT, which begins after the exhibition to a certain antigen and offer a

long-term protection.

The GALT is distributed in two different ways:

- In an organised manner: in lymphoid follicles, distributed in the so called Peyer’s

patches, the vermiform appendix and sacculus rotundus.

- In diffused form: both in the lamina propria (lymphocytes of the lamina propria) as well

as in between the epithelial cells of the intestinal mucosa (intraepithelial lymphocytes).

Mucosal immune system can be functionally and physically divided into inductive

site: The Peyer’s patches and effector sites: mainly the lamina propria but also the intraepi-


45

thelial spaces (Figure 2). All they are communicated through the selective migration of lym-

phocytes.

Figure 2. Organization of the mucosal immunes system (The Immune System in

Health and Disease, 5th

edition. Janeway C.A. et al. 2001)

The organized lymphoid tissue contains many follicles where part of the intestinal

immune cells are made and where the immune response begins. In the former and better

characterized scenario, the M cell, a specialized epithelial cell type, transports antigen to an

underlying aggregation of lymphoid cells known as the Peyer’s patch, in which antigen

presentation and affinity maturation occur (McGhee et al., 1999; Owen, 1999). Activated,

antigen-specific lymphocytes then re-enter the circulation and migrate to effector sites in the

intestine and other mucosal sites via specific homing receptors. There is also and other

routes of entry which involve antigen transport via a transcellular epithelial pathway or, less

commonly, via a paracellular route (Perdue, 1999). Antigens enter by antigen-presenting

cells in the lamina propria and are presented to lamina propria cells (McGhee et al., 1999).


46

The lamina propria is populated by resident immune cells, including antibody-

secreting B cells (plasma cells), T cells (primary the CD4 helper T-cell type) and macro-

phages, which constitute over 60% of the cellular population in the lamina propria (McGhee

et all., 1999). The most common immunoglobulin produced in the intestine is IgA which

keep mucosa integrity against possible infections and toxic agents. Secretory IgA (SIgA) can

bind antigens present in the lamina propria and in the lumen, and can therefore prevent anti-

gens from traslocating the epithelium or within the lamina propria (McGhee et al., 1999).

Additionally, IgA is relatively non-inflammatory, as it does not activate complete, thus pre-

venting and excessive inflammatory response to ubiquitous dietary and microbial antigens.

Another important immunoglobulin that it is important in the intestine is IgE which it is syn-

thesized during allergic reactions.

The intraepithelial lymphocytes are the first line of defence from mucosal infections.

A larger proportion of intraepithelial lymphocytes (IELs) expresses CD8 and has cytolytic

activity (James and Kiyono, 1999; Lefrancëois and Puddington, 1999). The T cytotoxic

lymphocytes play an important role in the defense from viral and toxin agents and in recov-

ery of usual intestinal mucosa infections.

After recognition of the antigens, the macrophages and dendritic cells by means of

the proteolytic enzymes, degrade the antigen in immunogenic peptides and present it to the

cooperating T lymphocytes (Th) or CD4+. The Th lymphocytes modulate the response by

segregating cytokines (soluble proteins) that will activate the humoral response (Th2 com-

plex) or a cellular type response (Th1 complex). The humoral response activates the B lym-

phocytes, which will secrete the immunoglobulin (IgA, IgM, IgG) against the specific anti-

gens and will lead to a tolerance response. The cell response is mediated by the Th1 cyto-

kine complex and will lead to the activation (IL2: Interleukin 2) of the cytotoxic lympho-

cytes or CD8+ and to the death of cells which contain the antigen. In addition the cytotoxic

lymphocytes can also be activated by the mucosal cells (enterocytes) via the class I immune-

histocompatibility complex (MHCI). As well as in the humoral response, T cytotoxic answer

is antigen specific, therefore only antigens join to immune-histocompatibility complex will

unleash the immune response (Kagnoff, 1987). Humoral response are typical of the extracel-

lular infection processes, while cellular response use to appear in viral processes which use

the host cellular mechanisms for their own replication and requires destruction of the infect-

ed cells.


47

According to Knight and Crane (1994), the development of the rabbit immune sys-

tem and particularly of the B cells, can be divided into three stages. The first stage, foetal

and neonatal consists in a lymphopoiesis that will create the neonatal lymphocyte repertory

and will be mostly carried out in the medulla. The second phase consist in the creation of a

primary repertory of antibodies between week 3 and 8 of life by proliferation and diversifi-

cation of the GALT lymphocytes. The last stage corresponds to the formation of a second

repertory of antibodies in adults, which will be mainly centred on the proliferation of B cells

in the secondary lymph organs.

First stage in the development of the immune system depends of genetic factors and

placental transfer during gestation. Nonetheless, it seems that the primary repertory devel-

opment depends on intestinal microbiota. Several authors have found and abnormal devel-

opment of the GALT and a reduction in the number of lymphocytes in animals rose free of

bacteria (Stepankova and Kovaru, 1978; Tlaskalova and Stepankova, 1980). This lack of

development has also been observed in pigs. Rothkötter et al. (1994) found in germ free

pigs, that the maturity of the immune system at 45-d was similar to that of normal 5-d old

pigs, concluding that the stimulation of bacterial flora, as well as the presence of food anti-

gens, is necessary for a normal development of the GALT. A number of reviews, both in

rabbits (Knight and Winstead, 1997; Lanning et al., 2000) and in humans (Kelly et al., 2005)

indicate that the presence of saprophyte flora and, possibly of some specific genera, can be

crucial for the development of the primary repertoire.

2.4.1.3.-The intestinal microbiota

The intestinal host microbiota is composed by a high number of bacteria that play an

important role in the nutrition and in the health status of the animal by promoting nutrient

supply, preventing pathogen colonization and shaping and maintaining normal mucosal im-

munity. In these sense, it can be assured that the presence of certain saprophyte bacteria is

needed to maintain the immune equilibrium either in the adult intestine or in the one that is

developing (Kelly, 2005).

Mucosal immune responses to pathogenic bacteria and the mechanisms that govern

disease progression and outcome have been intensively researched. Recently, the influence


48

of the resident non-pathogenic o or ―commensal‖ microflora on mucosal immune function

and gut health has emerged. Major differences occur in the mucosal immune response to

pathogens and commensals. In part, this functional dichotomy is explained by the presence

of virulence factors in pathogenic species, which are generally absent in commensals. Addi-

tionally, immunological ―unresponsiveness‖ towards the resident commensals microflora is

thought to permit their successful colonization and co-existence within the host gut. Howev-

er, evidence of an active dialogue between members of the commensal microflora and the

host mucosal immune system is rapidly unfolding. This crosstalk is likely to affect immuno-

logical tolerance and homeostasis within the gut and to explain some of the differential host

responses to commensal and pathogenic bacteria (Kelly et al., 2005).

With the initiation of bacterial colonization, gross changes in the immune architec-

ture occur with the expansion and phenotypic differentiation of specific cell lineages of the

mucosal immune system. An important concept, in this regard, is that commensal bacteria

differ in their ability both to promote development of the gut-associated lymphoid tissues

and to maintain its function. It can thus be postulated that functionally significant bacteria

of the normal commensal microflora, are required to maintain immune homeostasis in both

developing and adult gut (Kelly, 2005).

There are several hypotheses which try to explain the immunological unresponsive-

ness to the resident commensal microbiota:

- Synthesis of specific polysaccharides and glycoproteins might permit close contact be-

tween viable bacteria and host epithelial cells.

- The expression of specific surfaces structures, such as elongation factors, flagellums,

etc. Could be important for intimate contact with the host.

- Quorum-sensing autoinducers (AIs), which are communication molecules released by

bacteria at high densities, might also modulate host responses through regulation of

commensal genes involved in gut colonization and host signalling.

- Another possibility is that the pro-inflammatory response induced by the commensal

microbiota was rapidly attenuated so much by the host defense systems and well as by

those of the intestinal microbiota.


49

Saprophyte bacteria in humans and monogastrics are involved in the competitive ex-

clusion processes (Bacteroides, Lactobacilli, and Salmonella) and seem to have approxima-

tion mechanisms to the mucosa and recognition (Toll-like receptors) that allows to modulate

the immune response towards its tolerance in the gastrointestinal tract (Kelly et al., 2005).

Adequate characteristic of the mucosal barrier not only improve the immune system,

but also the digestive and absorptive efficiency, decreasing the flow of nutrients to the cae-

cum which can favour growth of pathogens. Those strategies are not easy to develop since

there are many interactions among the factors involved, but, as our knowledge increases, it

appears that they are a useful way to control digestive diseases.

In rabbits, the existing information respect to the grade of characterization and identi-

fication of the intestinal microbiota was few and scarce until the recent development of the

new molecular techniques.

The originality of rabbit microbiota is that slowly established and presents a simple

composition dominated by strictly non-sporulated anaerobic bacteria (Carabaño et al., 2006)

with important individual variations, especially in the high parts of the intestine (Gouet and

Fonty, 1979).

Some authors (Penney et al. 1986; Fonty and Gouet, 1989; Padilha et al., 1996; Hul-

lar et al., 1996 and Canzi et al., 2000) have observed that most intestinal microbiota in rab-

bits is developed in caecum (1010

-1012

bacteria/gr. of digesta), proximal colon (107

bacte-

ria/gr. of digesta) and population is also found in the stomach (103-10

5 bacteria/gr. of diges-

ta ) (Penney et al., 1986). Nevertheless, environmental conditions made that they leave to be

viable 6-7 weeks post-ingestion (Jilge and Meyer, 1975).

Microbial composition do not rest constant throughout rabbit life, being strongly in-

fluenced by the time that weaning takes place (Padilha et al., 1995). During the first days

that follow to birth, the digestive tract of the rabbits is almost sterile, resulting impossible to

isolate any bacteria in young rabbits of less than days (Smith, 1965). After the first week of

life, the gastrointestinal tract is quickly colonizated by strictly non-sporulated anaerobic

Gram-positive bacterias, mainly Bacteroides (Gouet and Fonty, 1973; 1979; Penney et al.,

1986). Till 15 days of age, the facultative anaerobic bacteria in rabbit have a simple compo-


50

sition dominated by Streptococcus, whereas enterobacteriace are detected only occasionally

(Gouet and Fonty, 1979). As soon as solid food is consumed this situation is inverted.

After weaning, the main facultative anaerobic bacteria isolated in the gastrointestinal

belong to the Gram-positive genera Bacillus, Enterococcus and Staphylococcus and Gram-

negative Enterobacter or Escherichia coli (Forsythe and Parker, 1985; Canganella et al.,

1992). Respect to the strictly non-sporulated anaerobes four cellulolytic species usually

identified in ruminants are the Species dominant: Fibrobacter succinogenes, F. intestinalis,

Ruminococcus albus and R. Flavefaciens. Lactobacili are generally absent (Cole et al., 1983;

Gouet and Fonty, 1973; 1979; Penney et al., 1986) or exceptionally found (Yu and Tsen,

1993) in the gastrointestinal tract of rabbits.

Respect to the metabolic activities of the bacteria described, classified according to

their importance are: ammonia use, ureolytic, proteolytic and cellulotytic. Amynolytic flora

is present as soon as day 15 of life, before the rabbit consumes starch and then, does not de-

crease (Padilha et al., 1995). Fibrolytic activity of rabbit microbiota take place in the small

intestine and in the caecum, dominating in both segments pectinolytic activity compared to

xilanolytic and cellulolytic (Marounek et al., 1995).

The new molecular techniques have permitted a deeper knowledge of intestinal mi-

crobiota, although these methodologies are evolving continuously in order to obtain a more

robust information. There is an evolution of caecal microbiota from neonatal period, simple

and unstable community, into a more complex one at the end of the fattening period

(Combes et al., 2011). At weaning period it is observed in caecal microbiota a similar im-

portance of the Firmicutes and Bacteroidetes phylum, but few days after weaning (26 d of

age) there is an evolution increasing Firmicutes and decreasing Bacteroidetes phylum in the

caecum (Delgado et al., 2012). In adult rabbits, caecal microbiota shows a high diversity

and is dominated by the Firmicutes phylum (Monteils et al., 2008). The trends in the ileal

microbiota are not so clear like in the caecum (Delgado et al., 2012).


51

3 REFERENCES

Abecia, L., Fondevila, M., Balcells, J., Edwards, J.E., Newbold, C.J., McEwan, N.R (2007).

Effect of antibiotics on the bacterial population of the rabbit caecum. FEMS Microbiol.

Lett. 272:144-153

Abad, R., Ibáñez, M.A., Carabaño., R., García, J. (2013). Quantification of soluble fibre in

feedstuffs for rabbits and evaluation of the interference between the determinations of

soluble fibre and intestinal mucin. Anim. Feed Sci. Technol. (submitted).

Abad, R., Gómez-Conde, M.S., Carabaño, R., García, J. (2012) Efecto del tipo de fibra sobre la

digestibilidad ileal y fecal de la fibra. XXXVII Symposium de Cunicultura de ASESCU,

pp. 51-54. Barbastro.

Asp, N-G. (1992). Resistant Starch. Eur. J. Clin, Nutr. 46 (Suppl. 2): 1.

AOAC, Official Methods of Analysis (17th ed.), Association of Official Analytical Chemists,

Arlington, VA, 2000.

Bauer, E., Williams, B.A., Smidt, H., Mosenthin, R ., Verstegen, M.W.A. (2006). Influence of

dietary components on development of microbiota in singles-stomached species. Nutr. Res.

Rew. 19: 63-78.

Blas, E. (1986): El almidón en la nutrición del conejo: utilización digestiva e implicaciones

prácticas. PhD Doctoral Thesis. Zaragoza University, Spain.

Blas, E., Cervera, C. and Fernández-Carmona, J. (1994). Effect of two diets with varied starch

and fibre levels on the performances of 4-7 weeks old rabbits. World Rabbit Sci. 2: 117-

121.

Bach Knudsen, K.E. (1997). Carbohydrates and lignin content of plant materials used in animal

feeding. Anim. Feed Sci. Technol. 67: 319-338.

Bach Knudsen, K.E.. (2001). The nutritional significance of ―dietary fiber‖ analysis. Anim.

Feed Sci. and Technol. 90: 3-20.


52

Bellier, R. and Gidenne, T. (1996). Consequences of reduced fibre intake on digestion, rate of

passage and caecal microbial activity in the young rabbit. Brit. J. Nutr. 75: 353-363.

Bennegadi, N. Fonty, G., Millet, L., Gidenne, T. and Licois, D. (2003). Effects of age and

dietary fibre level on caecal microbial communities of conventional and specific pathogen-

free rabbits. Microb. Ecol. Health Dis. 15: 23-32.

Berry, C. (1986). Resistant starch: formation and measurement of starch that survives

exhaustive digestion with amylolytic enzymes during the determination of dietary fibre. J.

Cereal Sci. 4: 301-314.

Björnhag, G. (1972). Separation and delay of contents in the rabbit colon. Swedish Journal of

Agricultural Research 2: 125-136.

Brooks, D.L. (1978). Endemic Diarrhoea of domestic rabbits in California. PhD Doctoral

Thesis. California, Davis, USA.

Burkitt, D.P., Walker, A.R.P and Painter, N.S. (1972). Effect of dietary fibre on stools and

transit times, and its role in the causation of disease. The Lancet, Volume 300, Issue 7792:

1408-1412.

Campín, J., Eiras, P., Rebollar, P.G. and Carabaño, R. (2003). Estudio del tejido linfoide

asociado a intestino en gazapos en torno al destete. ITEA 24: 660-662.

Canzi, E., Zanchi, R., Camaschella, P., Cresci, A., Greppi, G.F., Orpianesi, C., Serrantoni, M.

and Ferrari, A. (2000). Modulation by lactic acid bacteria of the intestinal ecosystem and

plasma cholesterol in rabbits fed a casein diet. Nutrition Research. 20: 1329-1340.

Carabaño, R. (1995). Valor nutritivo de los cereales para Conejos. XI Curso de Especialización.

Avances en Nutrición y Alimentación Animal. Pp: 40-46.

Carabaño, R., de Blas, J.C. and García, A.I. (2000). Recent advances in nitrogen nutrition in

rabbits. World Rabbit Science 8: 14-24.


53

Carabaño, R., García, J. and de Blas J.C. (2001). Effect of fiber source on ileal apparent

digestibility of non-starch polysaccharides in rabbits. Anim. Sci. 72: 343-350.

Carabaño, R., Badiola, I., Licois, D. and Gidenne, T. (2006). The digestive ecosystem and its

control though nutritional or feeding strategies. In: Maertens, L., Coudert, P. (Eds). Recent

Advances in Rabbit Sciences. COST adn ILVO Publication, Merelbeke, Belgium, pp. 211-

227.

Carabaño, R., Villamide, M.J., García, J., Nicodemus, N., Llorente, A., Chamorro, S., Menoyo,

D., García-Rebollar, P., García-Ruiz A.I., de Blas J.C. (2009). New concepts and objectives

for protein-amino acid nutrition in rabbits: a review. World Rabbit Sci. 17:1-14.

Champ, M. and Colonna, P. (1993). Importance de l’endommagement de l’almidon dans les

aliments pour animaux. INRA Production Animales 6: 185-198.

Chiou, P. W.S, Yu, B. and Lin, C.H. (1994). Effect of different components of dietary fibre on

the intestinal morphology of domestic rabbits. Compt. Biochem. Physiol. 108A: 629-638.

Colonna, P. and Mercier, C. (1985). Gelatinization and melting of maize and pea starches with

normal and high-amylose genotipes. Phytochemistry 24: 1667-1674.

Combes., S., Michelland, R.J., Monteils, V., Cauquil, L., Soulie, V., Tran, N.U., Gidenne, T.,

Fortun-Lamothe, L. (2011). Postnatal development of the rabbit caecal microbiota

composition and activity. FEMS Microbiol. Ecol. 77, 680-689.

Corring, T., Lebas, F. and Courtout, D. (1972). Contrôle de l’evolution de l’equipement

enzymatique du pancréas exocrine de la nasissance à 6 semanines. Annales de Biologie

Animale, Biochemie et Biophysique 12: 221-231.

Dasso, J.F., Obiakor, H., Bach, H., Anderson, A.O., Mage, R.G. (2000). A morphological and

immunohistological study of the human and rabbit appendix for comparison with the avian

bursa. Dev Comp Immunol 24: 797-814.


54

De Blas, J.C., Pérez, E., Fraga, M.J., Rodríguez, J.M. and Gálvez, J. (1981). Effect of diet on

feed intake and growth of rabbits from weaning to slaughter at different ages and weights.

J. Anim. Sci. 52: 1225-1232.

De Blas, J.C., Santomá, G., Carabaño, R. and Fraga, M.J. (1986). Fiber and starch levels in

fattening rabbit diets. J. Anim. Sci. 63: 1897-1904.

De Blas, J.C., Taboada, E., Mateos, G.G., Nicodemus, N. and Méndez, J. (1995). J. Anim. Sci.

73: 1131-1137.

De blas, J.C. and Mateos, G.G. (2010). Feed formulation. De Blas, J. and Wiseman, J., Eds.

2nd ed. The nutrition of the rabbit. CABI Publishing. Wallingford, UK. pp: 222-232.

De blas, J.C., García, J. and Carabaño, R. (1999). Role of fibre in rabbits. A review. Ann.

Zootech. 48: 3-13.

Debray, L., Fortun-Lamothe, L. and Gidenne, T. (2002). Influence of low dietary starch/fibre

ration around weaning on intake behaviour, performance and health status of young rabbit

does. Anim. Res. 51: 63-75.

Delgado, R., Menoyo, D., Badiola, I., Perez de Rozas, A., Carabaño, R., García, J. (2010)

Evolución con la edad de los mecanismos de barrera intestinal. 2. Mofología intestinal y

sistema inmune. XXXV Symposium de Cunicultura de ASESCU. Pp. 95-98.

Delgado, R., Badiola, I., Perez de Rozas, A., Menoyo, D., García, J. and Carabaño, R., (2012)

Caracterización de la microbiota intestinal en gazapos tras el destete. XXXVII Symposium

de Cunicultura de ASESCU. Pp. 55-59.

De Vries, J.M. and Rader, J.I. (2005). Historical perspective as a guide for identifiying and

developing applicable methods for dietary fiber. J. AOAC Int. 88: 1349-1366.

De vries J.W. 2010. Validating oficial methodology commensurate with dietary fibre research

and definitions. In Van der Kamp J.W, Jones J., McCleary B., Topping D. (Eds). Dietary


55

Fibre, New Frontiers for Food and Health. Wageningen Academic Publishers. The

Netherlands. Pp. 29-48.

Dojana, N., Costache, M., Dinischiotu, A. (1998). The activity of some digestive enzymes in

domestic rabbits before and after weaning. Anim. Sci. 66: 501-507.

Englyst, H.N., Kingsman, S.M. and Cumming J.H. (1992). Classification and measurement of

nutritionally important starch fractions. Eur. J. Clin. Nutr. 46: 33-50.

Fabre, C., Juvero, M.A., Blas, E., Fernández-Carmona, J. and Pascual, J.J. (2006). Utilización

de un pienso rico en fibra digestible e indigestible y pobre en almidón en conejos de

engorde: ensayo en condiciones de campo. In: Actas del XXXI Symposium de Cunicultura.

Lorca. Pp. 67-72.

Falcao-e-Cunha, L., Peres, H., Freire, J.P.B. and Castro-Solla, L. (2004). Effects of alfafa,

wheat bran or beet pulp, with or without sunflower oil, on caecal fermentation and on

digestibility in the rabbit. Animal Feed Science and Technology 117: 131-149.

Fanning, A.S., Mitic, L.L., Anderson, J.M. (1999). Transmembrane proteins in the tight

junction barrier. J. Am. Soc. Nephrol. 10:1337-1345.

Feugier, A., Smit, M.N., Fortun-Lamothe, L. and Gidenne, T. (2006). Fibre and protein

requirements of early weaned rabbits and the interaction with weaning age: effect on

digestive health and growth performance. Anim. Sci. 82: 493-500.

Fonty, G. and Gouet, P.H. (1989). Fiber degrading microorganisms in the monogastric

digestive tract. Animal Feed Science and Technology 23: 91-107.

Forsythe, S.J. and Parker, D.S. ((1985). Nitrogen metabolism by the microbial flora of the

rabbit. J. App. Bacteriol., 58: 363-369.

Fraga, M.J., Pérez De Ayala, P., Carabaño, R. and De Blas, J.C. (1991). Effect of type of fiber

on the rate of passage and on the contribution of soft feces to nutrient intake of finishing

rabbits. J. Anim. Sci. 69: 1566-1574.


56

Gálfi, P. and Bokori, J. (1990). Feeding trial in pigs with a diet containing sodium n-butyrate.

Acta Vet. Hung. 38: 3-17.

Gallant, D.J., Bouchet, B., Buleon, A. and Pérez, S. (1992). Physical characteristic of starch

granules and susceptibility to enzymatic degradation. Eur. J. Clin. Nutr. 46: S3-S16.

Gallois, M., Fortun-Lamothe, L., Michelan, A. and Gidenne, T., (1998). Adaptability of the

digestive function according to age at weaning in the rabbit: II. Effect on nutrient digestion

in the small intestine and in the whole digestive tract. Animal 2: 536-547.

Gallois, M., Gidenne, T., Fortun-Lamothe, L., Le Huerou-Luron, M. and Lalles, J.P. (2005a).

An early stimulation of solid feed intake slightly influences the morphological gut

maturation in the rabbit. Reprod. Nutr. Dev. 45: 109-122.

Gallois, M., Boullier, S., Milon, A., Gidenne, T. (2005b). Age au sevrage et sensibilité a une

infection expérimentale par une souche d’E coli 0103. 11èmes Journées de la Reserche

Cunicole,INRA, Paris.

Gallois, M., Gidenne, T., Tasca, C., Coudert, C., Milon, A., Boullier, S. (2007). Maternal milk

contains antimicrobial factors that protect young rabbits from enteropathogenic Escherichia

coli infection. Clin. Vac. Immunol.: 585-592.

García, G., Gálvez, J.F., De Blas, J.C. (1992). Substitution of sugar beet pulp for barley in diets

for finishing rabbits. 2. Effect of growth performance. Journal of Applied Rabbit Research

15: 1017-1024.

García, J., Gálvez, J.F. and De Blas, J.C. (1993). Effect of substitution of sugrabeet pulp for

barley in rabbits for finishing rabbits on growth performance and on energy and nitrogen

efficiency. J. Anim. Sci. 71: 1823-1830.

García, J., Villamide, M.J. and De Blas, J.C. (1997). Energy, protein and fiber digestibility of

soybean hulls for rabbits. World Rabbit Sci. 5: 111-113.


57

García, J., Carabaño, R. and De Blas, J.C. (1999). Effect of fiber source on cell Wall

digestibility and rate of passage in rabbits. J. Anim. Sci. 77: 898-905.

García, J., Carabaño, R., pérez-alba, L. and De Blas, J.C. (2000). Effect of fiber source on cecal

fermentation and nitrogen recycled through cecotrohy in rabbits. J. Anim. Sic. 78: 638-646.

García, J., Gidenne, T., Falcao-E-Cunha, L. and De Blas, J.C. (2002a). Identification of the

main factors that influence caecal fermentation traits in growing rabbits. Anim. Res. 51:

165-173.

García, J., Nicodemus, N., Carabaño, R. and De Blas (2002b). Effect of inclusión of defatted

seed meal in the diet on digestión and performance of growing rabbits. J. Anim. Sci. 80:

!62-170.

Garrido, S., Nicodemus, N., Chamorro, S. and De Blas, J.C. (2006). Influencia de la edad al

destete (25 vs 35), la nave y el período sobre la mortalidad y parámetros productivos de

gazapos. XXXI Symposium Cunicultura Asescu, Lorca. pp: 95-101.

Gidenne, T. (1992). Effect of fibre level, particle size and adaptation period on digestibility and

rate of passage as measured at the ileum and in the faeces in the adult rabbit. Br. J. Nutr. 67:

133-146.

Gidenne, T. (1993). Measurement of the the rate of passage in restricted-fed rabbits. Effect of

dietary cell wall level on the transit of fiber particles of different sizes. Anim. Feed Sci.

Technol. 42: 151-163.

Gidenne, T. (1994). Estimation de la production d’acides gras volatils et de leur apport

energetique, chez le lapin: effets de la teneur en fibres du regime. Viémes Journées de la

Recherche Cunicole, 2:293-299. La Rochelle.

Gidenne, T. (2000). Recent advances in rabbit nutrition: emphasis on fibre requirements. A

review. World Rabbit Science 8: 23-32.


58

Gidenne, T. (2003). Fibres in rabbit feeding for digestive troubles prevention: respective role of

low-digested and digestible fibre. Livest. Prod. Sci. 81:105-117.

Gidenne, T. and Ruckebusch, Y. (1989). Flow and rate of passage studies at the ileal level in

the rabbit. Reproduction Nutrition Development 29: 403-412.

Gidenne, T. and Pérez, J.M. (1993). Effect of dietary starch origen on digestion in the rabbit. 2:

starch hydrolysis in the small intestine, cell wall degradation and rate of passage

measurements. Anim. Feed Sci. and Technol. 42: pp. 249-257.

Gidenne, T. and Bellier, R. (2000). Use of digestible fibre in replacemente to available

carbohydrates. Effect on digestion, rate of passage and caecal fermentation pattern during

the growth of the rabbit. Livest. Prod. Sci. 63: 141-152.

Gidenne, T. and Pérez, J.M. (2000). Replacement of digestible fibre by starch in the diet of the

growing rabbit. I. Effects on digestion, rate of passage and retention of nutrients. Annales

de Zootechnie 49: 357-368.

Gidenne, T., Pinheiro, V., Falcao-E-Cunha, L. (2000). A comprehensive approach of the rabbit

digestion: consequences of a reduction in dietary fibre supply. Livest. Prod. Sci. 64: 225-

237.

Gidenne, T., Pérez, J.M., Xiccato, G., Trocino, A., Carabaño, R., Villamide, M.J., Blas, E.,

Cervera, C., Falcao-E-Cunha, L. and Maertens, L. (2001). Technical note: Attempts to

harmonize chemical analyses of feeds and faeces for rabbit feed evaluation. World Rabbit

Sci. 9: 57-64.

Gidenne, T., Jehl, N., Segura, M. and Michalet-Doreau, B. (2002). Anim. Feed Sci. Techn. 99:

107-118.

Gidenne, T., Jehl, N., Lapanouse, A. and Segura, M. (2004a). Inter-relationship of microbial

activity, digestion and gut health in the rabbit: effect of substituting fibre by starch in diets


59

having a high proportion of rapidly fermentable polysaccharides. British Journal of

Nutrition 92: 95-104.

Gidenne, T., Mirabito, L., Jehl, N., Pérez, J.M., Arveux, P., Bourdillon, A., Briens, C.,

Duperray, J. and Corrent, E. (2004b). Impact of replacing starch by digestible fibrfe, at two

levels of lignocellulose, on digestion, growth and digestive health of the rabbit. Anim. Sci.

78: 389-398.

Gidenne, T. and Licois, D. (2005). Effect of high fibre intake on the resistance of the growing

rabbit to an experimental inoculation with an enteropathogenic strain of Escherechia coli.

Anim. Sci. 80: 281-288.

Gidenne, T, Segura, M. and Lapanouse, A. (2005). Effect of cereal sources and processing in

dietgs for the growing rabbits. I. Effects on digestion and fementative activity in the

caecum. Animal Researc h 54: 55-64.

Gidenne, T. and García, J. (2006). Nutritional strategies improving the digestive health of the

weaned rabbit. In: Maertens, L., Coudert, P. (Eds). Recent Advances in Rabbit Sciences.

COST adn ILVO Publication, Merelbeke, Belgium, pp. 229-238.

Gidenne, T., Debray, L., Fortun-Lamothe, L. and Le Huërou-Luron, I. (2007). Maturation of

the intestinal digestion and of microbial activity in the young rabbit: impact of the dietar

fibre:starch ratio. Comparative Biochemestry and Physiology part A 148, 834-844.

Gidenne, T., Carabaño, R. García, J. and De Blas, J.C. (2010). Fibre digestion. De Blas, J. and

Wiseman, J., Eds. 2nd ed. The nutrition of the rabbit. CABI Publishing. Wallingford, UK.

pp.: 66-82.

Gutiérrez, I., Espinosa, A., García, J., Carabaño, R. and De Blas, C. (2002a). Effects of level of

starch, fiber and lactose on digestion and growth performance of early-weaned rabbits. J.

Anim. Sci. 80: 1029-1037.


60

Gutiérrez, I., Espinosa, A., García, J., Carabaño, R. and De Blas, C. (2002b). Effects of starch

and protein sources, heat processing and exogenous enzymes in starter diets for early

weaned rabbits. Anim. Feed Sci. and Technol. 98: 175-186.

Gouet, P.H.and Fonty, G. (1973). Evolution de la microflore digistive du lapin hooxénique de

la naissance au sevrage. Ann. Biol. Anim. Bioch. Biophys. 19: 553-563.

Gouet, P.H.and Fonty, G. (1979). Changes in the digestive microflora of holoxenic rabbits from

birth until adulthood. Ann. Biol. Anim. Biochim. Biophys. 19:553-566.

Hall, M.B., Lewis, B.A., Van Soest, T.J. and Chase, L.E. (1997). A simple method for

estimation of neutral detergent-soluble fiber. J. Sci. Food Agric. 74: 441-449.

Hall, M.B. (2003). Challenges with nonfiber carbohydrate methods. J. Anim. Sci. 81: 3226-

3232.

Hampson, D.J., Pluske, J.R. and Pethick, D.W. (2001): Proceedings of the VIIIth International

Symposium on Digestive Physiology in Pigs. (Eds. A. Piva, K.E. Bach Knudsen y J.E.

Lindberg), pp: 247-261. CAB International, Wallingford, UK.

Hezcko, U., Abe, A. and Finlay, B.B. (2000). In vivo interactions of rabbit enteropathogenic

Escherichia coli 0103 with host: and electron microscopic and histopathologic study.

Microbes Infec. 2: 5-16.

Hipsley, E.H. (1953). Dietary ―fibre‖ and pregnancy toxemia. Br. Med. J. 2: 420-422.

Hizukuri, S. (1996). Starch: Analytical aspects. Eliasson, A.C. (ed.). Carbohydrates in food.

Marcel Dekker Inc. Basel, Switzerland, pp. 347-429.

Hoseney, R.C. (1991). Principios de Ciencia y Tecnología de los cereales. Acribia.

Hoover., W.H., Heitman., R.N. (1972). Effects of dietary fiber levels on weight gains, cecal

volume and volatile fatty acid production in rabbits. J. Nutr., 102: 373-380.

Hullar, I., Fekete, S., Szigeti, G. and Bokori, J. (1996). Sodium butyrate as a natural growth

promoter for rabbits. 6th World Rabbit Congress, Toulouse, France, Vol. 2: 175-179.


61

Janeway C.A., Travers, P. and Walport, M. (2001). Immunobiology: The Immune System in

health and disease. 5th edition. New York: Garland Science.

Kelly, D., Conway, S. and Aminov, R. (2005). Commensal gut bacteria: mechanisms of

immune modulation. Trend in Inmunology 26: 326-333.

Knight, K.L. and Crane, M.A. (1994). Generating the antibody repertoire in rabbit. Adv.

Inmunol. 56: 179-218.

Knight, K.L. and Winstead, C.R. (1997). Generation of antibody in rabbits.Curr. Opin.

Immunol. 9: 228-232.

Kripke, S.A., Fox, A.D., Berman, J.M. Settle, R.G. and Rombeau, J.L. (1989). Stimulation of

intestinal mucosal growth with intracolonic infusion of short chain fatty acids. J. Parent.

Enteral Nutr. 13:109-116.

Lanning D., Sethupathi, P. Rhee, K.J., Zhai, S.K. and Knight, K.L. (2000). Intestinal microflora

and diversification the rabbit antibody repertoire. J. Immunol. 165: 2009-2012.

Lebas, F., Gidenne, T., Pérez, J.M. and Licois, D. (1998). Nutrition and pathology. De Blas, C.,

Wiseman, J. (Eds). The nutrition of the rabbit CABI Publishing. CAB International,

Wallingford Oxon, UK. Pp. 197-213.

Lebas, F. and Lamboley, B. (1999). Méthode de determination par tamisage en phase liquide de

la taille des particules contenues dans un aliment granule pour lapin. World Rabbit Sci.

7:229-235.

Lebas, F. and Laplace, J.P. (1972). Mensurations viscérales chez le lapin. I. Croissance du foie,

des reins et des divers segments intestinaux entre 3 et 11 semaines d’age. Ann Zootech 21:

337-347.

Livesey, G. (1990). Energy values of unavailable carbohydrate and diets: an inquiry and

analysis. Am. J. Clin. Nutr. 51: 617-637.

http://www.garlandscience.com/textbooks/0815341237.asp


62

Maertens, L. and Luzi, F. (1995). The effect of extrusion in diets with different starch levels on

the performance and digestibility of young rabbits. Proceedings 9th Symposium on

Housing and Diseases of Rabbits, Furbearing Animals and Pet Animals. DVG, Celle,

Germany, pp. 131-138.

Maertens, L., Lebas, F., Szendro, Z. (2006). Rabbit milk: a review of quantity, quality and non-

dietary affecting factors. World Rabbit Sci. 14: 205-230.

Makkar, H.P.S and Singh, B. (1987). Comparative enzymatic profiles of rabbit caecum and

bovine rumen contents. Journal of Applied Rabbit Research 10: 172-174.

Mantle, M. and Thakore, E. (1988). Rabbit Intestinal and colonic mucins: isolation, partial

characterization, and measurement of secretion using an enzyme-linked immunoassay.

Biochem. Cell. Biol. 66: 1045-1054.

Margüenda, I., Carabaño, R., García-Rebollar, P., De Blas, C. and García-Ruíz, A.I. (2006).

Effect of the substitution of starch sources or wheat straw with beet pulp on growth

performance, mortality and carcass yield, under field conditions. In: Actas del 3er Congreso

de cunicultura de las américas. maringá, brasil.

Martínez-Vallespín, B., Navarrete, C., Martínez-Paredes, E., Ródenas, L., Cervera, C. and

Blas, E. (2011) Determinación de la fibra soluble en detergente neutro: modificaciones del

método original. XIV Jornadas sobre Producción Animal - AIDA, Tomo I, 292-293.

Marty, J. and Vernay, M. (1984). Absorption and metabolism of the volatile fatty acids in the

hindgut of the rabbit. Brit. J. Nutr. 51: 265-277.

Marounek, M., Vook, S.J. and Skrivanová, V. (1995). Distribution of the activity of hydrolytic

enzymes in the digestive tract of rabbits. British Journal of Nutrition 73: 463-469.

McBurney, M.I. (1994). The gut: central organ in nutrient requirements and metabolism. Can.

J. Physiol. Pharmacol. 72:260-265.


63

Mc.Crake n, V.J. and Lorenz, R.G. (2001). The gastrointestinal ecosystem a precarious alliance

among epithelium, immunity and microbiota. Microreview. Cellular Microbiology 3(1): 1-

11.

Mc.Ghee, J.R., Lamm, M.E. and Strober, W. (1999). Mucosal immune responses: an overview.

Mucosal Immunology. Ogra, P.L. (ed.). San Diego: Academic Press. pp: 485-506.

McGuckin, M.A., Linden, S.A., Sutton, P., Florin, T.H. (2011). Mucin dynamics and enteric

pathogens. Nat. Rew. Microbiol. 9:265-278.

Méndez, J., De Blas, J.C. and Fraga, M.J. (1986). The effects of diet and remating interval after

parturition on the reproductive performance of the comercial doe rabbit. J. Anim. Sci. 62:

1624-1634.

Merino, J.M and Carabaño, R. (1992). Effect of type of fibre on ileal and fecal digestibilities. J.

Appl. Rabbit Res. 15: 931-937.

Mertens, D.R. (2002). Gravimetric determination of amylase-treated neutral detergent fibre in

feed with refluxing beakers or crucibles: collaborative study.J. AOAC Int. 85: 1217-1240.

Mertens, D.R. (2003). Challenges in measuring insoluble dietary fibre. J. Anim. Sci. 81: 3233-

3249.

Miles, M.J., Morris, V.J., Orford, P.D. and Ring, S.G. (1985). The roles of amylose and

amylopectin in the gelation and retrogradation of starch. Carbohydr. Res. 135: 271-281.

Monteils., V., Cauquil., L., Combes, S., Godon, J.J. and Gidenne, T. (2008). Potential cores

species and satellite species in the bacterial community within the rabbit caecum. FEMS

Microbiol. Ecol. 66:620-629.

Morrison, F.B. (1956). Feeds and feeding. 22nd ed. Morrison Publishing Co., Ithaca, N.Y.

Nicodemus, N., García, J., Carabaño, R. and De Blas, J.C. (1999). Performance response of

lactating and growing rabbits to dietary lignin content. Anim. Feed Sci. and Technol. 80:

43-54.


64

Nicodemus, N., Redondo, R., Carabaño, R., De Blas, C. and García, J. (2003). Effect of level

of fibre and type of ground of fibre sources on digestión and performance of growing

rabbits. Proceedings of the 3rd Meeting of Nutrition and Pathology and Meat quality.

Working groups of COST ACTION 848. Prague, Czech Republic. P. 22

Nicodemus, N., Pérez-Alba, L., Carabaño, R., De Blas, C., Badiola, I., Perez De Rozas, A. and

García, J. (2004). Effect of the fibre and level of ground of fibre sources on digestion and

ileal and caecal characterization of microbiota of early weaned rabbits. Proceedings 8th

World Rabbit Congress, Puebla, México. Becerril, C., Pro, A. (Eds), Colegio de

Postgraduados for WRSA publ. Pp 143. (http://www.dcam.upv.es(8wrc/).

Nicodemus, N., García, J., Carabaño, R. and De Blas, J.C (2006). Effect of a reduction of

dietary particle size by substituting a mixture of fibrous by-products by lucerne hay on

performance and digestión of growing rabbits and lactating does. Livest. Sci. 100: 242-250.

O’Neil, D.A., Porter, E.M., Elewaut, D., Anderson, G.M., Eckmann, L., Ganz, T. and Kagnoff,

M.F. (1999). Espression and regulation of the human β-defensins hBD-1 and hBD-2 in

intestinal epithelium. J. Immunol. 163: 6718-6724.

Owen, R.L. (1999). Uptake and transport of intestinal macro-molecules and microorganisms by

M cells in Peyer’s patches- A personal and historical perspective. Semin. Immunol. 11:

157-163.

Padilha, M.T.S., Licois, D., Gidenne, T., Carré, B. and Fonty, G. (1995). Relationship between

microflora and caecal fermentation in rabbits before and after weaning. Reproduction

Nutrition Development 35: 375-386.

Padilha, M.T.S., Licois, D., Gidenne, T., Carre, B., Coudert, P. and Lebas, F. (1996). Caecal

microflora and fermentation pattern in exclusively milk-feed young rabbits. 6th World

Rabbit Congress, Toulouse. Francia, pp: 247-251.

http://www.dcam.upv.es(8wrc/)


65

Parker, D.S. (1976). The measurement of production rates of volatile fatty acids in the caecum

od the conscious rabbit. Br. J. Nutr. 36: 61-70.

Peeters, J.E., Licois, D., Su, M., Badiola, J.I. and Rosell, J.M. (2000): Enfermedades del

conejo. Rosell, J.M. (Ed.). Mundiprensa, Tomo II. pp: 163-213.

Penney, R.L, Folk, G.E., Galask, R.P. and Petzold, C.R. (1986). The microflora of the

alimentary tract of rabbits in relation to pH diet and cold. J. Appl. Rabbit Res. 9: 152-156.

Perdue, M.H. (1999). Mucosal immunity and inflammation. III. The mucosal antigen barrier:

cross talk with mucosal cytokines. Am. J. Physiolo. 277: G1-G5.

Pérez De Rozas, A.M., Carabaño, R., García, J., Rosell, J., Díaz, J.V., Pascual, J.J. and Badiola,

I. (2005). Proyecto de Investigación sobre la etiopatogenia de la Enteropatía Epizoótica del

Conejo. XXX Symposium de Cunicultura. Valladolid. España. Pp: 167-174.

Pluske, J.R. (2001). Morphological and functional changes in the small intestine of the newly-

weaned pig. (A. Piva, K.E., Bach Knudsen and J.E. Lindberg. Ed. Gut Enviroment of Pigs.

Nottingham Univ. Press. Nottingham, U.K. pp: 1-27.

Prohaszka, L. (1980). Antibacterial effect of volatile fatty acids in enteric Escherichia coli

infections of rabbits. Zentralbl. Veteri-naermed. 27: 631-639.

Robertson, J.B. and Van Soest, P.J. (1980). The detergent system of analysis. James, W.P.T.,

Theander, O. (Eds), The Analysis of Dietary fibre in food. Marcel Dekker, NY, Chapter 9.

pp: 123-158.

Rodriguez-Romero, N., Abecia, L., Fondevila, M., Balcells, J. (2011). Effects of levels of

insoluble and soluble fibre in diets for growing rabbits on faecal digestibility, nitrogen

recycling and in vitro fermentation. World Rabbit Sci. 19:85-94.

Romero, C., Nicodemus, N., Corujo, A., Astillero, J.R. and De Blas, J.C. (2007). Effet du

rythme reproductif, la teneur en fibre insoluble de l’aliment, l’âge et l’hygiène sur la


66

composition microbienne caecale et iléale chez le lapereau. 12èmes Journées de la

Reserche Cunicole. INRA, Mans.

Rosell, J.M. (2000): Enfermedades del conejo. (Ed.). Mundiprensa, Tomo II.

Rothkötter, H.J., Kirshoff, T. and Pabst, R. (1994). Lymphoid and nonlymphoide cells in the

epithelium and lamina propria of intestinal mucosa of pigs. Gut 35: 1582-1589.

Russ, A., Barnett, M., McNabb, W., Anderson, R., Reynolds, G. and Roy, N. (2010). Post-

weaning effects of milk and milk component s on the intestinal mucosa in inflammation.

Mut. Res. 690: 64-70.

Sánchez, M. (2006). Efecto de la edad al destete sobre la composición corporal y los

rendimientos de conejos y gazapos. Proyecto fin de carrera, EUITA, UPM, Madrid.

Sequeira, J., García Ruíz, A. and Villamide, M.J. (2000). Effect of grinding and estrusion on

the digestibility of wheat and corn by rabbits. VII World Rabbit Congress. SPAIN

Scapinello, C., Gidenne, T. and Fortun-Lamothe, L. (1999). Digestive capacity of the rabbit

during the post-weaning period, according to the milk/solid feed intake pattern before

weaning. Reprod. Nutr. Dev. 39: 423-432.

Stepankova, R. and Kovaru, F. (1978). Development of lymphatic tissues in germ free and

conventionally reared rabbits. Lymphology (Malek, P., Bartos, V., Weisssleder, H., Witte

M.H., eds). G. Thieme Publ., Stuttgart. pp. : 290-294.

Strugala, V., Allen, A., Dettmar, P.W., Pearson, J.P. (2003). Colonic mucin: methods of

measuring mucus thickness. Proc. Nutr. Soc. 62, 237-243.

Tlaskalova-Hogenova, H. and Stepankova, R. (1980). Development of antibody formation in

germfree and conventionally reared rabbits: the role of intestinal lymphoid tissue in

antibody formation to E. coli antigens. Folia Biol. 26: 81-93.

Theander, O., Aman, P., Westerlund, E. and Graham, H. (1994). Enzymatic/chemical analysis

of dietary fiber. J. AOAC Int. 77: 703-709.


67

Trowell, H. (1974). Editorial: Definition of dietary fibre. Lancet 1: 503.

Tsuboi, K.K., Kwong, L.K., Neu, J. and Sunshine, P. (1981). A proposed mechanism of

normal intestinal lactase decline in the post-weaned mammal. Biochem. Biophy. Res.

Commun. 101: 645-652.

Tsuboi, K.K., Kwong, L.K., D’Harlingue, A.E., Stevenson, J., Kerner, A. and Sunshine, P.

(1985). The nature of maturational decline of intestinal lactase activity. Biochem. Biophy.

Acta 840: 69-78.

Uden, P., Robinson, P.H. and Wiseman, J. (2005). Use of detergent system terminology and

criteria for submission of manuscripts on new, or revised, analytical methods as well as

descriptive information on feed analysis and/or variability. Anim. Feed Sci. and Technol.

118: 181-186.

Van Soest, P.J. and Wine, R.H. (1967). Uses of detergent in the analysis of farmer feeds. IV.

Determination of plant cell wall constituents. J. AOAC 50: 50-55.

Vernay, M. (1987). Origin and utilization of volatile fatty acids and lactate in the rabbit:

influence of the faecal excretion pattern. Br. J. of Nutrition 57: 371-381.

Villamide, M.J. and De Blas, C. (1991). Nutritive value of cereal grains for rabbits. J. Appl.

Rabbit Res. 14: 144-147.

Wallace, R.J., Falconer, M.L. and Bhargava, P.K. (1989). Toxicity of volatile fatty acids at

rumen pH prevents enrichment of Escherichia coli by sorbitol in rumen contents. Current

Microbiol. 19: 277-281.

Westengen, M.W.A. and Van del Roel, A.F.B. (2009). XXV Curso Especialización FEDNA:

Avances Nutrición y Alimentación Animal. FEDNA. Eds.: P.Gª. Rebollar, C. de Blas y

G.G. Mateos: 281-289.


68

Wiseman, J., Villamide, M.J., De Blas, C. and Carabaño, R. M. (1992). Prediction of the

digestible energy and digestibility of gross energy of feeds for rabbits. I. Individual classes

of feeds. Anim. Feed Sci. Technol. 39: 27-38.

Wiseman, J. (2006). Variations in starch digestibility in non-ruminants. Anim. Feed Sci.

Technology 130: 66-77.

Xiccato, G., Carazzolo, A., Cervera, C., Falcao-E-Cunha, L, Gidenne, T., Maertens, L., Pérez,

J.M. and Villamide, M.J. (1996). European ring test on the chemical analyses of feed and

faeces: influence on the calculation of nutrient digestibility in rabbits. Proceedings of the

6th World Rabbit Congress, vol., 1, ITAVI, Paris, Toulouse, Francia, pp. 293-297.

Xiccato, G., Trocino, A., De Boever, J.L., Maertens, L., Carabaño, R., Pascual, J.J., Pérez, J.M.,

Gidenne, T. and Falcao-E-Cunha. L. (2003). Prediction of chemical composition, nutritive

value and ingredient composition of European compounds feeds for rabbits by Near

Infrared Reflectance Spectroscopy. Anim. Feed Sci. technol. 104: 153-168.

Yoshida, T., Pleasants, J.R., Reddy, B.S. and Wostmann, B.S. (1968). Efficiency of digestion

in germ-free and conventional rabbits. Br. J. Nutr. 22: 723-737.

[Escriba texto]

Chapter 2

Neutral Detergent Soluble Fiber improves gut

barrier function in twenty–five-day-old rabbits

Published in: 2007 J. Anim. Sci. 85: 3313-3321

Chapter 2: NDSF on gut barrier function

71

1. ABSTRACT

The effect of level of soluble fiber on gut barrier function and intestinal microbiota

was examined in weaned rabbits. A control diet (AH) containing 103 g of neutral detergent-

soluble fiber/kg of DM, included alfalfa as main source of fiber. Another diet (B-AP) was

formulated by replacing half of the alfalfa hay by a mixture of beet and apple pulp resulting

in 131 g soluble fiber/kg of DM. A third diet (OH) was obtained by substituting half of the

alfalfa hay with a mix of oat hulls and a soybean protein concentrate and contained 79 g

soluble fiber/kg of DM. Rabbits weaned at 25 d and slaughtered at 35 d were used to deter-

mine ileal digestibility, jejunal morphology and sucrase activity, lamina propria lympho-

cytes, and intestinal microbiota. Suckling 35-d-old rabbits were used to assess mucosal mor-

phology. Mortality (from weaning to 63 d of age) was algo determined. Villous height of

jejunal mucosa increased with dietary soluble fiber (P = 0.001). Rabbits fed the highest level

of soluble fiber (BA-P diet) showed the highest villous height/crypth depth ratio (8.14; P =

0.001), sucrase specific activity (8671 µmol glucose/ g protein; P = 0.019), and the greatest

ileal starch digestibility (96.8%; P = 0.002). The opposite effects were observed in rabbits

fed decreased levels of soluble fiber (AH and OH diets; 4.70, 5,848 µmol of glucose/g of

protein, as average, respectively). The lowest ileal starch digestibility was detected for ani-

mal fed OH diet (93.2%). Suckling rabbits of the same age showed a lower villous

height/crypt depth ratio (6.70) compared with the B-AP diet group, but this ration was high-

er that the AH or OH diet groups. Lower levels of soluble fiber tended (P = 0.074) to in-

crease the cellular immune response (CD8+ lymphocytes). Diet affected IL-2 production

(CD25+, P = 0.029; CD5+CD25+, P = 0.057), with no clear relationship between soluble

fiber and IL-2. The intestinal microbiota biodiversity was not affected by diets (P ≥ 0.38).

Animals fed B-AP and AH diets had a reduced cecal frequency of detection compatible with

Campylobacter spp. (20.3 vs. 37.8, P = 0.074), and Clostridium perfringens (4.3 vs. 17.6%,

P = 0.047), compared with the OH diet group. Moreover, the mortality rates decreased from

14.4 (OH diet) to 5.1% (B-AP diet) with the increased presence of soluble fiber in the diet.

In conclusion, increased levels of dietary soluble fiber improve mucosal integrity and func-

tionality.

Key words: immune response, intestinal microbiota, mucosa integrity, rabbit, soluble fiber.


72

2. INTRODUCTION

Infectious diseases of the digestive system represent the main cause of mortality dur-

ing the fattening period in rabbits (Rosell, 2003). This has increased the demand for nutri-

tional strategies to reduce their incidence and the use of antibiotics, particularly in the post-

weanig period. At weaning, rabbits have a higher risk of disease because the colonization of

the gut by mic microbiota (Gouet and Fonty, 1979; Padilha et al., 1995) and the develop-

ment of gut associated lymphoid tissue is not completed at this stage (Dasso et al., 2000,

Lanning et al., 2000; Brown et al., 2006). Furthermore, a reduction of both the vil-

lous/crypth height ratio and the activity of the jejunal brush border enzymes (sucrose and

maltase) has been observed just after weaning (Gutiérrez et al., 2002), and this may indicate

an impairment of mucosal functional capacity (Henning, 1985; Hampson and Kidder, 1986;

Tang et al. 1999).

The dietary fraction most related to digestive troubles in rabbits is insoluble fiber

which is the most important determinant of rate of passage and growth of microbes (de Blas

et al., 1999; García et al., 2000; Gidenne, 2003). Furthermore, at same concentration dietary

fiber, the dietary inclusion of soluble and fermentable fiber decreases the pH and increases

the total cecal concentration of VFA and the total mean retention time (García et al., 1993;

Carabaño et al., 1997). Previous studies in rabbits have also shown that pectin compared

with highly lignified fibers improved morphology of the intestinal mucosa and increased the

activity of intestinal cells (Chiou el al. 1994). However, the implications of these changes in

the intestinal microbiota and their relationship to intestinal health in rabbits are currently

unknown. The objective of this study was to analyze the effects of dietary soluble fiber on

gut barrier function in rabbits during the postweaning period.

3. MATERIALS AND METHODS

3.1 Animals

This study was approved by the Committee of Ethics of the Departamento de

Producción Animal of the Departamento de Producción Animal of the Universidad Politéc-


73

nica de Madrid. All animals were handled according to the principles of animal care pub-

lished by Spanish Royal Decree 1201/2005 (BOE, 2005).

Crossbred (New Zealand White Californian) rabbits obtained from a farm affected

by Epizootic Rabbit Enteropathy (ERE) were used in all the experiments. Each of the ani-

mals appeared healthy at the beginning of the study, and no mortality was observed during

the 10-d period from 25 to 35 d old. Animals were individually caged in flat-deck cages of

600 250 330 mm, and housing conditions were controlled during the whole experimental

period as follows: a 12-h light-dark cycle was established and temperature conditions were

maintained between 15 and 24ºC.

3.2 Diets

Three starter diets with different soluble fiber content (measured as neutral detergent

soluble fiber) were formulated (Table 1). A control diet (AH) containing 103 g/kg (DM ba-

sis) of soluble fiber was established by including alfalfa hay as the main source of fiber. A

diet containing greater soluble fiber (B-AP) was formulated by replacing half of the alfalfa

hay used in the AH diet with a mixture of beet and apple pulp (75:25), resulting in a soluble

fiber content of 131 g/kg DM. A third lower soluble fiber diet (OH) was developed by sub-

stituting half of the alfalfa hay by a mixture of oat hulls and a soybean protein concentrate

(88:12, wt/wt; Soycomil P-economy, Loders Croklaan, Wormerveer; The Neatherlands) and

contained 79 g of soluble fiber/kg of DM.

These diets were formulated to contain 11.3 MJ of DE/kg, and 12 g of digestible

CP/MJ digestible energy, according to the nutrient requirements of growing rabbits (De Blas

and Mateos, 1998; Gutiérrez et al., 2002). They also contained similar concentrations of

total dietary fiber (NDF + soluble fiber), starch and CP (Table 1). To determine the apparent

ileal digestibility, 5g of DM/kg of alfalfa labeled wiht Yb2O3 was included in all diets, and

analyzed according to the procedures described by García et al. (1999). Rabbits had ad libi-

tum access to the feed and water, and a mixture of 100 ppm of apramicine sulphate and 120

ppm of tylosine tartrate was supplied by water to all animals from 25 to 35 days of age. Be-

fore the experimental period (before weaning), both weanling rabbits and their mothers were

given during lactation ad libitum access to a commercial feed (Cunilactal, NANTA, S.A.,

Spain) containing (g/kg DM) 182 CP, 359 NDF, 45 ADL, 161 starch and 53 ether extract.


74

Experiment 1: Determination of Ileal Digestibility, Mucosa Morphology and

Sucrase Activity

Eighty-four weanling rabbits of both sexes, 25 d in age, and a BW of 535 8.6 g

BW were blocked by litter and randomly assigned to the 3 experimental diets (28 rab-

bits/diet) to determine the apparent ileal digestibility of DM, CP and starch. After a 10-d

experimental period animals, 35-d-old animals with a BW of 1034 14.9 g BW were killed

between 1900 and 2100 to minimize the influence of cecotrophy. The caudal 20 cm of the

ileum in each case was excised, emptied, and the digesta were frozen in dry ice. For chemi-

cal analyses of the ileal digesta, samples were freeze-dried and ground. Due to the small

amount of sample, ileal digesta from 3 to 5 rabbits for each treatment were pooled, resulting

in 7 samples/treatment. The iIleal digestibility of DM, starch and CP was determined by the

dilution technique using ytterbium as a marker as follows:

100ionconcentrat ytterbium ileal

ion/concentrat ytterbiumdietary 1DMid

100

ionconcentratstarch dietary

ionconcentrat ytterbium ion/ilealconcentratstarch

ilealionconcentrat ytterbiumdietary

1starch id

100

ionconcentratprotein dietary

ionconcentrat ytterbium ion/ilealconcentratprotein


1CPid

The Ileal flow of DM, starch and CP was calculated by multiplying the apparent ileal

digestibilities obtained for each pool of samples and the average daily intake of DM, starch

and CP recorded for the corresponding animals.

Two 6-cm samples were excised from the middle part of the jejunum for 30 of the

killed rabbits. One of these samples from each animal was the flushed with saline solution,

frozen in dry ice and immediately stored at –20ºC to determine sucrase activity

(10/treatment). The second of the 6-cm sample (taken in 8 rabbits/treatment) was rinsed with


75

KCl solution and placed into a 10% buffered neutral formaldehyde solution (pH 7.2 to 7.4)

to evaluate the mucosal morphology.

In a parallel experiment, samples from a control group of 19 suckling rabbits (35 d

old) with a BW of 876 17.0 g BW were processed in the same way to assess their mucosal

morphologies. The jejunal samples used in these analyses were gradually dehydrated in an

ethanol series (50 to 100%), embedded in paraffin, sectioned at 6 μm and stained with hema-

toxylin and eosin (Armed Forces Institute of Pathology, 1968). Three to five slides contain-

ing jejunal cross sections were prepared for each sample and were view at 40x magnification

using an Olympus BX-40 light microscope. Images were digitally captured for later analysis

using Soft software version 3.2 C4040Z (Soft Imaging System, Olympus GmbH, Hamburg,

Germany. The villous height and crypt depth were determined according to the procedure

described by Hampson (1986), and an average of the measurements for 3 independent cross-

sectional jejunal samples (for which at least 10 different measurements were done) was ob-

tained for each animal.

Experiment 2: Characterization of Lamina Propria Lymphocytes

Thirty-six weanling rabbits of both sexes mixed-sex rabbits (12/treatment) of 25 d in

age, and weighing 529 33 g BW were blocked by litter and randomly assigned to the 3

experimental diets. Having reached 35 d in age and a BW of 972 46 g, the animals were

euthanized and 1-mm samples were excised from the duodenum just caudal to the pancreatic

flexure and processed immediately. The isolation of lymphocytes in lamina propria was

conducted according to the procedure described by Eiras et al. (1998). Duodenal samples

were longitudinally opened and incubated in 5 ml of a 10% RPMI/Fetal bovine serum solu-

tion containing 50 l of collagenase (1.5 mg/mL. Cymbus Bioscience, Eastleigh, United

Kingdom). For phenotypic characterization of lamina propria lymphocytes and measuring of

their activation grade, commercial monoclonal antibodies were used (Cymbus Biotechnolo-

gy. Eastleigh. United Kingdom). Anti-rabbit CD45 was used for the localization of all types

of lymphocytes, CD5 antibodies were employed to distinguish T from B lymphocytes,

CD25 antibodies were used to indetify T lymphocytes that express the interleukin-2 (IL-2)

receptor, CD4 antibodies was used as a marker for T helper lymphocytes, and CD8 antibody

was used to identify suppressor T lymphocytes. Data analysis was done by using flow cy-


76

tometry (Tricolour flux in FACScan standard. Becton Dickinson. S. Agustín de Guadalix.

Spain).

Experiment 3: Characterization of Intestinal Microbiota and Determination of Mortal-

ity Rate

One hundred eleven 25-d-old weanling rabbits of both sexes weighing 423 ± 11 g of

BW were blocked by litter and randomly assigned to the 3 experimental diets. The animals

were euthanized at 35 d between 1900 and 2100, and approximately 1 g of both ileal (from

18 to 22 rabbits/treatment) and cecal samples (from 33 to 37 rabbits/treatment) were

collected in sterile plastic tubes containing 3 mL of 98% molecular biology grade ethanol.

The collected samples were maintained at 4ºC until use. Several rabbits had no ileal content,

and the number of ileal samples was therefore lower than the cecal sample number.

Another 354 rabbits, weighing 485 ± 6 g, were blocked by litter and assigned at

random to the three treatments (118 animals per diet). Each experimental diets was offered

ad libitum for 2wk after weaning (at 25 d of age). After this period, all of these animals were

given a commercial feed (Cuniunic, Nanta, S.A.), containing (g/kg as fed): 160 CP, 345

NDF, 50 ADL, and 144 g starch/kg. Mortality was recorded from the time of weaning until

63 d of age.

3.3 Chemical Analysis

Procedures of the AOAC (2000) were used to determine the concentrations of DM

(934.01), ash (967.05), CP (968.06), ether extract (920.39), and starch (amyloglucosidase--

amylase method, 996.11). Dietary NDF, ADF and ADL were determined sequentially by

using the filter bag system (Ankom Technology, New York) according to Mertens (2002),

AOAC (2000; procedure 973.187) and Van Soest et al. (1991), respectively. Soluble dietary

fiber was analyzed as a neutral detergent soluble fiber according to Hall et al. (1997), and

included fructans, galactans, β-glucans and pectic substances.

For the determination of sucrase activity (EC 3.2.1.48) jejunal samples were thawed

and the mucosa was scraped using a blunt spatula and placed into vials containing distilled


77

water to form a 1/20 (w/v) mucosal homogenate. This was followed by homogenization for

30 s in a polytron (MODEL RE 16, Janke&Kunkel, IKA-Labortechnik, Germany). The

supernatant was decanted and stored in vials at –20ºC. Sucrase activity (EC 3.2.1.48) was

determined using sucrose (Sigma, St. Louis; MO) as a substrate. The concentration of

glucose liberated at 37ºC for 30 minutes were determined by using the UV-Method, using

the glucose-6-phosphate dehydrogenase (EC 1.1.1.49)-hexoquinase (EC 2.7.1.1) assay

(Boehringer-Mannehein Biochemica, Germany) (Schmidt, 1973). The protein content of the

mucosal homogenates were determined using a commercial kit (Sigma Procedure no. P-

5656, Sigma-Aldrich, Tres Cantos, Spain) based on Lowry’s method as modified by

Peterson (1977).

Microbiota biodiversity and detection frequency was determined by 16S rRNA

RFLP according to the following procedure: 400 mg of gut contents were processed for total

DNA extraction using the QIAamp DNA Stool Mini Kit (Qiagen Inc., Chatsworth, CA)

according to the manufacturer’s instructions, with additional lysozyme and proteinase K

steps. The purified DNA was maintained at -20ºC until use. The primers 5’-

CTACGGGAGGCAGCAGT-3’ and 5’-CCGTCWATTCMTTTGAGTTT-3, corresponding

to regions I and II of the 16S rRNA gene (Lane, 1991), were used to amplify a DNA

segment of 500-600 bp product. Amplifications were performed in a final volume of 50 µL

using a PCR-Master Mix (Applied Biosystems) contained 1.25 IU of Taq polymerase, 50 ng

of DNA template, 0.2 µM of each primer, and the following cycling conditions: 94°C for 5

min, followed by 35 cycles of 94°C for 1 min, 45°C for 1 min and 72°C for 1m 15s. The last

extension cycle was continued for 5 min. Aliquots of the amplified DNA fragments were

digested, in separated tubes, with Alu I, Rsa I, Hpa II, Sau 3A I or Cfo I restriction

endonucleases (Sigma-Aldrich) in accordance with manufacturer specifications. The

endonuclease fragments were solved in 2% agarose gels at 150 V for 60 min, and bands

were visualized in an UV Chemigenious Image System at a 4.63-s exposure (SynGene)

using GeneSnap software (SynGene, Cambridge, UK).

Using the relative size of the RFLP bands obtained in our analyses and the

information stored in the ―SSU_Una.gb‖ file from the Ribosomal Database Project (Maidak

et al., 1997), we could identify the bacterial genus or species compatible with the obtained

RFLP profile using specific software developed in our Institute. From each animal, a


78

biodiversity degree, defined as the number of 16S r-DNA (genes that codify the rRNA)

sequences deposited in the Ribosomal Database Project, that was compatible with the RFLP

profile obtained from the total DNA extracted from the gut samples was recorded (Andrés-

Elias et al., 2007). In addition, the frequency of detection of each bacterial strain examined

was used to define the presence or absence of a specific genus or bacterial species for each

animal.

3.4 Statistical Analysis

The results obtained in this study for apparent ileal digestibility, ADFI, ileal flow,

mucosa morphology traits, sucrase activity, intestinal microbiota and mortality rates were

analyzed as a completely randomized block, with the type of diet used as the main source of

variation and the litter as a block effect using the GLM procedure (SAS Inst. Inc., Cary,

NC). Weaning weight was included as a linear covariate. Means were compared using a pro-

tected t-test and differences were considered significant at P < 0.05.

4 RESULTS


Activity

Daily feed intake, and the apparent ileal digestibility of ileal flow of DM did not dif-

fer among the 3 experimental diets tested (P ≥ 0.16), which measured on average: 79.7 g of

DM, 46.8% and 42.1 g of DM/d, respectively (Table 2). Starch ileal digestibility increased

(P = 0.002) with dietary soluble fiber by 3.9% between the extreme diets, which resulted in

a 58% reduction of ileal starch flow (P = 0.001) in rabbits fed B-AP compared to those fed

OH diet. In contrast, the apparent ileal digestibility of CP showed a tendency for a 15% de-

crease with soluble fiber by 15% (P = 0.074), with no effects on the ileal flow of CP.

The villous height in the jejunum increased by 46% with the dietary level of soluble

fiber (P = 0.001) between the extreme diets (Table 3). In addition, the animals who received

the greatest level of soluble fiber (B-AP diet) showed a 22% reduction (P = 0.001) in crypt

depth compared with those on both the AH and OH diets, which did not differ. According-


79

ly, animals given the B-AP diet showed the highest ratio villous height/crypt depth (P =

0.001), whereas no differences were detected between animals who received the AH and OH

diets. Suckling rabbits of the same age showed shorter villous compared wih animals fed

with the greatest level of soluble fiber (B-AP diet), and these values were similar and greater

than those obtained for the AH and OH diets, respectively. Suckling rabbits showed the

same crypt depth as the animals who had been fed with the B-AP diet, and these were short-

er than the AH and OH animals. As a result, the villous height/crypt depth ratio for suckling

rabbits was lower compared with the animals given the B-AP diet, but higher than the AH

and OH groups.

The sucrase specific activities in jejunum (Table 3) had increased (P = 0.019) in rab-

bits on the B-AP diet, but no differences were evident between the AH and OH diet groups.

No differences were observed between the protein contents of the jejunal mucosa in any of

the diet groups, and this value was 70.7 mg/g of tissue on average (data not shown).

Experiment 2: Characterization of Lamina Propria Lymphocytes

Differences in the level of soluble dietary fiber did not affect the percentage of lym-

phocytes in the lamina propria, which averaged 6.22% of the total cells (Table 4). In addi-

tion, no effects of the different diets upon the proportions of T lymphocytes (CD5+), B lym-

phocytes (CD5-), T helper lymphocytes (CD4+) or CD4+CD8+ lymphocytes were detecta-

ble, which measured 75.6, 24.4, 31.7 and 23.7%, respectively. However, the proportion of T

lymphocytes harboring an activated IL-2 receptor (CD25+) was decreased (P = 0.029) in

animals fed with the lowest level of soluble fiber (OH diet). The rabbist in the OH group

also tended (P = 0.057) to show a lower activation of T lymphocytes (CD5+CD25+). In ad-

dition, the proportion of CD8+ lymphocytes tended to increase (P = 0.074) with soluble fi-

ber reduction.

Experiment 3: Characterization of Intestinal Microbiota and Determination of Mortal-

ity

The dietary treatments did not affect the biodiversity of the ileal microbiota, which

showed an average of 591 sequences recognized by SSU_Unal.gb (Ribosomal Database


80

Project), and an ileal detection frequency compatible with Bacteroides spp., Bacteroides

fragilis, Clostridium spp., Clostridium difficile, Escherichia coli, Propionibacterium spp.

and Ruminococus spp. that averaged 67.1, 7.03, 85.0, 5.37, 6.55, 28.2 and 29.0%, respec-

tively (Table 5). However, the ileal frequency profile compatible with Clostridium

perfringens, Butirivibrio fibrosolvens and Campylobacter spp. tended to decrease in animals

given the highest level of soluble fiber (P < 0.079).

Caecal biodiversity was greater compared with the ileum (an average of 1,044 se-

quences were recognized by SSU_Unal.gb (Ribosomal Database Project); (P<0.05) and was

also not affected by the different dietary treatments. The cecal frequency of detection of

Bacteroides spp., Bacteroides fragilis, Butirivibrio fibrosolvens, Clostridium spp., Clostridi-

um difficile, Escherichia coli, Propionibacterium spp. and Ruminococus spp. did not vary

with the levels of soluble fiber and averaged 64.8, 14.9, 72.0, 97.9, 27.1, 14.0, 68.4 and

62.1%, respectively. However the detection frequency of Clostridium perfringens decreased

(P = 0.047) and that of Campylobacter spp. tended to decrease (P = 0.074) with increasing

levels of soluble fiber.

Mortality was affected by the diet (SEM = 2.7; P = 0.050) and decreased from 14.4

(OH diet) to 5.1 % (B-AP diet) with increased dietary soluble fiber. Rabbits fed the AH diet

showed an intermediate result (8.5 %). Most of the dead rabbits showed symptoms of borbo-

rygmus (stomach rumbling), the presence of mucus in colon and ileum, and cecal impacta-

tion, indicating that the ERE was the underlying cause of death (Pérez de Rozas et al., 2005;

Marlier et al., 2006).

5. DISCUSSION

Epizootic Rabbit Enteropathy is a primary cause of postweaning mortality in rabbits

in most European countries (Rosell, 2003; Pérez de Rozas et al., 2005; Licois et al., 2005).

The etiology of ERE has not yet been elucidated, but proper medication does reduce the in-

cidence of death. Therefore, most farmed rabbits are medicated under field conditions. At

present, ERE is the main cause of mortality in our research facility and the present experi-

ments were carried out under conditions in which this disease can occur, similar to commer-

cial farms.


81

The intestinal barrier plays a key role in the protection of rabbits and other animals

against pathogens because it prevents the colonization and the translocation of bacteria and

toxins. Several criteria have been proposed to characterize mucosal integrity. The villous

height/crypth ratio and the activities of one or more mucosal enzymes (sucrose-isomaltose,

aminopeptidase, or alkaline phosphatase) are the most widely used of these to estimate both

mucosal integrity and enterocyte maturity in nonruminants (Van der Kils and Jansman,

2002). In our current study, an increase in dietary soluble fiber increased villous height of

the jejunal mucosa in weaned rabbits. In addition, rabbits fed the greatest levels of soluble

fiber (BA-P diet) showed a reduction in their crypth depth and an increase in the villous

height/crypth depth ratio. Therefore, our current data are in agreement with the earlier find-

ings of Chiou et al. (1994), which showed that the substitution of lignin with pectin im-

proved villous height/crypth depth ratio in purified diets.

In addition to the improvement in the mucosal morphology with increasing levels of

soluble fiber, rabbits given the BA-P diet also showed the greatest sucrase activities and ileal

starch digestibility. These findings are consistent with the studies of Chun et al. (1989) and

Lizardo et al. (1997) who reported an increase in brush border enzyme activity with increas-

ing levels of soluble fiber in rats and pigs, respectively.

The beneficial effects of soluble fiber were further evident when 35-d-old rabbits in

the B-AP diet group were compared with suckling rabbits of the same and are in agreement

with Gallois et al. (2005). We previously showed also that low soluble fiber diets are associ-

ated with the impairment of the mucosal morphology in weaned rabbits (Gutiérrez et al.,

2002). These results indicate the potential benefits of formulating specific postweaning diets

for rabbits based on the inclusion of moderate levels of soluble fiber.

Mucosal atrophy and decreased enterocytes functionality were observed in the ani-

mals fed with a low soluble fiber diet, and these might cause an enhanced permeability of

the mucosa to pathogens. We also observed in our present analyses that there was a trend to

increase the cellular immune response (CD8 lymphocytes) associated with lower levels of

soluble fiber (Lim et al., 1997; Field et al., 1999). We thus hypothesize that dietary soluble

fiber has a protective effect upon the mucosa that favors an immune response. Furthermore,


82

this possibility and the improvement of mucosa functionality is consistent also with our ob-

servation that there is a reduction in the mortality rate for rabbits fed with moderate levels of

soluble fiber. However, further investigations are necessary to clarify the role of soluble

fiber on Th1 cytokines as previously suggested Field et al. (1999). We have observed an

effect of diet on IL-2 production (CD25+ and CD5+CD25+), but we did not detect a clear

relationship between soluble fiber and IL-2.

The influence of the dietary levels of soluble fiber on mucosal integrity and on the

immune response might also be mediated by their effects on the proliferation of microbiota.

In our current study, rabbits fed with the highest levels of soluble fiber tended to show a

reduction in the ileal frequency of detection that is compatible with Clostridium perfringens.

In the cecum of rabbits fed with normal and moderate soluble fiber levels (AH and B-AP

diets) also decreased the frequency of detection that is compatible with Clostridium

perfringens. In fact, recent reports suggest that Clostridium perfringens might be implicated

in the incidence of ERE (Pérez de Rozas et al., 2005; Marlier et al., 2006). The negative ef-

fects of these bacteria are exacerbated by existing intestinal damage, as demonstrated in

broilers affected by necrotic enteritis (Van Inmerseel et al., 2004). Differences observed

among diets that differ in the substrate available at the end of the ileum might also favor the

proliferation of some pathogens or commensal bacteria. An increase in CP flow at the ileum

has been shown to favor C. perfringens proliferation (Wilkie et al., 2005; Chamorro et al.,

2006), but in our study no differences were detectable in the ileal CP flow. The decrease in

the ileal flow of starch observed in rabbits fed the B-AP diet might have reduced the sporu-

lation and toxin formation of Clostridium perfringens (Labbe and Duncan, 1975). However,

other factors might also been implicated because the differences in ileal starch flow between

animals fed AH and B-AP diets were not observed in the presence of Clostridium

perfringens. The enterotoxin produced by Clostridium perfringens might also induce cellular

damage of the intestinal mucosa and alter its membrane permeability (McClane, 2001;

Berkes et al., 2003). In our present analyses, a decrease of the level of soluble fiber also

tended to increase the frequency of detection of Campylobacter spp.

Commensal bacteria are poorly described in rabbits but some genera (e.g., Bac-

teroides and Clostridium) seem to be predominant (Gouet and Fonty, 1979). The main mi-

crobial enzyme activity in the ileum and ceacum corresponds to pectin-degrading enzymes

(Marounek et al., 1995). Furthermore, bacteria belonging to the genus Bacteroides are the


83

principal pectinolytic organisms in the rabbit caecum (Sirotek et al., 2001). However, no

clear relationship has been observed between soluble fiber and the presence of some fibro-

lytic bacteria that might act as a probiotic (Bacteriodes spp., Butirivibrio fibrosolvens, Pro-

pionibacterium spp. and Ruminococus spp.) (Michel et al., 2005; Ohkawara et al., 2005).

In conclusion, an increase in soluble dietary fiber in rabbits improves mucosal integ-

rity and functionality. However, the changes detected in the immune response in the lamina

propria and in the profile of intestinal microbiota are not consistent with the changes in sol-

uble fiber.


84

6. LITERATURE CITED

Andrés-Elias, N., Pujols, J., Badiola, I., and Torrallardona, D. 2007. Effect of nucleotides and

carob pulp on gut health and performance of weanling piglets. Livest. Sci. 108:280-283.

AOAC. 2000. Official Methods of Analysis. 17th ed. Assoc. Off. Anal. Chem., Arlington, VA.

Armed Forces Institute of Pathology. 1968. Routine staining procedures. In: L.G. Luna (ed).

Manual of Histologic Staining Methods. pp 32-46. Mcgraw-Hill Book Co., New York.

Association of Official Analytical Chemists. 2000. Official Methods of Analysis, 17th Ed.

Association of Official Analytical Chemists, Arlington,VA.

Berkes, J., V.K. Viswanathan, S.D. Savkovic, and G. Hecht. 2003. Intestinal epithelial

responses to enteric pathogens: effects on the tight junction barrier, iron transport, and

inflammation. Gut 52:439-451.

Boletín Oficial del Estado. 2005. Real Decreto 1201/2005. Sobre protección de los animales

utilizados para experimentación y otros fines científicos. B.O.E. 252:34367-34391.

Brown, D.C., C.V. Maxwell, G.F. Erf, M.E. Davis, S. Singh, and Z.B. Johnson. 2006. The

influence of different management systems and age on intestinal morphology, immune

cell numbers and mucin production from globet cells in post-weaning pigs. Vet.

Immunol. Immunopathol. 111: 187-198.

Carabaño, R., W. Motta Ferreira, J.C. de Blas, and M.J. Fraga. 1997. Substitution of sugarbeet

pulp for alfalfa hay in diets for growing rabbits. Anim. Feed Sci. Technol. 65:249-256.

Chamorro S., M.S. Gómez-Conde, A.M. Pérez de Rozas, I. Badiola, R. Carabaño, and C. De

Blas. 2006. Effect of the level and type of protein in diets for kits on their performance and

intestinal health. World Rabbit Science 14:7-8.

Chiou, P.W., B. Yu, and C. Lin. 1994. Effect of different components of dietary fiber on the

intestinal morphology of domestic rabbits. Comp. Biochem. Physiol. 108:629-638.


85

Chun, W., T. Bamba, and S. Hosoda. 1989. Effect of pectin, a soluble dietary fiber, on

functional and morphological parameters of the small intestine in rats. Digestion 42:22-29.

Dasso, J.F., H. Obiakor, H. Bach, A.O. Anderson, and R.G. Mage. 2000. A morphological and

immunohistological study of the human and rabbit appendix for comparison with the avian

bursa. Devolp. Comp. Immol. 24: 797-814.

De Blas, J.C., and G.G. Mateos. 1998. Feed formulation. In: J.C. De Blas, and J. Wiseman

(Eds) The nutrition of the rabbit. pp 241-253. Commonwealth Agricultural Bureau,

Wallingford, UK.

De Blas, C., J. Garcia, and R. Carabaño. 1999. Role of fibre in rabbit diets. A review. Ann.

Zootech. 48:3-13.

Eiras, P., E. Roldán, C. Camarero, F. Oliveares, A. Bootello, and G. Roy. 1998. Flow

cytometry description of a novel CD3-/CD7+ intraepithelial lymphocyte subset in human

duodenal biopsies: Potential diagnostic value in celiac disease. Cytometry. 34:95-102.

FEDNA, 2003. Tablas FEDNA de composición y valor nutritivo de alimentos para la

fabricación de piensos compuestos (2ª ed). J.C. De Blas, G.G. Mateos, P.G. Rebollar (Eds).

Fundación Española para el Desarrollo de la Nutrición Animal, Madrid, Spain.

Field, C.J., M. I. McBurneya, S. Massiminob, M.G. Hayekb, and G.D. Sunvold. 1999. The

fermentable fiber content of the diet alters the function and composition of canine gut

associated lymphoid tissue. Vet. Immunol. Immunop. 72:325-341.

Gallois, M., T. Gidenne, L. Fortun-Lamothe, I. Le Huerou-Luron, and J.P. Lallès. 2005. An

early stimulation of solid feed intake slightly influences the morphological gut maturation

in the rabbit. Reprod. Nutr. Develop.45:109-122.

García, J., R. Carabaño, and J. C. de Blas. 1999. Effect of fiber source on cell wall digestibility

and rate of passage in rabbits. J. Anim. Sci. 77:898-905.

http://wos.isiknowledge.com/?SID=X2MAOglb3npcg4O2m52&Func=Abstract&doc=2/12


86

García, J., R. Carabaño, L. Pérez-Alba, and J. C. de Blas. 2000. Effect of fiber source on cecal

fermentation and nitrogen recycled through cecotrophy in rabbits. J. Anim. Sci. 78:638–

646.

García, G., J. F. Gálvez, and J. C. de Blas. 1993. Effect of substitution of sugarbeet pulp for

barley in diets for finishing rabbits on growth performance and on energy and nitrogen

efficiency. J. Anim. Sci. 71:1823-1830.

Gidenne, T. 2003. Fibres in rabbit feeding for digestive troubles prevention: respective role of

low-digested and digestible fibre. Livest. Prod. Sci. 81:105-117.

Gouet, Ph., and G. Fonty. 1979. Changes in the digestive microflora of holoxenic rabbits from

birth until adulthood. Ann. Biol. Anim. Biochim. Biophys. 19:553-566.

Gutiérrez, I., A. Espinosa, J. García, R. Carabaño, and J.C. De Blas. 2002. Effect of levels of

starch, fiber, and lactose on digestion and growth performance of early-weaned rabbits. J.

Anim. Sci. 80:1029-1037.

Hall, M.B., B.A. Lewis, P.J. Van Soest, and L.E. Chase. 1997. A simple method for estimation

of neutral detergent-soluble fiber. J. Sci. Food Agric.74:441-449.

Hampson, D.J. 1986. Alteration in piglet small intestinal structure at weaning. Res. Vet. Sci.

40:32-40.

Hampson, D.J., and D.E. Kidder. 1986. Influence of creep feeding and weaning on brush

border enzyme activities in the piglet small intestine. Res. Vet. Sci. 40: 24-31.

Henning, S.J. 1985. Ontogeny of enzymes in the small intestine. Annu. Rev. Physiol. 47: 231-

245.

Labbe G.R., and C.L. Duncan. 1975. Influence of carbohydrates on growth and sporulation of

Clostridium perfringens Type A. Appl. Microbiol. 29:345-351.


87

Lane, D.J. 1991. 16S/23S rRNA sequencing. In: E. Stackebrandt and M. Goodfellow (Eds)

Nucleic acid techniques in bacterial systematics. pp 115-175. John Wiley and Sons, New

York.

Lanning, D., P. Sethupathi, K. J. Rhee, S. K. Zhai, and K.L. Knight. 2000. Intestinal microflora

and diversification of the rabbit antibody repertoire. J. Immunol. 165: 2012-2019.

Licois, D., M. Wyers, and P. Coudert. 2005. Epizootic Rabbit Enteropathy: experimental

transmission and clinical characteristics. Vet. Res. 36:601-613.

Lim, B.O., K. Yamada, M. Nonaka, K. Yuichiro, P. Hung, and M. Sugano. 1997. Dietary fiber

modulates indices of intestinal immune function in rats. J. Nutr. 127:663-667.

Lizardo, R., J. Peiniau, and A. Aumaitre. 1997. Inclusion of sugar-beet pulp and change of

protein source in the diet of the weaned piglet and their effects on digestive performance

and enzymatic activities. Anim. Feed Sci. Technol. 66:1-14.

Maidak, B.L., G.J. Olsen, N. Larsen, R. Overbeek, M.J. McCaughey, and C.R. Woese. 1997.

The RDP (Ribosomal Database Project). Nucleic Acid Res. 25:109-110.

Marlier, D., Dewree, R., Lassence, C., Licois, D., Mainil, J., Coudert, P., Meulemans, L.,

Ducatelle, R., Vindevogel, H. 2006. Infectious agents associated with epizootic rabbit

enteropathy: Isolation and attempts to reproduce the syndrome. Vet. Journal. 172 (3): 493-

500.

Marounek, M., S. J. Vook, and V. Skrivanova . 1995. Distribution of activity of hydrolitic

enzymes in the digestive tract of rabbits. Br. J. Nutr. 73:463–469.

McClane B.A. 2001. The complex interactions between Clostridium perfringens enterotoxin

and epithelial tight junctions. Toxicon 39:1781-1791.

Mertens, D.R. 2002. Gravimetric determination of amylase-treated neutral detergent fibre in

feeds with refluxing beakers or crucibles: collaborative study. J. Assoc. Off. Assoc. Chem.

Int. 85, 1217-1240.


88

Michel, C., N. Roland, G. Lecannu, C. Hervte, J.C. Avice, M. Rival, and C. Cherbut. 2005.

Colonic infusion with Propionibacterium acidipropionici reduces severity of chemically-

induced colitis in rats. Lait 85:99-111.

Ohkawara, S., H. Furuya, K. Nagashima, N. Asanuma, and T. Hino. 2005. ral administration of

Butyrivibrio fibrisolvens, a butyrate-producing bacterium, decreases the formation of

aberrant crypt foci in the colon and rectum of mice. J. Nutr. 135: 2878–2883.

Padilha, M.T.S., D. Licois, T. Gidenne, B. Carre, G. Fonty. 1995. Relationships between

microflora and caecal fermentation in rabbits before and after weaning. Reprod. Nutr. Dev.

35:375-386.

Peréz de Rozas, A.M., R. Carabaño, J. García, J. Rosell, J.V. Díaz, J. Barbé, J.J. Pascual, and I.

Badiola. 2005. Proyecto de Investigación sobre la etiopatogenia de la Enteropatía

Epizoótica del Conejo. In: ITACyL and ASESCU (Eds) XXX Symposium de Cunicultura.

pp 167-174. Valladolid. Spain.

Peterson, G.L. 1977. A simplification of the protein assay method of Lowry et al. which is

more generally applicable. Anal. Biochem. 83:346-356.

Rosell, J.M. 2003. Technical note: health status of commercial rabbitries in the iberian

peninsula. A practitioners study. World Rabbit Sci. 11:157-169.

Schmidt F.H. 1973. Blood glucose levels in capillary blood of adults assessed by the

hexokinase method. Klin. Wochenschr. 51:520–522.

Sirotek, K., E. Santos, V. Benda, and M. Marounek. 2003. Isolation, identification and

characterization of rabbit caecal mucinolytic bacteria. Acta Vet. Brno 72:365-370.

Tang, M., B. Laarveld, A.G. Van Kessel, D.L. Hamilton, A. Estrada and J.F. Patience. 1999.

Effect of segregated early weaning on postweaning small intestine development in pigs. J.

Anim. Sci. 77: 3191-3200.


89

Van der Kils, J.D., and A.J.M. Jansman. 2002. Optimising nutrient digestion, absorption and

gut barrier in monogastric. Reality or illusion?. In: M.C. black. H.A. Vahl, L. de Lange

A.E. Van de Braak, G. Hemke, M. Hessing (Eds). Nutrition and health of the intestinal

tract. pp 15-36. Wageningen Academic Publishers. The Netherlands.

Van Immerseel, F., De Buck, J., Pasmans, F., Huyghebaert, G., Haesebrouck, F., and Ducatelle,

R. 2004. Clostridium perfringens in poultry: an emerging threat for animal and public

health. Avian Pathology 33:537-549.

Van Soest, J.P., J.B. Robertson, and B.A. Lewis. 1991. Methods for dietary fiber, neutral

detergent fiber and nonstarch polysaccharides in relation to animal nutrition. J. Dairy Sci.

74:3583-3597.

Wilkie D.C., A.G. Van Kessel, L..J. White, B. Laarveld, and M.D. Drew. 2005. Dietary amino

acids affect intestinal Clostridium perfringens populations in broiler chickens. Can. J.

Anim. Sci. 85: 185-193.


90

Table 1. Composition of the experimental diets

Experimental diets

Item Oat hulls Alfalfa hay Beet-Apple pulp

Ingredients, g/kg (as-fed)

Alfalfa hay 139 283 139

Oat hulls 147 - -

Beet pulp - 23 150

Apple pulp - - 50

Soybean meal concentrate 20 - -

Heat treated wheat 323 323 323

Wheat bran 84 84 84

Sunflower meal 71 71 71

Soybean meal 111 111 111

Sunflower hulls 44 60 44

Lard 33 23 5

Alfalfa hay mordanced with Yb 5 5 5

L-Lysine HCL 4.5 4 4

DL-Methionine 1 1 1

L-Threonine 1.5 1 1.5

Sodium Chloride 5 6 4

Calcium Carbonate 6 - 2

Vitamin/mineral premix1 5 5 5

Analyzed composition, g/kg DM

DM 918 914 917

Ash 65 71 67

CP 199 200 197

Ether extract 59 55 37

Starch 211 208 205

NDF 358 335 331

ADF 162 164 169

ADL 39 47 37

Neutral detergent-soluble fiber

79 103 131


91

Calculated composition, g/kg DM2

Sugars

39 43 57

Lysine 12.6 11.8 12.2

Methionine 4.1 4.1 4

Methionine+cystine 7.2 7.2 7

Threonine 8.5 7.9 8.4

Calcium 6.8 6.9 6.8

Phosphorus 4.5 4.8 4.5

Sodium 2.3 2.9 2.2

1Provided by Ibérica de Nutrición Animal S.L. (Madrid, Spain). Mineral and vitamin composition (per

kg of complete diet): 40 mg of Mn as MnO; 50 mg of Zn as ZnO; 1.25 mg of Ias KI; 40 mg of Fe ad Fe

SO4; 25 mg of Cu as Cu SO4; 0.5 mg of Co as Co SO4; 250 mg of choline chloride; 2 mg of Riboflavin;

5 mg of calcium d-pantothenate; 15 mg of nicotinic acid; 1mg of menadione sodium bisulphite; 9,000

IU of vitamin A as retinyl acetate; 1,800 IU of vitamin D3 as cholecalciferol; 12.5 IU of vitamin E as α-

tocopherol acetate; 0.01 mg of cyanocobalamin; 1 mg of thiamine; and 66 mg of Cycostat (contained 66

g/kg of the active substance robenidine hydrochloride).

2 Calculated values according to Fundacion Española para el Desarrollo de la Nutrición Animal

(FEDNA, 2003)


92

Table 2. Effects of fiber soured and soluble fiber levels1 on ADFI, apparent ileal digestibility

and ileal flow of nutrients in 35 d old rabbits

Experimental diets

SEM2

P

Oat hulls Alfalfa hay Beet-Apple pulp

ADFI 25-35 d, g/d

83.3 77.6 78.1 2.88 0.43

Apparent ileal digestibility, %

DM 46.8 44.8 48.7 1.91 0.37

Starch 93.2a 95.0

b 96.8

c 0.58 0.002

CP 66.9 59.8 57.0 2.94 0.074

Ileal flow, g/day

DM 44.3 41.9 40.1 1.51 0.16

Starch 1.2a

0.8b

0.5c

0.099 0.001

CP 5.5 6.2 6.6 0.46 0.24

a-c Mean values in the same raw with different superscript differ, P < 0.05.

1Soluble fiber content of the diets (g/kg of DM) was: oat hull, 79, alfalfa hay, 103, and beet apple pulp,

131.

2 n = 28 for ADFI, and

n = 7 for apparent ileal digestibility and ileal flow of nutrients.


93

Table 3. Effects of fiber source, soluble fiber levels1, and weaning on the morphology and

sucrase activity of the jejunal mucosa in 35-d-old rabbits

Weaned rabbits

Suckling

rabbits SEM

2 P

Experimental diets

Oat hulls Alfalfa

hay

Beet-Apple

pulp

Villous height, m 493a 567

bc 722

c 566

b 28.0 0.001

Crypt depth, m 113a 115

a 89.0

b 89.6

b 4.35 0.001

Villous height/crypt depth 4.41a 5.00

a 8.14

c 6.70

b 0.39 0.001

Sucrase specific activity, mol

of glucose/g of protein

5,202a 6,495

a 8,671

b – 580 0.019


1Soluble fiber content of the diets (g/kg of DM) was: oat hull, 79, alfalfa hay, 103, and beet apple

pulp, 131.

2 n = 8 in all cases except for 35-d-old suckling rabbits (n=19) and sucrose specific activity (n=10).


94

Table 4. Effects of fiber source and soluble-fiber levels1 on the percentage of lymphocytes

and phenotypic (given as percentage of the total lymphocytes) in the duodenal lamina pro-

pria in 35-d-old rabbits

Experimental diets

SEM2 P

Oat

hulls

Alfalfa

hay

Beet-Apple

pulp

Total lymphocytes, % total cells in lamina

propia

8.69 5.14 4.83 1.95 0.35

Lymphocyte profile3, % total lymphocytes

CD5+ 76.8 74.2 75.9 3.80 0.89

CD5- 23.2 25.8 24.1 3.80 0.89

CD25+ 11.2a 19.0

b 14.8

ab 1.85 0.029

CD5+CD25+ 8.36 15.3 11.6 1.84 0.057

CD4+ 26.2 33.9 35.1 3.87 0.25

CD8+ 30.3 26.9 21.3 2.61 0.074

CD4+CD8+ 25.0 22.5 23.7 3.39 0.87


1Soluble fiber content of the diets (g/kg of DM) was: oat hull, 79, alfalfa hay, 103, and beet

apple pulp, 131.

2 n = 12.

3CD5+ = T lymphocytes; CD5+ = B and natural killer lymphocytes; CD25+ = activated lym-

phocytes (expressing IL-2 receptor); CD5+CD25+ = activated T lymphocytes (expressing IL-2

receptor); CD4+ = T helper lymphocytes; CD8+ = T cytotoxic lymphocytes, and CD4+CD8+ =

T lymphocytes expressing CD4+ and CD8+ receptors.


95

Table 5. Effect of fiber source and soluble fiber levels1 on ileal and cecal biodiversity, and

the detection frequency (%) of the indicated bacteria in 35-d-old rabbits

Experimental diets

SEM2

P

Oat hulls Alfalfa hay Beet-Apple pulp

Ileum

No. of rabbits 22 18 20

Ileal biodiversity3

616 712 446 97.7 0.38

Bacteroides spp. 66.5 82.1 52.7 9.2 0.17

Bacteroides fragilis 0 11.1 10.0 5.5 0.25

Butirivibrio fibrosolvens 22.7 38.9 10.0 8.9 0.079

Campylobacter spp. 27.3 16.7 5.00 7.7 0.057

Clostridium spp. 83.1 94.1 77.8 8.0 0.40

Clostridium difficile 0 11.1 5.00 5.5 0.28

Clostridium perfringens 9.09 22.2 0 6,1 0.062

Escherichia Coli 9.09 5.55 5.00 5.6 0.89

Propionibacterium spp. 31.8 27.8 25.0 10.2 0.90

Ruminococus spp. 22.7 44.4 20.0 9.2 0.18

Caecum

No. of rabbits 37 33 36

Caecal biodiversity3

1,064 1,098 971 131 0.79

Bacteroides spp. 63.3 64.3 66.9 9.8 0.94

Bacteroides fragilis 13.5 9.09 22.2 6.5 0.44

Butirivibrio fibrosolvens 59.5 78.8 77.8 7.1 0.18

Campylobacter spp. 37.8 21.2 19.4 6.7 0.074


96

Clostridium spp. 96.3 97.4 100.0 3.0 0.42

Clostridium difficile 40.5 24.2 16.7 7.4 0.18

Clostridium perfringens 17.6a

2.87b

5.7b

4.2 0.047

Escherichia Coli 18.2 18.2 5.56 5.7 0.19

Propionibacterium spp. 62.2 81.8 61.1 7.6 0.22

Ruminococus spp. 59.4 57.6 69.4 7.9 0.33


1Soluble fiber content of the diets (g/kg of DM) was: oat hull, 79, alfalfa hay, 103, and beet apple

pulp, 131.

2 n = 19.9 and 35.2 for ileal and cecal samples, respectively.

3 number of sequences recognized in the SSU_Unal.gb (Ribosomal Database Project, Maidak et al.,

1997).

CHAPTER 3

EFFECT OF NEUTRAL DETERGENT

SOLUBE FIBRE ON DIGESTION, INTESTINAL

MICROBIOTA AND PERFORMANCE IN TWENTY

FIVE DAY OLD WEANED RABBITS

Published in: 2009 Livestock Science 125: 192-198

Chapter 3: NDSF on digestión, intestinal microbiota and performance

99

1 ABSTRACT.

The effect of neutral detergent soluble fibre (NDSF) level on digestibility, fermenta-

tion traits, intestinal microbiota and performance was studied in weaned rabbits. A control

diet (DA) containing 103 g NDSF/kg DM included dehydrated alfalfa as the main source of

fibre. Another diet (B-AP) was formulated by replacing half of the dehydrated alfalfa with a

mixture of beet and apple pulp resulting in 131 g NDSF/kg DM. A third diet (OH) was ob-

tained by substituting half of the dehydrated alfalfa with a mix of oat hulls and a soybean

protein concentrate and contained 79 g NDSF/kg DM. All diets contained similar levels of

total fibre (NDSF + neutral detergent fibre), starch and protein (446, 208, and 199 g/kg DM,

respectively). Forty-two rabbits (14/diet) weaned at 25 d were used to determine faecal di-

gestibility from 32 to 35 d of age. This group, plus another nine rabbits/diet (23/diet), were

used to determine weight of stomach and caecum and their contents, caecal fermentation

traits and similarity rate (SR) of intestinal microbiota. Another 105 and 245 weaned rabbits

were used to determine growth traits and mortality, respectively. From 25 to 39 d of age,

rabbits were fed the experimental diets and from 39 to 60 d they were fed a commercial diet,

including robenidine hydrochloride in all diets. Drinking water was supplemented with

apramicine sulfate and tylosine tartrate throughout the experimental period. Faecal DM and

energy digestibility increased linearly by 8% and NDF digestibility by 43% between ex-

treme diets with NDSF inclusion (P < 0.001). Weight of total gastrointestinal tract decreased

linearly and quadratically with NDSF reduction (P = 0.008 and P = 0.089, respectively).

Stomach pH decreased linearly with increasing levels of NDSF (P ≤ 0.041). Weight of cae-

cal contents increased linearly between animals fed OH and B-AP diets (P < 0.001). Level

of inclusion of NDSF had no effect (P ≥ 0.12) on pH, VFA concentration and VFA molar

proportions in caecal contents. Treatments appeared to influence the SR of caecal microbiota

but a lesser effect was observed on ileal microbiota. Post weaning feed efficiency (25-39 d)


100

increased linearly (P < 0.001) with NDSF inclusion by 10% between extreme diets, and by

3% in the whole fattening period (P = 0.027). Average feed intake during the post weaning

(25-39 d) and the whole fattening period increased with NDSF reduction (P ≤ 0.079). No

effect of NDSF was detected on average daily gain (P ≥ 0.15). Mortality decreased linearly

with increasing levels of NDSF in the post weaning and in the whole fattening period (P =

0.086 and 0.016, respectively).

Keywords: neutral detergent soluble fibre, performance, digestion, intestinal microbiota,

growing rabbits


101

2. INTRODUCTION

The dietary fraction most related to digestive troubles in rabbits is insoluble fibre

(neutral detergent fibre, NDF) which is the most important factor in regulating rate of pas-

sage and microbial activity (Gidenne, 1994; de Blas et al., 1999; García et al., 2002). A level

of fibre below 30% NDF increases mortality which is usually attributed to an accumulation

of digesta in the caecum (Laplace, 1978; De Blas et al., 1986; Nicodemus et al., 2004). Fur-

thermore, in diets with the same level of dietary fibre, the type of fibre alters the time and

the characteristics of fermentation in the cecum. The dietary inclusion of soluble and fer-

mentable fibre sources (as beet and citrus pulps) and/or small particle size source of fibre

(rice hulls, paprika meal) causes accumulation of digesta in the caecum, which might lead to

a reduction of feed intake and dressing out percentage, increases in caecal microbial activity,

especially of the fibrolytic saprophyte microbiota, and increases of the caecal acidity (Fraga

et al., 1991; Carabaño et al., 1997; García et al., 2002; Falcao-e-Cunha et al., 2004; Nicode-

mus et al., 2006). The opposite effect has been observed with the dietary inclusion of insol-

uble and low fermentable sources of fibre as cereal straw, sunflower hulls and grape marc

(Fraga et al., 1991; Motta-Ferreira et al., 1996; García et al., 2000). The inclusion of increas-

ing levels of beet pulp seems to decrease mortality (Soler et al., 2004). However, fermenta-

ble sources of fibre (as beet pulp) contain a low lignin proportion, which deficiency seems to

increase mortality (Nicodemus et al., 1999; Gidenne et al., 2001), and consequently requires

to be complemented with lignified fibre sources.

In spite of its nutritional significance, the information related to the effect of the sol-

uble fibre fraction is scarce and insufficient to formulate practical recommendations, mainly

due to the difficulty of quantifying it. In a previous study, in which we used the same diets

as the present work, the inclusion of moderate levels of neutral detergent soluble fibre


102

(NDSF, 12%) in the starter (25-39 d) diet improved the gut barrier function and tended to

reduce the frequency of detection of Clostridium perfringens in the ileum and caecum of

rabbits in the post weaning period (Gómez-Conde et al., 2007). The objective of this study

was to determine the effect of increasing levels of NDSF in the starter diet on performance,

digestion, and intestinal microbiota of growing rabbits.

3. MATERIAL AND METHODS

3.1 Diets

Three starter (25-39 d) diets with different NDSF contents were formulated (Table

1). A control diet (DA) containing 103 g/kg (dry matter basis; DM) of NDSF was made in-

cluding dehydrated alfalfa as the main source of fibre. Two other diets were formulated by

replacing half of the dehydrated alfalfa used in the DA diet by a mixture of beet and apple

pulp (75:25) (B-AP diet) or by a mix of oat hulls and soya bean protein concentrate (88:12)

(Soycomil P-economy®, Loders Croklaan, Wormerveer; The Netherlands) (OH diet), which

resulted in a higher and lower NDSF content (131 and 79 g/kg of dry matter, respectively).

These three diets were formulated to contain 11.3 MJ digestible energy/kg, and 12 g digesti-

ble crude protein/MJ digestible energy, according to the nutrient requirements of growing

rabbits (De Blas and Mateos, 1998; Gutiérrez et al., 2002). They also contained similar lev-

els of total dietary fibre (NDF + NDSF), starch and crude protein (CP) (Table 1). Rabbits

had ad libitum access to feed and water, and a mixture of 100 ppm of apramicine sulfate

(Girolan®

) and 120 ppm of tylosine tartrate (Tailan®) was supplied in the water to all ani-

mals during the whole experimental period (25 to 60 d of age). Prior to the experimental

period (before weaning), both suckling rabbits and their mothers were given ad libitum ac-

cess to a commercial feed (Cunilactal, NANTA, S.A., Spain) containing (g/kg DM) 182 CP,

359 NDF, 45 acid detergent lignin, 161 starch and 53 ether extract. From 25 (weaning) to 39


103

d of age rabbits were offered the experimental diets. From 39 to 60 d of age all rabbits were

fed with a commercial feed (CUNIUNIC, NANTA, S.A.: 160 g CP, 144 g starch, 345 g

NDF and 50 g acid detergent lignin per kg). All diets contained robenidine hydrochloride as

Cycostat®

.

3.2 Animals and housing

Crossbred (New Zealand White Californian) healthy mixed-sex rabbits weaned at

25 days of age obtained from a farm affected by Epizootic Rabbit Enteropathy were used in

all the experiments. Animals were housed in individual flat-deck cages of 600 250 330

mm dimensions, except during faecal digestibility trials in which animals were kept in wire

metabolism cages (405 510 320 mm) that allowed separation of feces and urine. Farm-

ing conditions were controlled during the whole experimental period. A cycle of 12 hours of

light (07:00-19:00 h) and 12 h of dark was used. Temperature conditions were kept between

15 and 24ºC by heating and cooling systems combined with continuous forced ventilation.

Rabbits were handled according to the principles for experimental animal care published by

Spanish Royal Decree 1201/2005 (B.O.E., 2005), and experiments were approved by the

Ethics Committee from the Department of Animal Production of the Polytechnic University

of Madrid.

3.3 Digestibility trial

Forty-two (14/diet) weanling mixed-sex rabbits of 25 d of age, with a BW of 53770

g (mean standard deviation) were blocked by litter and randomly assigned to the three

experimental diets to determine the faecal apparent digestibility of DM, gross energy, CP,

NDF, acid detergent fibre (ADF) and starch. Following a 7-d adaptation according to the

method of Gómez-Conde et al. (2006), dry matter intake and total faecal output (cecotrophy


104

was not prevented) were recorded for each rabbit over a 3-d collection period (from 32 to 35

days of age). Feces produced daily were collected in labeled polyethylene bags and stored at

-20ºC. When the faecal digestibility trial was finished, feces were dried at 80ºC for 48 h and

ground to 1 mm for further analysis.

3.4 Stomach, caecal fermentation and similarity rate of intestinal microbiota

trial

Sixty-nine mixed-sex rabbits (23/diet) weaned at 25 days of age with a BW of

53970 g were blocked by litter and assigned at random to the three experimental diets. This

group included the rabbits used in the faecal digestibility trial. After a 10-d period consum-

ing the feed, and with an average BW of 1040134 g, they were slaughtered by cervical dis-

location between 19:00 and 21:00 h in order to avoid the caecotrophy period. After slaugh-

ter, the whole gastrointestinal tract was removed and weighed, and the length of the gastro-

intestinal tract from the pylorus to the caecum was recorded for 17 rabbits/treatment.

Weights of the stomach and caecum, with and without their contents, were taken individual-

ly. Stomach and caecal pH were measured. In the stomach, pH was recorded at the pyloric

and fundus areas and also in the mix of the entire stomach digesta (Gutiérrez et al., 2002).

Twelve samples of caecal content were centrifuged at 25,000 g at 0ºC for 10 min. The

supernatant fluid was used to determine ammonia (12 animals/treatment) and total volatile

fatty acids concentrations (VFA, 6 animals/treatment). For ammonia determination the su-

pernatant was acidified with a solution of 0.2 M hydrochloric acid (1 ml/ml) and frozen. For

VFA determination, a solution of 5% orthophosphoric acid (vol/vol) plus 1% mercury cho-

ride (wt/vol) was added (0.1 ml/ml) to the supernatant. Only six samples per treatment out of

twelve were available for VFA analysis as we had problems with the conservation of the

other six samples. To study the similarity rate (SR) of microbiota among animals and treat-


105

ments, approximately 1 g of ileal and caecal samples from each animal (there were no ileal

contents in four rabbits) were collected in sterile plastic tubes containing 3 ml of 98% mo-

lecular biology grade ethanol. The collected samples were maintained at 4ºC until use. Re-

striction fragment length polymorphism (RFLP) was used to build the dendograms (Gómez-

Conde et al., 2007) using the similarity matrix derived from the Manhattan distances (Kauf-

mann and Rousseeuw, 1990). This coefficient uses the size and intensities of the bands as

judged by peak heights in the densitometric curves and allows the generation of dendro-

grams with the neighbour-joining method. Animals grouped in different clusters indicate

that the similarity rate between them is lower than the similarity rate of animals grouped in

the same cluster. Samples from diseased rabbits were not collected (5 ileal and caecal sam-

ples) and another 4 ileal samples were not collected due to the absence of ileal digesta.

3.5 Growth trial

One hundred-five rabbits weaned at 25 days of age with a BW of 48568 g were

blocked by litter and assigned at random to the three experimental diets (35/diet). Animals

were housed individually and fed with the experimental diets during a 14-d period after

weaning. After that (at 39 days of age), all the animals received a commercial feed with

robenidin (CUNIUNIC

, NANTA, S.A.) until 60 days of age. Average feed intake, weight

gain and mortality from 25 to 39 days of age and from 39 d to the end of the experimental

period (60 d) were recorded. Data from dead animals was not included. To study mortality,

another group of 245 rabbits weaned at 25 days of age with a BW of 47585 g were blocked

by litter, caged individually and assigned at random to the three experimental diets (83, 83

and 79 rabbits for OH, DA and B-AP groups, respectively). Total mortality of both groups

together (118, 118 and 114 rabbits for OH, DA and B-AP groups) is presented.


106

3.6 Analytical methods


(934.01), ash (967.05), crude protein (Dumas method, 968.06), ether extract (920.39), and

starch (amyloglucosidase--amylase method, 996.11). Dietary NDF, ADF and ADL were

determined sequentially by using the filter bag system (Ankom Technology, New York)

according to Mertens (2002), AOAC (2000; procedure 973.187) and Van Soest et al (1991),

respectively. Dietary soluble fibre was analyzed as neutral detergent soluble fibre according

to Hall et al. (1997), it included fructans, galactans, β-glucans, and pectic substances. The

NDSF was calculated as the difference in OM mass between the ethanol-insoluble residue

and NDF after correction for CP and starch. Gross energy was determined with adiabatic

calorimetry. Caecal ammonia was analyzed using an autoevaluation distillation unit. Sam-

ples were distilled with a solution of sodium tetraborate (2.5%), collected on boric acid solu-

tion (1%), and titrated with hydrochoric acid (0.05M) including a color indicator. Caecal

VFA concentration was determined in a Hewlett-Packard (5710 Å) chromatograph, with a

flame ionization detector, a Hewlett-Packard (3390 Å) recorder integrator, and a steel col-

umn: free fatty acids and phenols (FFAP) 10% H3PO4, 1% acid-washed chromosorb W,

100-120 mesh. The carrier gas was nitrogen with a flow rate of 30 ml/min; hydrogen and air

flows to the detector were 30 and 200 ml/min, respectively. Injector and detector tempera-

tures were 250ºC. The oven temperature was increased during the analysis from 110 to

160ºC at a rate of 8ºC/min.

3.7 Statistical analysis

Linear and quadratic effects of dietary NDSF were studied by using polynomial con-

trasts. In the model, litter was also included as block effect. Weaning weight was also in-

cluded as linear covariate in the statistical model to analyse growth traits. These analyses


107

were performed with the GLM procedure of SAS (2008). Mortality rate was analysed using

logistic regression (GENMOD procedure of SAS considering a binomial distribution) and

results were transformed from the logit scale. All data are presented as least-squares means.

4. RESULTS

4.1 Faecal digestibility trial

Daily feed intake of DM and faecal starch digestibility were not affected in this study

by dietary NDSF (P ≥ 0.29), being on average 79.7 g DM/d and 99.4%, respectively (Table

2). A linear effect of NDSF inclusion was detected for faecal DM and energy digestibility (P

< 0.001), both were 8% higher in rabbits fed the B-AP diet than those fed OH diet. Faecal

digestibility of NDF increased linearly by 43% between the dietary extremes (P < 0.001),

whereas ADF digestibility increased linearly with NDSF inclusion (P < 0.001). Faecal ap-

parent CP digestibility was affected quadratically by NDSF inclusion (P = 0.045). Animals

fed the DA diet had lower values compared to those fed OH and B-AP diets.

4.2. Digestion traits

Average feed intake in the days before slaughter decreased linearly with NDSF in-

crement (P = 0.040) (Table 3), whereas weight of total full gastrointestinal tract increased

linearly and quadratically with the NDSF increment (P = 0.008 and P = 0.089, respectively).

No effect of diet was detected on the gastrointestinal tract length and on the proportional

weight of the stomach contents (215 cm and 4.81%BW on average, respectively. P ≥ 0.52).

Empty weight of stomach tended to decrease linearly with NDSF (P = 0.055). Stomach pH

at the pylorus, at the fundus and in the mixture of the whole stomach content decreased line-

arly by 21, 34 and 21% with increasing levels of NDSF (P ≤ 0.041). The weight of caecal


108

contents and empty caecum increased linearly between animals fed OH and B-AP diets (P <

0.001), by 14 and 32%, respectively. Level of inclusion of NDSF had no effect (P ≥ 0.12) on

pH, VFA concentration and molar proportions of acetic acid, propionic acid, butyric acid,

valeric acid and isovaleric acid in caecal contents. These values were on average: 5.39, 66

mmol/l, and 78.7, 6.7, 13, 0.79 and 1.23%, respectively. A quadratic trend of NDSF inclu-

sion on caecal ammonia concentration was detected, showing that animals fed the DA diet

had the highest caecal ammonia concentration (P = 0.088). The effect of treatments on the

SR of ileal microbiota (Figure 1) appeared to be less than that observed for the caecal mi-

crobiota (Figure 2, because the caecal microbiota of rabbits fed the same diet tended to

group together in the same clusters much more than was seen with the ileal microbiota. In

this study, the treatments seem to have a higher influence than the mother on SR of caecal

microbiota.

4.3 Growth trial

A linear positive effect of NDSF inclusion was observed for post weaning feed effi-

ciency (25-39 d) (P < 0.001), that increased by 12% from the OH to the B-AP diet (Table 4).

A trend toward reduced mortality with increasing levels of NDSF (P = 0.086) was also ob-

served in the post weaning period. Linear and quadratic effects of NDSF inclusion (P =

0.077 and P = 0.017, respectively) were observed on average daily feed intake in the first

two weeks after weaning. Animals fed the DA diet had the lowest value. For this same peri-

od, NDSF level did not affect average daily gain (P ≥ 0.15). Treatments had no effect (P ≥

0.25) on weight gain, feed intake and feed efficiency from 39- 60 d of age. These values

were on average 47.3±4.26 g/d, 151±13.4 g/d, and 0.317±0.024 g/g, respectively. Consider-

ing the whole fattening period, feed efficiency increased linearly by 3% and mortality de-

creased linearly by 63% (P = 0.027 and P = 0.016, respectively) between the dietary ex-


109

tremes with NDSF. Average feed intake tended to increase with NDSF reduction (P =

0.079). No effect of NDSF was detected on average daily gain.

5. DISCUSSION

The main interest in soluble/fermentable fibre (mainly supplied by sugar beet pulp)

in rabbit diets has been its use as starch replacement (García et al., 1993; Gidenne and

Bellier, 2000; Gidenne et al., 2004; Volek et al., 2005), in order to limit the mortality at-

tributed to the latter (Blas and Gidenne, 1998). Accordingly, its effects usually cannot be

separated from those derived from different starch levels. Besides, the lack of a simple tech-

nique to quantify soluble fibre has meant that this fraction has normally been ignored, and

little information is available about its effects in rabbits or in other non ruminants.

In this work, the increase of NDSF level improved feed efficiency in the starter peri-

od (25-39 d of age) which was reflected in the feed efficiency of the whole fattening period,

with no changes in growth rate. This effect is in accordance with the linear increase of faecal

DM and energy digestibility with NDSF level. Similar results has been reported previously,

using diets with similar fibre and starch levels but differing in soluble fibre content (Pérez de

Ayala et al., 1991; García et al., 1999; Falcao-e-Cunha et al., 2004). Our results are mainly

due to the increase of soluble fibre, and the digestibility of insoluble fibre (NDF and ADF)

with increasing levels of NDSF. In fact, the monomers usually related to pectic substances,

an important fraction of soluble fibre, accounted for 66% of faecal digestibility of non-starch

polysaccharides, the latter being closely related to faecal NDF digestibility (García et al.,

1999). Falcao-e-Cunha et al. (2004) obtained similar results, but also observed an increase of

the pectinolytic and cellulolytic activities in soft faeces when soluble fibre increased. It


110

would suggest a higher fermentation activity and possibly a change in the microbiota popu-

lation in the animals fed increasing levels of soluble fibre (NDSF). This is in agreement with

the apparent change of caecal microbiota indicated by the close similarity of caecal microbi-

ota among animals of the same treatment. Moreover, not only did the fibrous substrate for

caecal microbiota change, but also the availability of protein and starch in the caecum, as

caecal protein and starch digestibility changed with NDSF level. Apparent caecal protein

digestibility (calculated from the faecal digestibility values obtained in this experiment and

the ileal digestibility values of Gómez-Conde et al., 2007) increased (13.2, 17.3 and 22.3 %,

for OH, DA and B-AP diets, respectively) with NDSF, whereas that of starch decreased (6.2,

4.5 and 2.5%, for OH, DA and B-AP diets, respectively). Consequently, it might have been

involved in the changes observed in caecal microbiota among treatments as well. These

changes were reflected in neither the caecal pH and VFA concentrations, nor in the molar

VFA proportions, and only minor effects were detected on caecal N-NH3. This might put

into question the value of measuring caecal VFA and pH as indicators of the effect of diet on

caecal microbiota. In spite of the significant fibre proportion that is digested before the cae-

cum (around 20%) mostly (80%) constituted by the monomers usually contained in soluble

fibre (Gidenne, 1992; Carabaño et al., 2001), a lower effect of NDSF level was detected on

SR of ileal microbiota compared to that observed in the caecum.

The increase of NDSF level was related to the corresponding linear increases in cae-

cal contents weight and linear and quadratic increases in total gastrointestinal tract weight.

These effects might account for the linear and quadratic decrease observed in feed intake. It

would be partly related to the increase of dietary digestible energy content when NDSF in-

creased, and the negative effect of an accumulation of digesta in the caecum on feed intake

(García et al., 2002) obtained previously when the effects of 54 diets were reviewed.


111

In our work, the increase of NDSF also linearly reduced mortality, which is in

agreement with the reduction of frequency of detection of several potential pathogens as C.

perfringens and Campylobacter spp. observed by Gómez-Conde et al. (2007) using the same

diets. These results suggest a positive influence of NDSF on intestinal health. It could be

explained not only by the improved gut barrier function (jejunal morphology and functional-

ity and immune response) and positive influence of moderated levels of NDSF on intestinal

microbiota (Gómez-Conde et al., 2007), but also by the protective effect derived from the

acidification of stomach contents, and the changes produced in the intestinal microbiota ob-

tained in our work with NDSF inclusion.

6. CONCLUSIONS

Increasing NDSF level from 79 to 131 g/kg DM improved rabbit growth perfor-

mance (reduced mortality and increased feed efficiency) and digestibility, increased weight

of caecal contents, and influenced caecal microbiota.

7. ACKNOWLEDGEMENT

We are grateful to the Comisión Interministerial de Ciencia y Tecnología (CICYT)

project AGL2001-2796 for financial support, and to Luis Pérez Alba, ELANCO Valquímica

S.A., Esteve Santiago S.A., Ibérica de Nutrición Animal S.L. and Pascual de Aranda S.A.

for their contribution to this study.


112

8. REFERENCES

Association of Official Analytical Chemists. 2000. Official Methods of Analysis, 17th Ed.


Blas E., Gidenne T. 1998. Digestión of starch and sugars. In: J.C. De Blas, and J. Wiseman

(Eds) The nutrition of the rabbit. pp 17-38. Commonwealth Agricultural Bureau,

Wallingford, UK.

Boletín Oficial del Estado. 2005. Real Decreto 1201/2005. Sobre protección de los animales

utilizados para experimentación y otros fines científicos. B.O.E. 252, 34367-34391.

Carabaño, R., Motta Ferreira W., de Blas J.C., Fraga M.J. 1997. Substitution of sugarbeet pulp

for alfalfa hay in diets for growing rabbits. Anim. Feed Sci. Technol. 65, 249-256.

Carabaño, R., García, J., De Blas, C. 2001. Effect of fibre source on ileal apparent digestibility

of non-starch polysaccharides in rabbits. Anim. Sci. 72, 343-350.

De Blas, J.C., Santomá, G., Carabaño, R., Fraga, M.J. 1986. Fiber and starch levels in fattening

rabbit diets. J. Anim. Sci. 63,1897-1904.

De Blas, J.C., Mateos G.G. 1998. Feed formulation. In: J.C. De Blas, and J. Wiseman (Eds)

The nutrition of the rabbit. pp 241-253. Commonwealth Agricultural Bureau, Wallingford,

UK.

De Blas, C., Garcia J., Carabaño R. 1999. Role of fibre in rabbit diets. A review. Ann. Zootech.

48, 3-13.

Falcao-e-Cunha, L., Peres, H., Freire, J.P.B., Castro-Solla, L. 2004. Effects of alfalfa, wheat

bran or beet pulp, with or without sunflower oil, on caecal fermentation and on digestibility

in the rabbit. Anim. Feed Sci. Technol. 117, 131-149.

FEDNA, 2003. Tablas FEDNA de composición y valor nutritivo de alimentos para la

fabricación de piensos compuestos (2ª ed). J.C. De Blas, G.G. Mateos, P.G. Rebollar (Eds).

Fundación Española para el Desarrollo de la Nutrición Animal, Madrid, Spain.

http://wos.isiknowledge.com/?SID=X2MAOglb3npcg4O2m52&Func=Abstract&doc=2/12


113

Ferreira, W.M., Fraga, M.J., Carabaño, R. 1996. Inclusion of grape pomace in substitution for

alfalfa hay, in diets for growing rabbits. Anim. Sci. 63, 167-174.

Fraga, M.J., Pérez de Ayala, P., Carabaño, R., De Blas, J.C. 1991. Effect of type of fiber on the

rate of passage and on the contribution of soft feces to nutrient intake of finishing rabbits. J.

Anim. Sci. 69, 1566-1574.

García, G., Gálvez, J.F., De Blas, J.C. 1993. Effect of substitution of sugarbeet pulp for barley

in diets for finishing rabbits on growth performance and on energy and nitrogen efficiency.

J. Anim. Sci. 71, 1823-1830.

García, J., Carabaño, R., De Blas, J.C. 1999. Effect of fiber source on cell wall digestibility and

rate of passage in rabbits. J. Anim. Sci. 77, 898-905.

García, J., Carabaño R., Pérez-Alba L., de Blas J.C. 2000. Effect of fiber source on cecal

fermentation and nitrogen recycled through cecotrophy in rabbits. J. Anim. Sci. 78, 638–

646.

García, J., Gidenne, T., Falcao-e-Cunha, L., De Blas, C. 2002. Identification of the main factors

that influence caecal fermentation traits in growing rabbits. Anim. Res. 51, 165-173.

Gidenne, T. 1992. Effect of fibre level, particle size and adaptation period on digestibility and

rate of passage as measured at the ileum and in the faeces in the adult rabbit. Br. J. Nutr. 67,

133-146.

Gidenne, T., 1994. Effets d’une réduction de la teneur en fibres alimentaires sur le transit

digestif du lapin. Comparaison et validation de modèles d’ajustement des cinétiques

d’excrétion fécales des marqueurs. Rep. Nutr. Dev. 34, 295-307.

Gidenne, T., Bellier, R. 2000. Use of digestible fibre in replacement to available

carbohydrates. Effect on digestion, rate of passage and caecal fermentation pattern

during the growth of the rabbit. Livest. Prod. Sci. 63, 141-152.

Gidenne, T., Arveux, P., Madec, O. 2001. The effect of the quality of dietary lignocellulose on


114

digestion, zootechnical performance and health of the growing rabbit. Anim. Sci. 73, 97-

104.

Gidenne, T., Mirabito, L., Jehl, N., Perez, J.M., Arveux, P., Bourdillon, A., Briens, C.,

Duperray, J., Corrent, E. 2004. Impact of replacing starch by digestible fibre, at two

levels of lignocellulose, on digestion, growth and digestive health of the rabbit. Anim.

Sci. 78, 389-398.

Gómez-Conde, M.S., Chamorro, S., Nicodemus, N., Villamide, M., García, J., De Blas, C.,

Carabaño, R. 2006. Determination of faecal dry matter digestibility in rabbits weaned at 25

days of age. World Rabbit Sci. 14, 61.

Gómez-Conde, M.S., García, J., Chamorro, S., Eiras, P., Rebollar, P.G., Pérez de Rozas, A.,

Badiola, I., De Blas, C., Carabaño, R. 2007. Neutral detergent-soluble fibre improves gut

barrier function in 25 d old weaned rabbits. J. Anim. Sci. 85, 3313–3321.

Gutiérrez, I., Espinosa A., García J., Carabaño R., De Blas J.C. 2002. Effect of levels of starch,

fiber, and lactose on digestion and growth performance of early-weaned rabbits. J. Anim.

Sci. 80, 1029-1037.

Hall, M.B., Lewis B.A., Van Soest, P.J., Chase L.E. 1997. A simple method for estimation of

neutral detergent-soluble fiber. J. Sci. Food Agric.74, 441-449.

Kaufmann, L., Rousseeuw, P.J. 1990. Finding Groups in Data. An Introduction to Cluster

Analysis. John Wiley & Sons, New York, NY.

Laplace, J.P. 1978. Le transit digestif chez monogastriques. III. Comportement (prise de

nourriture-caecotrophie), motricité et transit digestif et pathogenie des diarrheas chez le

lapin. Ann. Zootech. 27, 225-265.

Mertens, D.R. 2002. Gravimetric determination of amylase-treated neutral detergent fibre in

feeds with refluxing beakers or crucibles: collaborative study. J. Assoc. Off. Assoc. Chem.

Int. 85, 1217-1240.


115

Nicodemus, N., García, J., Carabaño, R., De Blas, C. 1999. Performance response of lactating

and growing rabbits to dietary lignin content. Anim. Feed Sci. Technol. 80, 43-54.

Nicodemus, N., Pérez-Alba, L., Carabaño, R., De Blas, C., Badiola, I., Pérez de Rozas, A.,

García, J. 2004. Effect of level of fibre and level of ground of fibre sources on digestión and

ileal and caecal characterization of microbiota of early weaned rabbits. In: Proceedings of

the 8th

World Rabbit Congress, p 143. Puebla, Mexico.

Nicodemus, N., García, J., Carabaño, R., De Blas, C. 2006. Effect of a reduction of dietary

particle size by substituting a mixture of fibrous by-products for lucerne hay on

performance and digestión of growing rabbits and lactating does. Livest. Sci. 100, 242-250.

Pérez de Ayala, P., Fraga, M.J., Carabaño, R., De Blas, J.C. 1991. Effect of fiber source on

diet digestibility and growth in fattening rabbits. J. Appl. Rabbit Res. 14, 159-165.

SAS Institute. 2008. SAS/STAT Users Guide, (Release 9.2) SAS Inst. Inc., Cary, NC, USA.

Soler, M.D., Blas, E., Cano, J.L., Pascual, J.J., Cervera, C., Fernández-Carmona, J. 2004

Effect of digestible fibre/starch ration and animal fat level in diets around weaning on

mortality rate of rabbits. In: Proceedings of the 8th

World Rabbit Congress, p 156.

Puebla, Mexico.

Van Soest, J.P., Robertson J.B., Lewis B.A. 1991. Methods for dietary fiber, neutral detergent

fiber and nonstarch polysaccharides in relation to animal nutrition. J. Dairy Sci. 74, 3583-

3597.

Volek, Z., Marounek, M., Skrivanová, V. 2005. Replacing starch by pectin and inulin in diet of

early-weaned rabbits: effect on performance, health and nutrient digestibility. J. Anim. Feed

Sci. 14, 327-337.


116

Table 1. Ingredient and chemical composition of experimental diets

Experimental diets

Oat hulls Dehydrated al-

falfa Beet-Apple pulp

Ingredients, g/kg

Dehydrated alfalfa 144 288 144

Oat hulls 147 - -

Beet pulp - 23 150

Apple pulp - - 50

Soybean meal concentrate 20 - -

Boiled wheat 323 323 323

Wheat bran 84 84 84




Lard 33 23 5.0

L-Lysine HCL 4.5 4.0 4.5

DL-methionine 1.0 1.0 1.0

L-threonine 1.5 1.0 1.5

Sodium chloride 5.0 6.0 4.0

Calcium oxide 6.0 - 2.0

Vitamin/mineral premixa 5.0 5.0 5.0


Dry matter 918 914 917

Ash 65 71 67

Crude protein 199 200 197


Starch 211 208 205

Neutral detergent fibre 358 335 331

Acid detergent fibre 162 164 169

Acid detergent lignin 39 47 37

Neutral detergent soluble fibre

79 103 131

Gross energy, MJ/kg DM 19.1 18.9 18.7


117

Calculated composition, g/kg DMb

Sugars

39 43 57

Lysine 12.6 11.8 12.2

Methionine 4.10 4.11 4.01

Methionine+Cystine

7.23 7.23 7.01

Threonine

8.53 7.89 8.35

Calcium

6.76 6.89 6.79

Phosphorus

4.54 4.78 4.45

Sodium

2.33 2.89 2.23

aProvided by Ibérica de Nutrición Animal S.L. (Madrid, Spain). Mineral and vitamin composition

(mg/kg of complete diet): 40: Mn (MnO); 50: Zn (ZnO); 1.25: I (KI); 40: Fe (Fe SO4); 25: Cu (Cu

SO4); 0.5: Co (Co SO4); 250: Choline chloride; 2: Riboflavin; 5: Calcium d-pantothenate; 15: Nico-

tinic acid; 1; Menadione sodium bisulphite; 12.5: α-tocopherol acetate; 0.01: Cyanocobalamin; 1:

Thiamine; 9,000 IU: Vitamin A; 1,800 IU: Vitamin D3; 66: Cycostat (contains 66 g/kg of the active

substance robenidine hydrochloride).

bCalculated values according to Fundacion Española para el Desarrollo de la Nutrición Animal

(FEDNA, 2003)


118

Table 2. Effect of neutral detergent soluble fibre level on feed intake and faecal apparent

digestibility of 35 d old rabbits

Experimental diets

SEM1

PLinear

PQuadratic

Oat hulls Dehydrated

alfalfa

Beet-Apple

pulp

Neutral detergent soluble fibre,

g/kg DM

79 103 131

Feed intake, g DM/d

83.3 77.6 78.1 2.88 0.29 0.45

Faecal apparent digestibility, %

Dry matter

65.1

67.2

70.3

0.79 0.001 0.78

Gross energy 64.9

66.5

69.7

0.77 0.001 0.54

Crude protein 80.1 77.1 79.3 0.84 0.66 0.045

Neutral detergent fibre 26.1

31.2

37.4

1.46 0.001 0.96

Acid detergent fibre 12.3

14.2

26.2

1.83 0.001 0.091

Starch 99.4 99.5 99.3 0.063 0.49 0.35

1 n = 14.


119

Table 3. Effect of neutral detergent soluble fibre level on digestive traits of 35 d old rabbits

Experimental diets

SEM1 PLinear

PQuadratic

Oat

hulls

Dehydrated

alfalfa

Beet-

Apple

pulp

Neutral detergent soluble fibre, g/kg DM 79 103 131

Live weight at slaughter, g 1059 1032 1030 19.3 0.31 0.62

Feed intake, g DM/d 87.5 75.6 76.4 3.16 0.040 0.15

Gastrointestinal tract length, cm

215 215 216 4.90 0.82 0.95

Total full gastrointestinal tract weight, as

% of BW 23.3 25.5 25.4 0.45 0.008 0.089

Stomach

Empty weight, % BW 1.46 1.43 1.37 0.028 0.055 0.76

Content, % BW 4.87 4.94 4.63 0.24 0.52 0.60

pH pylorus 1.77 1.49 1.39 0.092 0.019 0.43

pH fundus 2.60 2.12 1.72 0.19 0.041 0.75

pH pylorus + fundus 2.41 2.41 1.91 0.13 0.018 0.21

Cecum

Empty weight, % BW 1.77 1.90 2.01 0.069 0.037 0.83

Content, % BW 5.89 6.78 7.77 0.29 0.001 0.99

pH 5.52 5.42 5.23 0.17 0.30 0.89

Caecal VFA, mmol/l

63 66 69 7.8 0.59 0.95

Acetic acid, % 77 79 80 1.5 0.27 0.98

Propionic acid, % 7.6 6.7 5.8 1.5 0.43 0.97

Butyric acid, % 13 13 13 0.99 0.91 0.72

Valeric acid, % 0.89 0.68 0.79 0.2 0.75 0.52

Iso-valeric acid, % 1.5 1.1 1.1 0.17 0.12 0.35

Caecal NH3-N, mmol/l

12.2 13.2 11.4 0.61 0.28 0.088

1 n = 17, except for caecal VFA concentration (n = 6) and NH3-N (n = 12).


120

Table 4. Effect of neutral detergent soluble fibre level on fattening performance from wean-

ing (25 d) to 60 d of age

Experimental diets

SEM1 PLinear

PQuadratic

Oat hulls Dehydrated

alfalfa

Beet-Apple

pulp

Neutral detergent soluble fibre,

g/kg DM 79 103 131

From 25 (weaning) to 39 d of age

Weight gain, g/d 42.3 39.9 43.4 1.58 0.60 0.15

Feed intake, g/d 76.9

65.9

70.0

2.45 0.077 0.017

Feed efficiency, g/g 0.553

0.605

0.618

0.012 0.001 0.16

Mortality rate, % 10.2 6.78 4.39 0.086 0.89

Whole fattening period (25-60 d)

Weight gain, g/d 45.7 44.7 45.2 0.70 0.63 0.40

Feed intake, g/d 122 118 117 1.95 0.079 0.60

Feed efficiency, g/g 0.377 0.380 0.387 0.003 0.027 0.60

Mortality rate, % 14.4

8.47

5.26

0.016 0.86

1 n = 35, except for mortality rate where n = 118, except for beet-apple pulp group in which n = 114.


121

Fig.1. Effect of neutral detergent soluble fibre level on similarity rate of the ileal microbiota

(SR, expressed in percentage) of 35 d old rabbits (OH: oat hulls diet, DA: dehydrated alfalfa

diet, B-AP: Beet-apple pulp diet. The first number of each animal is its identification and the

second one the mother’s identification (M)).


122

Fig.2. Effect of neutral detergent soluble fibre level on similarity rate of the caecal microbio-

ta (SR, expressed in percentage) of 35 d old rabbits (OH: oat hulls diet, DA: dehydrated al-

falfa diet, B-AP: Beet-apple pulp diet. The first number of each animal is its identification

and the second one the mother’s identification (M)).

Chapter 4

Effect of source of starch on digestion, intes-

tinal microbiota and performance in twenty-five-day-

old weaned rabbits

Unpublished

Chapter 4: Source of starch on digestión, intestinal microbiota and performance

125

1 ABSTRACT

Three diets with raw wheat, boiled wheat and a combination of boiled wheat and

boiled rice as main source of starch were formulated (g/kg DM: 217 starch, 345 NDF and

194 CP on average). Two groups of 99 and 193 rabbits weaned at 25 days of age were

blocked by litter and assigned to the three diets. Animals were housed individually and fed

with the experimental diets during a 14-d period after weaning. At 39 days of age, all the

animals of the first group received a commercial feed until 60 days of age. The second group

was used to determine faecal digestibility (from 32 to 35 d) and ileal digestibility at 35 d,

jejunal mucosa morphology, caecal fermentation traits and characterization of intestinal mi-

crobiota. For mortality, two additional groups of 384 (medicated) and 177 (not medicated)

rabbits weaned at 25 days of age were blocked by litter, caged in groups of 3-4 animals and

assigned to the treatments. Heat processing of wheat improved slightly ileal digestibility of

starch (P = 0.020) but did not modify the flow of starch to the caecum. Heat processing of

wheat increased the frequency of detection of Campylobacter spp. and Ruminococcus spp. in

the caecum (P ≤ 0.023) with no changes at ileal level. Heat processing of wheat did not

modify growth performance, mortality, ileal or faecal digestibility, and mucosa morphology.

The partial substitution of boiled wheat for boiled rice in the diet impaired ileal starch di-

gestibility (P = 0.020). It increased the ileal flow of starch to the caecum (P = 0.007). How-

ever, it did not increase mortality rate, but changed intestinal microbiota decreasing the fre-

quency of detection of Campylobacter spp. (both at ileum and caecum), Helicobacter spp.

(at ileum) and Ruminococcus spp. (at caecum) and increasing that of Bacteroides spp. (at

caecum) (P ≤ 0.046). The effects of boiled rice supplementation did not alter growth perfor-

mance, mortality, ileal or faecal digestibility of other nutrients than starch, and mucosa mor-

phology. The medication of rabbits reduced the ileal frequency of detection of most bacteria

studied (P ≤ 0.048), whereas the effect at the caecum was lower and limited to a reduction of


126

the frequency of detection of Campylobacter spp., Clostridium perfringens and Propioni-

bacterium spp. (P ≤ 0.048). These effects were associated to a strong reduction of mortality

rate (P < 0.001). In conclusion, the use of heat processed wheat or boiled rice did not im-

prove the results obtained with raw wheat.

Key words: starch source, heat processing, medication, performance, digestion, intestinal

microbiota, growing rabbits.


127

2. INTRODUCTION

In the digestive tract of rabbit starch is almost completely digested (> 0.98; Blas and

Gidenne, 2010), accounting for 0.30 of the total digestible energy ingested. In adult rabbits,

starch digestibility in the small intestine is also high (> 0.97; Blas and Gidenne, 2010).

However, faecal and ileal starch digestibility seems to decrease moderately after weaning

due to the temporal impairment of mucosa functionality (Corring et al., 1972; Dojana et al.,

1998; Gutiérrez et al., 2002a; Gallois et al., 2005). This age related effect is more pro-

nounced for maize or peas, that usually showed higher ileal and faecal losses (Gidenne and

Perez, 1993a,b; Gutiérrez et al., 2002b), but it was also observed for wheat or barley

(Gidenne et al., 2005a). Heat processing of starch sources or dietary enzymes inclusion pro-

vides a chance to improve starch digestion and growth performance, even reducing in some

cases mortality in young rabbits, although this effect might be not related to the starch but

with other feed fractions (Gutierrez et al., 2002b; Cachaldora et al., 2004; Gidenne et al.,

2005ab). However, the nutritional benefits of starch processing may depend on the source of

starch, as the extrusion improved nutritive value of maize but impaired that of wheat (Se-

queira et al., 2000). The use of rice, with higher starch and lower fibre concentration than

other cereals, might improve starch digestibility in young rabbits. In piglets, feeding rice

improved the performance and digestibility with respect to other cereals and might protect

pigs against diarrhea (Pluske et al., 2003, Vicente et al., 2008; Che et al., 2012). The aim of

this work was to study the effect of heat processing (raw wheat vs. boiled wheat) and type of

cereal (boiled wheat vs. boiled rice) on growth performances and intestinal health in rabbits.


128


3.1 Animals and housing

This study was approved by the Committee of Ethics of The Departamento of

Producción Animal of The Universidad Politécnica of Madrid. All animals were handled

according to the principles of animal care published in The Spanish Royal Decree

1201/2005 (BOE, 2005).

Crossbred (New Zealand White Californian) obtained from a farm affected by epi-

zootic rabbit enteropathy (ERE) were used in all of the experiments. Animals had ad libitum

access to feed and water, and were water medicated from 25 to 35 days of age with a mix-

ture of 100 ppm of apramicine sulphate and 120 ppm of tylosine tartrate to minimize the

incidence of epizootic rabbit enteropathy. Animals were housed individually in flat-deck

cages of 610 250 300 mm, except during digestibility trial in which, they were kept in

wire metabolism cages (405 510 320 mm) that allowed separation of feces and urine.

Housing conditions were controlled during the whole experimental period, cycles of 12-h

light:dark were established and temperature were maintained between 15 and 24ºC by heat-

ing and cooling systems combined with continuous forced ventilation.

3.2 Experimental diets

Three starter diets based on different starch sources were formulated. First diet in-

cluded raw wheat as main source of starch. The other two were obtained by substituting the

whole raw wheat by boiled wheat or half of the boiled wheat by a mix of boiled rice and

wheat bran (80:20). Both wheat and rice were provided by Esasa (Cabezón de Pisuerga, Val-

ladolid, Spain). Diets were almost isonutritive and were formulated according to the recom-

mendations for starter diets of Gutiérrez et al. (2002a) and to meet or exceed all the essential


129

nutrient requirements of growing rabbits (De Blas and Mateos, 2010). Ingredients and chem-

ical composition are shown in Table 1. In order to determine ileal apparent digestibility, 5 g

of DM/kg of alfalfa hay labeled with Yb2O3 was included in each diet, and analyzed accord-

ing to the procedures described by García et al. (1999). Rabbits had ad libitum access to feed

and water. During nursing, before the experimental period, both weanling rabbits and their

mothers were given ad libitum access to a commercial feed during lactation (Cunilactal,

Nanta S.A., Spain) containing (g/kg of DM), 182 CP, 359 NDF, 45 ADL, 161 Starch and 53

ether extract.

3.3 Growth trial

Ninety-nine rabbits weaned at 25 days of age and weighing 482 7.01 g BW, were

blocked by litter and assigned at random to the three experimental diets (33 rabbits/diet).

Animals were housed individually and fed with the experimental diets for a 14-d period after

weaning. After that (at 39 days of age), all the animals received a commercial feed (Cuniu-

nic, Nanta, S.A.: 160 g CP, 144 g starch, 345 g NDF and 50 g ADF per kg) until 60 days

of age. In both experimental periods, animals had ad libitum access to feed and water. Aver-

age feed intake, weight gain and mortality from 25 to 39 days of age and from 39 to the end

of the experimental period (60 d) were recorded. To study mortality rate, an additional group

of four hundred sixty-two rabbits weaned at 25 days of age and weighing 487 3.97 g were

blocked by litter and caged in groups of 3-4 rabbits. Two hundred eighty-five were assigned

at random to the different treatments and received medication in water (95 rabbits/diet). The

other 177 rabbits were also assigned at random to the different treatments but received no

medication (59 rabbits/diet).


130

3.4 Digestion trial

Sixty-three rabbits weaned at 25 days of age and weighing 528 10.6 g BW were

blocked by litter, caged individually, and randomly assigned to the three experimental diets

(21 rabbits/diet). Total tract apparent digestibility of dry matter (DM), gross energy, crude

protein, NDF and starch was determined in 42 rabbits (14 rabbits/diet) from the original

group. All these animals had a 7-d diet adaptation period (from 25 to 31 days of age) after

which, daily dry matter intake and total fecal output were recorded for each rabbit over a 3-d

collection period (from 32 to 35 days of age) according to the Gómez-Conde et al. (2011).

Feces produced daily were collected and stored at –20ºC, dried at 80º C for 48 h and ground

at 1 mm for further analysis. All the 63 animals were slaughtered between 1900 and 2100 h

to minimize the influence of caecotrophy weighing on average 945 8.79 g. The whole gas-

trointestinal tract was removed and weighed, and the length of the small intestine (from the

pylorus to the caecum), stomach and caecum weights (with and without their contents) were

recorded individually in 19 rabbits/diet. The caudal 20 cm of the ileum was excised, and

ileal digesta frozen in dry ice to determine ileal apparent digestibility. For the chemical

analyses of the ileal digesta, samples were freeze-dried and ground. Due to the small

amounts of sample, ileal digesta from 3 rabbits of the same treatment were pooled, resulting

in 7 samples per treatment. The ileal digestibility of DM, starch and crude protein were de-

termined by the dilution technique using ytterbium as a marker as follows:

100ionconcentrat ytterbium ileal

ion/concentrat ytterbiumdietary 1matterDry id

100

ionconcentratstarch dietary

ionconcentrat ytterbium ion/ilealconcentratstarch


1starch id


131

100

ionconcentratprotein dietary

ionconcentrat ytterbium ion/ilealconcentratprotein


1Protein id

Besides pH of digestive contents were recorded in the caecum and in the stomach (at

the pyloric and at the fundus area, and in the mixture of stomach digesta (19 rabbits/diet).

Fourteen samples of the caecal contents were centrifuged at 25,000 g at 0ºC for 10 min.

The supernatant fluid was used to determine ammonia and total VFAs concentration. For

ammonia determination this supernatant was acidified with a solution of 0.2 M hydrochloric

acid (1 mL/mL) and frozen. While for VFA determination, a solution of 5% orthophosphoric

acid (vol/vol) plus 1% mercury choride (wt/vol) was added (0.1 mL/mL) to the supernatant,

but only six samples per diet were analyzed for VFA concentration. Three cm samples from

the middle part of the jejunum of 8 rabbits per diet were rinsed with KCl solution and placed

into a 10%, buffered, neutral formaldehyde solution (pH 7.2 to 7.4) to evaluate the mucosal

morphology. In a parallel experiment, samples from a control group of 19 suckling rabbits

(35 d old) with a BW of 876 17,0 g were processed in the same way to assess their muco-

sal morphology. The jejunal samples used in these analyses were gradually dehydrated in an

ethanol series (50 to 100%), embedded in paraffin, sectioned at 6 m and stained with hema-

toxylin and eosin (Armed Forces Institute of Pathology, 1968). Three to five slides contain-

ing jejunal cross sections were prepared for each sample and were viewed at 40× magnifica-

tion using an Olympus BX-40 light microscope. Images were digitally captured for later

analysis using Soft software version 3.2 C4040Z (Soft Imaging System, Olympus, GmbH,

Hamburg, Germany). Villous heights and crypt depths were determined according to the

procedure described by Hampson (1986), from 30 vertically-oriented villi per animal ob-

tained from 3 independent cross-sectional jejunal samples which at least 10 measurement

done in each case.


132

3.5 Microbiota trial

On hundred thirty rabbits weaned at 25 days of age and weighing 514 7.66 g BW

were blocked by litter, caged individually, and randomly assigned to the three experimental

diets (44 rabbits/diet) being half of them medicated. At 35 d of age rabbits were slaughtered

at 1900 h and one gram of ileal and caecal digesta from all the rabbits were collected in a

sterile plastic tube containing 3 ml of 98% molecular biology grade ethanol and stored at 4º

C for ileal and caecal microbiota characterization by RFLP.

3.6 Chemical analysis


(934.01), ash (967.05), CP (968.06), ether extract (920.39) and starch (amyloglucosidase--

amylase method, 996.11). Dietary aNDFom, ADFom and lignin (sa) were determined se-

quentially using the filter bag system (Ankom Technology, New York, NY) according to the

method of Mertens (2002), the AOAC (2000; procedure 973.187) and Van Soest et al.

(1981), respectively. Soluble fibre was analyzed as a neutral detergent-soluble fiber accord-

ing to Hall et al. (1997). Microbiota detection frequencies and similarity rate were deter-

mined by 16S rRNA RFLP according to the following procedure: 400 mg of gut contents

were processed for total DNA extraction using the QIAamp DNA Stool Mini Kit system

(Quiagen Inc., Chatsworth CA) according to the manufacturer’s instructions, with additional

lysozyme and proteinase K steps. The purified DNA was maintained at –20ºC until use. The

primers 5’-CTACGGGAGGCAGCAGT-3’ and 5’-CCGTCWATTCMTTTGAGTTT-3’,

corresponding to regions I and II of the 16S rRNA gene (Lane, 1991), were used to amplify

a 500- to 600-bp product. Amplifications were performed in a final volume of 50 L using a

PCR-Master Mix (Applied Biosystems) containing 1.25 IU of Taq polymerase, 50 ng of

DNA template, 0.2 M of each primer, and the following cycling condition: 94ºC for 5 min,


133

followed by 35 cycles of 94ºC for 1 min, 45ºC for 1 min, and 72ºC for 1 min 15s. The last

extension cycle was continued for 5 min. Aliquots of the amplified DNA fragments were

digested, in separated tubes, with Alu I, Rsa I, Hpa II, Sau 3A I, or Cfo I restriction endonu-

cleases (Sigma-Aldrich). The endonuclease fragments were resolved in 2% agarose gels at

150 V for 60 min, and DNA bands were visualized using a UV Chemigenious Image System

at a 4.63-s exposure (SynGene) using GeneSnap software (SynGene, Cambridge, UK). With

the information resulting from the relative size of RFLP bands, the information stored in the

SSU_Una.gb file from the Ribosomal Database Project (Maidak et al., 1997), and a specific

software developed in our institute (CReSA), we could identify the bacterial genus or spe-

cies compatible with the obtained RFLP profile. The frequency of the compatibility profile

of every bacteria studied defined as the presence or absence of RFLP bands compatible with

the theoretical RFLP bands for a certain genus or bacterial species was studied for every

animal (i.e. a combination of bands with 92 bp þ 467 bp in Alu I, plus 120 bp þ 404 bp þ 35

bp bands in Rsa I, plus 189 bp þ 58 bp þ312 bp bands in Hpa II, plus 82 bp þ 477 bp bands

in Sau3A, plus a band of 559 bp in Cfo I is compatible with C. perfringens. The absence of

any of the above mentioned bands in the RFLP profile infer that the level of C. perfringens

in the sample is below the detection level, as in other PCR-electrophoresis methods around

104-105 DNA copies per g of sample).

3.7 Statistical analysis

The results obtained in this study for growth traits, faecal digestibility, mucosa mor-

phology, weight of digestive organs, digesta pH and fermentation traits were analyzed as a

completely randomised block, with the type of diet used as the main source of variation and

the litter as a block effect using the GLM procedure (SAS Inst. Inc., Cary, NC). Weaning

weight was included as a linear covariate for growth traits, whereas for ileal digestibility


134

only diet effect was considered. Means were compared using a protected t-test, and differ-

ences were considered significant at P < 0.05. Mortality and frequency of detection of the

bacteria were analysed using logistic regression (GENMOD procedure of SAS, considering

a binomial distribution) and results were transformed from the logit scale.

4. RESULTS

4.1. Growth trial

Daily weight gain, feed intake, and feed efficiency of medicated rabbits were not af-

fected by source of starch either from 25 to 39 d of age (on average 46.9 g/d, 75.6 g/d, and

0.573 g/g, respectively) or during the whole fattening period (45.4 g/d, 120 g/d, and 0.380

g/g, respectively) (Table 2). Mortality rate was also not affected by source of starch being on

average 12.7 and 18.5% in the post-weaning period and in the whole fattening period, re-

spectively (Table 2). However, it was double (P ≤ 0.005) in not medicated animals com-

pared to medicated ones just in the post-weaning period (8.9 vs. 17.5%) or in the whole fat-

tening period (13.0 to 25.4%). No significant interaction was found between source of starch

and medication (P > 0.10).

4.2 . Digestion trial

Source of starch had no effect on dry matter intake (82.3 g/d), on faecal and ileal di-

gestibility of DM (46.0 and 65.1%) and crude protein (63.5 and 80.6%), and on faecal di-

gestibility of gross energy and NDFom (64.8 and 23.0%) (Table 4). Ileal digestibility of

starch increased by 4.7% in animals fed boiled wheat diet compared to those fed boiled rice

(P < 0.050), being those fed raw wheat intermediate. It resulted that rabbits fed boiled rice

increased ileal flow of starch by 54% compared to those fed wheat based diets (P < 0.050).


135

These differences tended to disappear at faecal level, as starch faecal digestibility only

showed a slight trend to increase in rabbits fed boiled wheat compared to those fed boiled

rice diet (P = 0.090).

Weaning and source of starch influenced mucosa morphology (Table 5). Villus

height was a 16% shorter in weaned rabbits fed wheat based diets compared to suckling rab-

bits of the same age, showing those fed boiled rice an intermediate value (P < 0.050). Rab-

bits fed boiled rice showed deeper crypts respect to those fed boiled rice, being those of rab-

bits fed raw wheat intermediate (P < 0.050). Crypt depth of suckling rabbits was shorter than

for any weaned group of rabbits (P < 0.050). It resulted in a similar villus height/crypt depth

ratio in weaned rabbits independently of source of starch (4.07) that was much lower than

that obtained for suckling rabbits (6.68) (P < 0.050).

Source of starch did not affect total gastrointestinal weight (24.1% BW), length of

the small intestine (220 cm), and stomach pH (1.62, 2.95 and 2.51 in the pylorus area, fun-

dus, and in the mixture of the whole stomach content, respectively). However, rabbits fed

raw wheat showed higher weight of empty stomach and caecal content than those fed boiled

wheat and rice (by 5.8 and 17.9%, respectively. P < 0.050), although exerted no influence on

stomach content and empty caecum weight (4.88 and 1.85% BW, respectively). Source of

starch had no influence on caecal pH (5.48), caecal VFA concentration (62.0 mmol/L), and

molar proportions of acetic, propionic, butyric and valeric acids (79, 6.5, 13, and 1.1%, re-

spectively). Caecal NH3-N increased by 31% from rabbits fed boiled rice to those fed raw

wheat, and iso-valeric caecal concentration tended to be higher (P = 0.090) in rabbits fed

boiled wheat compared the other two treatments.


136

4.3. Microbiota trial

Source of starch had minor effect of frequency of detection (FD) of the studied bac-

teria in the ileum (Table 7). The ileal FD recorded for Campylobacter spp. and Helicobacter

spp. was higher (P < 0.050) in rabbits fed boiled wheat compared to those fed boiled rice.

Those fed raw wheat showed intermediate values for Campylobacter spp. but similar value

to boiled wheat for Helicobacter spp. No other dietary effect was found in ileum. On the

opposite, medication decreased all the FD of the studied bacteria compared to not medicated

rabbits (P ≤ 0.048), except that of Helicobacter spp. This reduction was larger for Bac-

teroides fragilis and Clostridium perfringens (reduced by 89 and 72%) and smaller for Clos-

tridium spp. and Bacteroides spp. (by 15 and 32%). No interaction between diet and medica-

tion was found for ileal FD of bacteria.

Source of starch had a major influence on FD of bacteria in the caecum than in the il-

eum (Table 8). Rabbits fed boiled wheat showed higher caecal FD of Campylobacter spp.

and Ruminococcus spp. than the other two diets, whereas FD of Bacteroides spp. was higher

for rabbits fed boiled rice respect to those fed boiled wheat, being intermediate those fed raw

wheat (P < 0.050). The caecal FD of Butirivibrio fibrisolvens and Clostridium perfringens

tended to be higher in rabbits fed boiled wheat compared to those fed boiled rice (P ≤

0.083). Medication showed minor effect in caecal FD of bacteria compared to the ileum. It

only decreased the FD of Campylobacter spp., Clostridium perfringens and Propionibacte-

rium spp. (by 30, 68 and 21%, respectively. P ≤ 0.048).

5. DISCUSSION

Heat processing of wheat improved slightly ileal digestibility of starch. The high ileal

digestibility of raw wheat starch limited the effect of heat processing that is more pro-


137

nounced for less digestible starch sources like maize or peas (Gidenne and Perez, 1993a;

Gutiérrez et al., 2002b; Gidenne et al., 2005a). Heat processing of wheat also decreased the

weight of caecal contents, although DM and starch flow to the caecum was not modified,

and increased the frequency of detection of Campylobacter spp. and Ruminococcus spp. in

the caecum with no changes at ileal level, but tending to increase caecal isovalerate propor-

tion. These effects might be related to the influence of heat processing in other nutrients like

fibre, although NDF faecal digestibility was not modified. These effects of heat processing

of wheat did not modify growth performance, mortality, ileal or faecal digestibility, and mu-

cosa morphology.

The partial substitution of boiled wheat for boiled rice in the diet impaired ileal

starch digestibility. This result contrasts with the high starch digestibility of rice observed in

pigs (Mateos et al., 2006; Menoyo et al., 2011). It might be partially explained by an in-

crease of retrograded starch after cooling (not measured), which would agree with the trend

to reduce also faecal starch digestibility, as reported by Maertens and Luzi (1995) for ex-

truded maize. The reduction of ileal starch digestibility of rice increased the ileal flow of

starch to the caecum. However, it did not increase mortality rate as suggested by Cheeke and

Patton (1980), but changed intestinal microbiota decreasing the frequency of detection of

Campylobacter spp. (both at ileum and caecum), Helicobacter spp. (at ileum) and Rumino-

coccus spp. (at caecum) and increasing that of Bacteroides spp. (at caecum). In spite of the

changes detected in the intestinal microbiota, it was only observed a trend to reduce caecal

isovaleric acid proportion when boiled rice substituted boiled wheat, with no major changes

in caecal fermentation pattern as observed by Gidenne et al. (2005a) and Belenguer et al.

(2012) when compared different sources of starch. The effects of boiled rice supplementa-


138

tion did not alter growth performance, mortality, ileal or faecal digestibility of other nutri-

ents than starch, and mucosa morphology.

Mucosa morphology was impaired at 35 d of age in 25 d weaned rabbits compared to

those still suckling as previously observed (Dojana et al., 1998; Gutiérrez et al., 2002a),

which is in agreement with the higher sensivity to suffer digestive disorders during the post-

weaning period (Coudert et al., 2000; Peeters et al., 2000), and source of starch did not im-

prove it. For this reason, medication is required sometimes in this period. The medication of

rabbits had a great impact on microbiota at the ileum, reducing the ileal frequency of detec-

tion of most bacteria studied, whereas the effect at the caecum was lower and limited to a

reduction of the frequency of detection of Campylobacter spp., Clostridium perfringens and

Propionibacterium spp. These effects were associated to a strong reduction of mortality rate,

and are in agreement with the reduction of of mortality rate and ileal frequency of detection

of different bacteria reported by Chamorro et al. (2007).

5.1 Conclusions

The heat processing of wheat has no advantage for rabbit growth performance and

health. The partial substitution of boiled wheat by boiled rice impaired starch digestibility

but had no practical effect on growth performance. Source of starch influenced intestinal

microbiota but had no impact on rabbit health. Medication reduced frequency of detection of

bacteria especially at the ileum and strongly reduced mortality.

Acknowledgements

We are grateful to the Comisión Interministerial de Ciencia y Tecnología (CICYT)

project AGL2001-2796 for financial support, and to ELANCO Valquímica S.A., Esteve

Santiago S.A., and Ibérica de Nutrición Animal S.L. for their contribution to this study.


139

6. REFERENCES

Armed Forces Institute of Pathology. 1968. Routine staining procedures. Pages 32–46 in L.

G. Luna, ed. Manual of Histologic Staining Methods. McGraw-Hill Book Co., New

York, NY.

Association of Official Analytical Chemists, 2000. Official Methods of Analysis. 17th Ed.


Belenguer, A., Abecia, L., Belanche, A., Milne, E., Balcells, J. 2012. Effect of carbohydrate

source on microbial nitrogen recycling in growing rabbits. Livest. Sci. 150:94-101.

Blas, E., Gidenne, T. 2010. Digestion of sugars and starch. In: The nutrition of the rabbit

(ed. JC de Blas and J Wiseman), pp. 19-38, Commonwealth Agricultural Bureau, Wall-

ingford, UK.

Cachaldora, P., Nicodemus, N., García, J., Carabaño, R., de Blas, J.C. 2004. Efficacy of

Amylofeed in growing rabbit diets. World Rabbit Sci. 12:23-31.

Chamorro, S., Gómez-Conde, M. S., Pérez de Rozas, A. M., Badiola, I., Carabaño, R., De

Blas, J. C. (2007). Effect on digestion and performance of dietary protein content and of

increased substitution of lucerne hay with soya-bean protein concentrate in starter diets

for young rabbits. Animal, 1 (5): 651-659.

Che, T.M., Perez, V.G., Song, M., Pettigrew, J.E. 2012. Effect of rice and other cereal grains

on growth performance, pig removal, and antibiotic treatment of weaned pigs under

commercial conditions. J. Anim. Sci. 90:4916-4924.

Cheeke, P.R and Patton, N.M. (1980). Carbohydrate-overload of the hindgut. A probable

cause of enteritis. Journal of Applied Rabbit Research. 3 (3): 20-23.

Corring, T., Lebas, F. and Courtot, T. (1972). Contrôle de l’evolution de l’equipament en-

zymatique du pancréss exocrine du lapin de la naissance a six semaines. Annales de Biol-

ogie Animale, Biochimie et Biophysique 12, 221-231.


140

Coudert, P., Peeters, J.E., Rosell, J.M., Argüello, J.L., Badiola, J.I., Blas, E., Cuervo, L.,

Drouet-Viard, F., Licois, D., Provot, F. 2000. Enfermedades del aparato digestivo: cau-

sas parasitarias y otras. In: Enfermedades del conejo (ed. Mundi-Prensa), pp 215-263.

De Blas, C., Mateos, G.G. 2010. Feed formulation. In: The nutrition of the rabbit (ed. JC de

Blas and J Wiseman), pp. 222-232, Commonwealth Agricultural Bureau, Wallingford,

UK.

Dojana, N., Costache, M., Dinischiotu, A. 1998. The activity of some digestive enzymes in

domestic rabbits before and after weaning. Anim. Sci. 66:501-507.

García, J., R. Carabaño, De Blas, J.C.. 1999. Effect of fiber source on cell wall digestibility

and rate of passage in rabbits. J. Anim. Sci. 77:898–905.

Gallois M., Gidenne T., Fortun-Lamothe L., Le Huerou-Luron, Lallés J.P. 2005. An early

stimulation of solid feed intake slightly influences the morphological gut maturation in

the rabbit. Reprod. Nutr. Dev., 45: 109-122. doi: 10.1051/rnd:2005008.

Gidenne, T., Perez, J.M. 1993a. Effect of dietary starch origin on digestión in the rabbit. 2.

Starch hydrolysis in the small intestine, cell wall degradation and rate of passage meas-

urements. Anim. Feed Sci. Technol. 42:237-247.

Gidenne, T., Perez, J.M. 1993b. Effect of dietary starch origin on digestión in the rabbit. 1.

Digestibility measurements from weaning to slaughter. Anim. Feed Sci. Technol. 42:249-

257.

Gidenne, T., Segura, M., Lapanouse, A., 2005a. Effect of cereal sources and processing in

diets for the growing rabbit. I. Effects on digestion and fermentative activity in the cae-

cum. Anim. Res. 54, 55-64.

Gidenne, T., Jehl, N., Perez, J.M., Arveux, P., Bourdillon, A., Mousset, J.L., Duperray, J.,

Stephan, S., Lamboley, B., 2005b. Effect of cereal sources and processing in diets for the


141

growing rabbit. II. Effects on performances and mortality by enteropathy. Anim. Res. 54,

55-64.

Gómez-Conde, M.S., García J., Villamide, M.J., Carabaño, R 2011. Determination of faecal

dry matter digestibility two weeks after weaning in twenty five day old weaned rabbits.

World Rabbit Sci. 19:57-62.

Gutiérrez I., Espinosa A., García J., Carabaño R., De Blas C. 2002a. Effects of levels of


Anim. Sci., 80: 1029-1037.

Gutiérrez I, Espinosa A, García J, Carabaño R, De Blas JC 2002b. Effect of starch and pro-

tein sources, heat processing, and exogenous enzymes in starter diets for early weaned

rabbits. Anim. Feed Sci. Technol. 98, 175-186.

Hall, M. B., Lewis B. A., Van Soest P. J., Chase L. E.. 1997. A simple method for estima-

tion of neutral detergent-soluble fiber. J. Sci. Food Agric. 74:441–449.

Hampson, D.J. 1986. Alteration in piglet small intestine structure at weaning. Res. Vet. Sci.

40, 32-40.

Lane, D.J. 1991. 16S/23S rRNA sequencing. In Nucleic acid techniques in bacterial system-

atics (ed. E Stackebrandt and M Goodfellow), pp. 115-175, John Wiley and Sons, New

York, USA.

Maertens, L., Luzi, F. 1995. The effect of extrusion in diets with different starch levels on

the performance and digestibility of young rabbits. In: Proceedings 9th Symposium on

Housing and Diseases of Rabbits, Furbearing Animals and Pet Animals. DVG, Celle,

Germany, pp. 131-138.

Maidak, B.L., Olsen, G.J., Larsen, N., Overbeek, R., McCaughey, M.J. Woese, C.R 1997.

The RDP (Ribosomal Database Project). Nucleic Acid Research 25,109-110.


142

Mateos, G.G., Martín, F., Latorre, M.A., Vicente, B., Lázaro, R. 2006. Inclusion of oat hulls

in diets for young pigs based on cooked maize or cooked rice. Anim. Sci. 82:57-63.

Menoyo, D., Serrano, M.P., Barrios, V., Valencia, D.G., Lázaro, R., Argente, J., Mateos,

G.G. 2011. Cereal type and heat processing of the cereal affect nutrient digestibility and

dynamics of serum insulin and ghrelin in weanling pigs. J. Anim. Sci. 89:2793-2800.

Mertens, D. R. 2002. Gravimetric determination of amylase-treated neutral detergent fibre in

feeds with refluxing beakers or crucibles: Collaborative study. J. Assoc. Off. Assoc.

Chem. Int. 85:1217–1240.

Peeters, J.E., Licois, D., Saco, M., Badiola, J.I., Rosell, J.M. 2000. Enfermedades del apara-

to digestive: causas infecciosas. In: Enfermedades del conejo (ed. Mundi-Prensa), pp

163-213.

Pluske, J.R., Black, B., Pethick, D.W., Mullan, B.P., Hampson, D.J. 2003. Effects of differ-

ent sources and levels of dietary fibre in diets on performance, digesta characteristics and

antibiotic treatment of pigs after weaning. Anim Feed Sci. Technol. 107:129-142.

Sequeira, J., García, A., Villamide, M.J., 2000. Effect of grinding and extrusion on the di-

gestibility of wheat and corn by rabbits. World Rabbit Sci. 8 (Suppl. 1, Vol. C), 429-435.

Vicente, B., Valencia, D.G., Pérez-Serrano, M., Lázaro, R., Mateos, G.G. 2008. The effects

of feeding rice in substitution of corn and the degree of starch gelatinization of rice on the

digestibility of dietary components and productive performance of young pigs. J. Anim.

Sci. 86:119-126.

Spanish Royal Decree 1201/2005. 2005. Sobre protección de los animales utilizados para

experimentación y otros fines científicos. Boletín Oficial del Estado 252, 34367-34391.

Van Soest, J.P., Robertson, J.B., Lewis, B.A. 1981. Methods for dietary fiber, detergent fi-

ber and nonstarch polysaccharides in relation to animal nutrition. J.Dairy Sci.74, 3583-

3597.


143

Table 1. Ingredient and chemical composition of experimental diets

Experimental diets

Raw wheat Boiled wheat Boiled rice

Ingredients, g/kg

Raw wheat 323 — —

Boiled wheat — 323 162

Boiled rice — — 129

Wheat bran 84 84 116


Soybean meal concentrate 20 20 20


Alfalfa hay 144 144 144

Oat hulls 147 147 147


Lard 33 33 33

L-Lysine HCL 4.5 4.5 4.5

DL-methionine 1.0 1.0 1.0

L-threonine 1.5 1.5 1.5

Sodium chloride 5.0 5.0 5.0

Calcium oxide 6.0 6.0 6.0

Vitamin/mineral premixa 5.0 5.0 5.0


Dry matter 914 912 914

Ash 65 65 66

Crude protein 193 200 190


Starch 215 207 230

Neutral detergent fibre 339 359 338

Acid detergent fibre 161 162 168

Acid detergent lignin 36 37 38

Neutral detergent soluble fibre

81 81 77


144

Calculated composition, g/kg DM

Sugarsb

39 39 36

Lysineb 12.5 12.5 12.5

Methionineb 4.10 4.06 4.05

Methionine+Cystineb

7.22 7.24 6.89

Threonineb

8.42 8.44 8.42

Calciumb

6.78 6.69 6.67

Phosphorusb

4.49 4.50 4.38

Sodiumb

2.30 2.30 2.30

aProvided by Ibérica de Nutrición Animal (S.L). INA LA 1 Conejos AC4-Único with Cycostat.

Supplied per kilogram of diet: retinyl acetate, 9,000 IU; cholecalciferol, 1,800 IU; dl--tocopheryl

acetate, 15.0 IU; menadione sodium bisulfite, 1 mg; riboflavin, 2 mg; cianocobalamine, 0.01 mg;

thiamine, 1 mg; calcium d-pantothenate, 5mg; Nicotic acid, 15 mg; Choline chloride, 250mg; Fe, 40

mg; Zn, 50 mg; Mn, 40 mg; Cu, 12 mg; I, 1.2 mg; Co, 0.5 mg; Cycostat 66G E 758, 66 mg.

b Calculated values according FEDNA (2003)


145

Table 2. Effect of source of starch on fattening performance of medicated rabbits

Experimental diets

SEM1

P

Raw wheat Boiled

wheat Boiled rice

Post weaning period (25-39 d)

Weight gain, g/d 44.4 43.5 42.9 2.05 0.88

Feed intake, g/d 78.9 77.5 71.2 3.07 0.23

Feed efficiency, g/d 0.563 0.566 0.594 0.015 0.53

Whole fattening period (25-60 d)

Weight gain, g/d 45.1 45.2 46.0 0.84 0.74

Feed intake, g/d 120 120 120 2.33 0.98

Feed efficiency, g/d 0.375 0.379 0.385 0.0031 0.19

1 n = 33. Means denoted with different letters differ at P < 0.05 level.


146

Table 3. Effect of source of starch and medication on mortality rate during fattening.

Experimental diets Medication P1

Raw

wheat

Boiled

wheat

Boiled

rice Yes No Diet Medication

No. of rabbits

Post weaning period

(25-39 d) 10.7 12.9 14.4 8.88 17.5 0.58 0.005

Whole fattening period

(25-60 d) 16.6 19.2 19.8 13.0 25.4 0.73 0.001

1 n = 128 medicated rabbits/diet and n = 59 not medicated rabbits/diet. Interaction between

diet and medication was not significant (P > 0.10).


147

Table 4. Effect of source of starch on feed intake, faecal and ileal apparent digestibility and

ileal flow of nutrients in 35-d-old medicated rabbits

Experimental diets

SEM1

P

Raw wheat Boiled wheat Boiled Rice

ADFI 32 to 35, g/d

82.8 82.8 81.4 2.63 0.94

Faecal apparent digestibility, %

Dry matter

64.8 64.7 65.8 0.69 0.58

Gross energy 63.9 64.6 65.8 0.68 0.25

Crude protein 80.7 80.4 80.7 0.74 0.96

Neutral detergent fibre 21.5 24.7 22.8 1.40 0.37

Starch 98.7 99.6 98.3 0.34 0.090

Ileal apparent digestibility, %

Dry matter 46.0 46.8 45.2 1.22 0.86

Starch 91.5b 93.2

a 89.0

c 0.56 0.020

Crude protein 63.6 66.9 60.0 1.40 0.15

Ileal flow, g/d

Dry matter 44.7 44.0 44.6 1.76 0.96

Starch 1.52b 1.16

b 2.07

a 0.17 0.007

Crude protein 5.81 5.48 6.20 0.38 0.44

1 n = 14 rabbit/diet, except for ileal apparent digestibility and ileal flow where n = 7

pooles/diet. Means denoted with different letters differ at P < 0.05 level.


148

Table 5. Effect of source of starch and weaning on the morphology of jejunal mucosa in 35-

d-old rabbits.

Weaned rabbits

Suckling

rabbits SEM

1 P Experimental diets


Villous height, µm 455b

493b

513ab

566a

28.4 0.010

Crypt depth, µm 122ab

113b 128

a 89.6

c 4.66 0.001

Villous height/Crypt

depth 3.75

b 4.41

b 4.04

b 6.68

a 0.37 0.001

1 n = 8 in all cases except for 35-d-old suckling rabbits (n = 19).


149

Table 6. Effect of source of starch on digestive organs, digesta pH and fermentation traits.

Experimental diets

SEM P


Total gastrointestinal tract weight, g 24.9 23.2 24.2 0.50 0.13

Small intestine length, cm

217 221 223 5.95 0.79

Stomach

Empty weight, % of body weight 1.55a

1.46b

1.47b

0.022 0.025

Content, % of body weight 4.90 4.78 4.96 0.20 0.86

pH Pylorus 1.51 1.75 1.61 0.074 0.14

pH Fundus 2.85 2.99 3.02 0.18 0.82

pH mix 2.49 2.56 2.49 0.13 0.94

Cecum

Empty weight, % of body weight 1.93 1.82 1.79 0.075 0.48

Content, % of body weight 7.27a 5.87

b 6.46

b 0.25 0.007

pH 5.47 5.48 5.49 0.046 0.99

Caecal VFA, mmol/L

59 63 64 6.1 0.89

Acetic acid, % 79 77 80 1.5 0.44

Propionic acid, % 6.7 7.6 5.1 1.2 0.39

Butyric acid, % 12 13 13 0.98 0.78

Valeric acid, % 0.71 0.89 0.64 0.101 0.25

Iso-valeric acid, % 1.1 1.5 1.1 0.14 0.090

Caecal NH3-N mmol/L

13.5a

12.2ab

10.3b

0.81 0.020

1 n = 19, except for VFA concentration (n = 6) and NH3-N (n = 14). Means denoted with

different letters differ at P < 0.05 level.


150

Table 7. Effect of source of starch and medication on ileal frequency of detection (%) of the

indicated bacteria in 35 d old rabbits1.

Experimental diets Medication P

2

Raw

wheat

Boiled

wheat

Boiled


Bacteroides spp. 80.5 76.9 64.5 60.8 89.9 0.42 0.002

Bacteroides fragilis 11.1 5.10 9.67 1.78 16.1 0.58 0.003

Butirivibrio fibrisolvens 33.3 41.0 35.3 21.5 54.1 0.73 0.001

Campylobacter spp. 19.2ab

38.4a

9.67b

12.5 36.1 0.046 0.002

Clostridium spp. 91.5 92.5 77.2 80.3 94.4 0.12 0.009

Clostridium perfrigens 19.5 33.3 16.1 10.8 38.0 0.19 0.001

Clostridium difficile 14.2 15.4 19.3 8.97 24.2 0.86 0.018

Escherichia coli 16.6

7.60

6.45

5.15 16.0 0.24 0.048

Helicobacter spp. 36.3a

46.1a

13.1b

25.0 40.1 0.004 0.11

Propionibacterium spp. 33.2 38.4 29.1 19.8 49.9 0.76 0.002

Ruminococcus spp. 30.4 46.2 32.4 19.6 55.8 0.68 0.001

1 n = 19, 21 and 16 for medicated raw wheat, boiled wheat and boiled rice groups, respec-

tively, and n = 17, 18 and 15 for not medicated raw wheat, boiled wheat and boiled rice

groups, respectively. 2 Means denoted with different letters differ at P < 0.05 level. No sig-

nificant interaction was detected between diet and medication.


151

Table 8. Effect of source of starch and medication on caecal frequency of detection (%) of

the indicated bacteria in 35 d old rabbits1.

Experimental diets Medication P

2

Raw

wheat

Boiled

wheat

Boiled


Bacteroides spp. 86.9ab

69.6b

91.0a

82.4 80.7 0.037 0.70

Bacteroides fragilis 36.6 23.3 30.1 26.4 33.2 0.43 0.45

Butirivibrio fibrisolvens 73.7 83.8 60.7 78.9 68.6 0.083 0.22

Campylobacter spp. 44.6b

72.1a

45.4b

45.6 64.8 0.023 0.036

Clostridium spp. 97.4 97.6 100 98.2 98.2 0.50 0.99

Clostridium perfrigens 36.7 46.5 21.0 17.5 54.3 0.068 0.001

Clostridium difficile 42.3 60.4 39.4 52.6 43.8 0.13 0.40

Escherichia coli 23.7 30.0 21.2 27.9 22.8 0.66 0.55

Helicobacter spp. 47.3 53.5 54.4 45.5 58.0 0.77 0.18

Propionibacterium spp. 76.3 78.9 70.0 66.7 84.2 0.72 0.048

Ruminococcus spp. 76.3b

95.3a

60.8b

80.7 77.2 0.001 0.42

1 n = 19, 22 and 16 for medicated raw wheat, boiled wheat and boiled rice groups, respec-

tively, and n = 19, 21 and 17 for not medicated raw wheat, boiled wheat and boiled rice

groups, respectively. 2 Means denoted with different letters differ at P < 0.05 level. No

significant interaction was detected between diet and medication.

Chapter 5

Determination of faecal dry matter digestibil-

ity two weeks after weaning in twenty-five-day-old

weaned rabbits

Published in: 2011 World Rabbit Science, 19: 57-62

Chapter 5: Determination of faecal dry matter digestibility

155

1 ABSTRACT

The aim of this work was to analyse the evolution from 26 to 40 d of age of apparent

faecal dry matter digestibility (DMd) in rabbits weaned at 25 d of age to define how to de-

termine nutrient digestibility in the post-weaning period. Fifteen New Zealand Californian

rabbits from 5 litters (3 rabbits/litter) weaned at 25 d of age and weighing 60275g were fed

ad libitum a commercial diet containing 20.0% crude protein and 33.5% NDF (on DM ba-

sis). Feed intake and faeces excretion were recorded daily from 25 to 40 d of age and DM

digestibility determined. Litter affected DM intake and excretion (P = 0.013 and 0.014, re-

spectively), and tended to affect DMd (P = 0.061), whereas age influenced all these traits (P

< 0.001). Dry matter intake and excretion increased from 26 to 40 d of age by 158 and

480%, respectively. During the first week after weaning, DM intake increased more slowly

than DM excretion (55 vs. 245%), but in the second week after weaning both increased by

67%. The correlation between daily feed intake was higher with the faeces excretion of the

same day than with faeces excretion of the next day, and the first values were used to deter-

mine daily DMd. A broken line regression model was fitted to daily DMd, which decreased

linearly from weaning to 32 d of age (2.17 0.25 percentage units per day), whereas from

32 to 40 d remained constant (69.4 0.47%). Accordingly, for 25-d old weaned rabbits it

would be advisable to begin a digestibility trial not before 32 d of age, using the first week

after weaning as adaptation period. Average standard deviation of DMd decreased by 54%

when the length of the collection period increased from 2 to 6 d. Consequently, the number

of animals required to detect a significant difference among means depends on the length of

the collection period. For a conventional collection period of four days a difference of 2 per-

centage units could be detected by using 14 animals/treatment.

Key words: dry matter digestibility, weaned rabbits.


156

2. INTRODUCTION

The post-weaning period is a critical time for rabbits, as their digestive function is

not completely mature and important changes takes place in terms of organ/mucosa devel-

opment, enzyme activity, microbiota colonisation, gut barrier mechanisms, and feed intake

(Carabaño et al., 2010). Some of these changes also seem to depend on the the composition

and nutritional value of the weaning diet (Gallois et al., 2005, 2008; Álvarez et al., 2007;

Gómez-Conde et al., 2007). However, different dietary digestibility values may be obtained

depending on the methodology used, the time after weaning and the age at weaning (Parigi

Bini et al., 1991; Gallois et al., 2008). In fact, recently weaned rabbits show a different di-

gestive pattern than older animals (> 49 d) normally used in in vivo digestibility trials as

recommended by the European reference method (Perez et al., 1995a). This method assumes

that 49 d old rabbits have a steady feed intake and faecal excretion during the 4 d collection

period. In contrast, at 25 d rabbits are changing from a mixed diet (milk + feed) to an exclu-

sively solid diet and a progressive development of the caecum is taking place (Laplace and

Lebas, 1972; Padilha et al., 1995; Gutiérrez et al., 2002; Xiccato et al., 2003), resulting in

changes in feed intake and faeces excretion throughout this period.

The aim of this work was to study the DM digestibility pattern in rabbits weaned at 25 days

during the two weeks after weaning and define the best procedure to determine faecal nutri-

ent digestibility by using total faecal collection.


3.1. Experimental procedure

Fifteen New Zealand Californian rabbits weaned at 25 d of age were taken at ran-

dom from 5 litters (3 rabbits/litter). Feed intake and total faeces excretion were recorded

daily from 25 to 40 d of age. Rabbit weight averaged 60275 g at weaning, and 1405156 g


157

at 40 d of age. Animals were housed individually in metabolism cages (405×510×320 mm)

and they had ad libitum access to the same commercial feed (crude protein 20.0%, starch

20.8%, NDF 33.5%, DM basis, with no antibiotic) throughout the experiment. Faeces were

collected daily, stored at -20ºC, and once the trial finished, dried at 80ºC for three days.

Samples of faeces dried at 80ºC and feed were dried at 103ºC for 24 h (AOAC, 2000; meth-

od 934.01). Apparent faecal dry matter digestibility (DMd) was calculated as: (DM intake –

DM excretion) 100/ DM intake.

3.2 .Statistical analysis

The effect of age on daily feed intake, faeces excretion and DMd was studied using a

repeated measures analysis by the MIXED procedure of SAS (Littell et al., 1998), including

the litter effect as a block. The NLIN procedure from SAS was used to fit a one-slope bro-

ken line model (Robbins et al., 2006) to the evolution of DMd with age. Number of repli-

cates required to detect a significant difference were calculated according to Kaps and Lam-

berson (2004).

4. RESULTS AND DISCUSSION

Average daily gain, feed intake and feed efficiency from 25 to 40 d of age were

53.56.04 g/d, 79.7±9.40 g/d and 0.6730.039, respectively, and no mortality was recorded.

Dry matter intake and excretion were influenced by litter (P = 0.013 and 0.014, respectively)

and age (P < 0.001 for both) and increased from 26 to 40 d of age by 158 and 480%, respec-

tively (Figure 1). The first week after weaning DM intake increased more slowly than DM

excretion (55 vs. 245%), but during the second week after weaning both increased by 67%.

Dry matter excretion was low just after weaning (6.523.38 g at 26 d of age) likely because


158

of the still high milk intake compared to feed intake (around 30 and 10 g, respectively, Nic-

odemus et al., 2010) in the last 4 d of lactation (21 to 25 d of age). However, in the first day

after weaning (26 d of age), feed intake seemed to increase sharply until 45.816.3 g DM/d,

compared to feed intake before weaning (10 g/d from 21 to 25 d of lactation, reported previ-

ously by Nicodemus et al., 2010), and kept on increasing until 29 d of age (81.310.8 g

DM/d. Figure 1). The increase in feed intake was not constant, and rabbits decreased feed

intake at 30 d of age and, although at a lower rate, at 33 and 37 d of age (Figure 1). This

behaviour probably depends on the adaptation of rabbits to solid feed intake. With regard to

faeces excretion, around weaning the caeucum is developing and increasing its capacity (Pa-

dilha et al., 1995; Gutiérrez et al., 2002) which might lead to a higher mean retention time of

digesta, with faeces excretion decreased and DMd consequently increasing. The situation

changed in the first week after weaning, as faeces excretion proportionally increased more

than feed intake (Figure 1).

Based on the evolution of feed intake and faeces excretion after weaning, Gallois et

al. (2008) compared DMd calculated either as in this work or by using the faeces excretion

delayed by one day, as initially proposed by Parigi Bini et al. (1991), since young rabbits

show a great increase in feed intake compared to a delayed faeces excretion due to the con-

temporary gut development. Gallois et al. (2008) found that DMd values were similar be-

tween 37 and 42 d of age but different between 23 and 28 d of age depending on the calcula-

tion procedure. Parigi Bini et al. (1991) did not find significant differences in DMd using the

two methods, while changes were found in fibre fractions digestibility in young rabbits still

nursing (from 20 to 25 d of age). In this work, we studied the correlation between daily feed

intake and daily faeces excretion of the same day or of the following day (Figure 2) to take

into account that around 12-24 h are required by the feed to complete its transit through the


159

gut. As the correlation was higher between feed intake and faeces excretion of the same day,

these data were used to determine daily DMd (Figure 3).

DMd calculated daily tended to be influenced by litter (P = 0.061) and was affected

by age (P < 0.001) (Figure 3). DMd linearly decreased from weaning to 32 d of age (2.17

0.25 percentage units per day), whereas it remained constant from 32 to 40 d (69.4

0.47%). The changes in DMd with age are in agreement with the observed feed intake and

excretion patterns. During the first week after weaning DMd was reduced as DM excretion

increased faster than feed intake. During the second week after weaning, DM intake and

excretion increased at a similar rate and therefore DMd remained unchanged. According to

these results it would be advisable in 25-d weaned rabbits to begin a digestibility trial not

before 32 d of age, using the first week after weaning as adaptation period. Similarly, Gal-

lois et al. (2008) found lower organic matter digestibility in rabbits from 37 to 42 d of age

(68.1%) compared to that determined from 23 to 28 d of age (74.5%), regardless of weaning

age.

The effect of the collection period length (no overlapping periods from 2 to 6 days)

and the initial day of the collection period is shown in Table 1. DMd values remained con-

stant in those periods starting from day 32 of age onwards. Average standard deviation of

DMd decreased by 20% when the first week after weaning was not considered. Moreover,

average standard deviation decreased by 54% when the length of the collection period in-

creased (3.15 vs. 1.45 from 2 to 6 d of collection), as previously observed by Villamide and

Ramos (1994). The standard deviation obtained with a 4-d digestibility period (2.62) was

higher than that reported by Perez et al. (1995b) for the European reference digestibility pro-

cedures obtained in 6 different laboratories (1.53) (using 49 d-old rabbits and 4 d collection


160

period). It indicates more variable results with younger rabbits that are only compensated

with a longer (6 d) collection period (reducing standard deviation until 1.45). Consequently,

the number of animals required to detect a significant difference among means depends on

the length of the collection period. In Table 2, the number of animals required per treatment

to detect a difference among DMd means at a significant level (P = 0.050 or 0.001) and 80%

power has been calculated using for each period the average standard deviation of periods

from day 32 onwards. For a conventional collection period of 4 d, 5 rabbits per treatment are

needed to detect a significant difference of 4 percentage units, but at least 14 and 52 rabbits

per treatment would be required to detect 2 or 1 percentage units of difference, respectively.


161

5. REFERENCES

Association of Official Analytical Chemists, 2000. Official Methods of Analysis, 17th Ed.


Álvarez J.L., Margüenda I., García-Rebollar P., Carabaño R., de Blas J.C., Corujo A., Gar-

cía-Ruiz A.I. 2007. Effects of type and level of fibre on digestive physiology and per-

formance in reproducing and growing rabbits. World Rabbit Sci., 15:9-17.

Carabaño R., Piquer J., Menoyo D., Badiola I. 2010. The digestive system of the rabbit. In:

Nutriton of the Rabbit. 2nd Ed. De Blas J.C., Wiseman J. (Eds). Commonwealth Agri-

cultural Bureau, Wallingford, UK, pp. 1-18.

Gallois M., Gidenne T., Fortun-Lamothe L., Le Huerou-Luron, Lallés J.P. 2005. An early

stimulation of solid feed intake slightly influences the morphological gut maturation in

the rabbit. Rep. Nutr. Dev., 45:109-122.

Gallois M., Fortun-Lamothe L., Michelan A., Gidenne T. 2008. Adaptability of the digestive

function according to age at weaning in the rabbit: II. Effect on nutrient digestion in the

small intestine and in the whole digestive tract. Animal, 2: 536-547.

Gómez-Conde M.S., García J., Chamorro S., Eiras P, Rebollar P.G., Pérez de Rozas A., Ba-

diola I., De Blas C., Carabaño R. 2007. Neutral detergent-soluble fiber improves gut bar-

rier function in twenty-five-day-old weaned rabbits. J. Anim. Sci., 85: 3313-3321.

Gutiérrez I., Espinosa A., García J., Carabaño R., De Blas C. 2002. Effects of levels of


Anim. Sci., 80: 1029-1037.

Kaps M., Lamberson W. 2004. Biostatistics for animal science. ). Commonwealth Agricul-

tural Bureau International publishing, Wallingford, UK.


162

Laplace J.P., Lebas F. 1972. Mensurations viscérales chez le lapin. II.- Principaux facteurs

déterminants des variations relatives de la croissance du foie, des reins et des segments

intestinaux entre 3 et 11 semaines d’age. Ann. Zootech., 21:505-524.

Littell, R.C., Henry, P.R., Ammerman, C.B. 1998. Statistical analysis of repeated

measures data using SAS procedures. J. Anim. Sci. 76, 1216–1231.

Nicodemus N., Redondo R., Pérez-Alba L., Carabaño R., De Blas J.C., García J. 2010. Ef-

fect of level of fibre and type of grinding on the performance of rabbit does and their lit-

ters during the first three lactations. Livest. Sci. 129:186-193.

Padilha M.T.S., Licois D., Gidenne T., Carre B., Fonty G. 1995. Relationships between mi-

croflora and caecal fermentation in rabbits before and after weaning. Reprod. Nutr. Dev.,

35:375-386.

Parigi Bini R., Xiccato G., Cinetto M., Dalle Zotte A. 1991. Efficienza digestiva e proteica

dei coniglietti durante l’allantamento e lo svezzamento. Zootecnica e Nutrizione Anima-

le, 17: 167-180.

Perez J.M., Lebas F., Gidenne T., Maertens L., Xiccato G., Parigi-Bini R., Dalle Zotte A.,

Cossu M.E., Carazzolo A., Villamide M.J., Carabaño R., Fraga M.J., Ramos M.A., Cer-

vera C., Blas E., Fernandez J., Falcao E Cunha L., Bengala Freire J. 1995a. European

reference method for in vivo determination of diet digestibility in rabbits. World Rabbit

Sci., 3:41-43.

Perez J.M., Cervera C., Falcao e Cunha L., Maertens L., Villamide M.J., Xiccato G. 1995b.

European ring-test on in vivo determination of digestibility in rabbits: reproducibility of

a reference method in comparison with domestic laboratory procedures. World Rabbit

Sci., 3:171-178.

Robbins K.R., Saxton A.M., Southern L.L. 2006. Estimation of nutrient requirements using

broken-line regression analysis. J. Anim. Sci., 84(E. Suppl.): E155-E165.


163

Villamide M.J., Ramos M.A. 1994. Length of collection period and number of rabbits in

digestibility assays. World Rabbit Sci., 2:29-35.

Xiccato G., Trocino A., Sartori A., Queaque P.I. 2003. Effect of starter diets and weaning

age on growth, body composition and caecal fermentation of young rabbits. Anim. Sci.

77, 101-111.


164

Figure 1. Daily dry matter intake and faeces excretion (g DM/d) from 26 to 40 d of age (n =

15). (bars indicate standard deviation. Page < 0.001 for DM intake and excretion).

0

20

40

60

80

100

120

140

25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

g D

M/d

Days of age

DM intake

DM excretion


165

Figure 2. Correlation between feed intake and faeces excretion (both on DM basis. n = 15)

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

Pears

on

co

rrela

tio

n c

oeff

icie

nt

Age of rabbits

Intake and faeces from the same day (P < 0.001 for all values)

Intake from day n and faeces from day n+1 (P < 0.05 for all values except for days 33 and 37)


166

Figure 3. Daily dry matter digestibility (%) from 26 to 40 d of age (n = 15). (bars indicate

standard deviation. Page < 0.001. Slope 2.17 0.25 from 26 to 32 d of age).

50

55

60

65

70

75

80

85

90

95

25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

Dry

ma

tte

r d

ige

sti

bil

ity, %

Days of age

32.1 0.48

69.4 0.47

Chapter 5: Determination of faecar dry matter digestibility

167

Table 1. Dry matter digestibility (mean ± standard deviation) determined on animals weaned at 25 d and on periods of different length (n = 15).

No consecutive

days

Independent periods1

Averaged standard deviation

1st 2

nd 3

rd 4

th 5

th 6

th 7

th All periods

Only from day

32 onwards

2

26-27 d 28-29 d 30-31 d 32-33 d 34-35 d 36-37 d 38-39 d

81.6±5.68 77.2±3.62 72.0±3.16 69.8±3.01 69.7±2.43 68.6±2.66 70.3±4.19 3.68 3.15

3

26-28 d 29-31 d 32-34 d 35-37 d 38-40 d

80.2±4.03 73.4±2.71 70.6±2.00 68.4±2.35 69.6±3.76 3.08 2.81

4

26-29 d 30-33 d 34-37 d

78.7±3.38 70.9±2.43 69.3±1.80 2.62 1.80

5

26-30 d 31-35 d 36-40 d

76.7±3.26 70.5±1.70 69.1±2.11 2.45 2.11

6

26-31 d 32-37 d

76.0±2.81 69.5±1.45 2.24 1.45

1 In the same row there is no overlapping between two consecutive periods.

Chapter 5: Determination of faecar dry matter digestibility

168

Table 2. Number of rabbits required (n) per treatment group to detect a significant dif-

ference (P = 0.05 or 0.001) between two means with a 80% power1 for dry matter di-

gestibility in a period between 32 and 40 d of age for rabbits weaned at 25 d of age.

Difference to

be detected

(percentage

units)

Length of collection period (days) and standard deviation (in brackets)2

P = 0.05 P = 0.001

2

[3.15]

3

[2.81]

4

[1.80]

5

[2.11]

6

[1.45]

2

[3.15]

3

[2.81]

4

[1.80]

5

[2.11]

6

[1.45]

5 8 7 4 4 3 17 14 8 9 6

4 11 9 5 6 4 24 20 10 13 8

3 19 15 7 9 5 41 33 16 20 11

2 40 32 14 19 10 88 71 31 41 21

1 157 125 52 71 34 342 273 114 155 75

1 For two-tailed tests with two-treatment experiments.

The value of standard deviation used corresponds to periods from day 32 onwards.

Chapter 6

Conclusions and general implications

Chapter 6: Conclusions and General Implications

171

1. Conclusions:

Increasing Neutral Detergent Soluble Fiber (NDSF) level from 79 to 131 g/kg of DM in

post-weaning diets:

- Improved mucosal integrity and functionality, as shows the higher villous height/crypt depth

ration, the greatest sucrose activities and the higher ileal digestibility of starch, which result-

ed in a lower ileal starch flow.

- However, the changes detected in the immune response in the lamina propria and in the pro-

file of the intestinal microbiota were not so consistent with the benefits obtained in gut barri-

er integrity with increasing levels of NDSF.

- Despite all, the weight of caecal content increased and the composition of the intestinal mi-

crobiota tended to be affected by the inclusion of NDSF, reducing the frequency of detection

of Clostridium perfringens; Butirivibrio fibrosolvens and Campylobacter spp with highest

levels of soluble fiber.

- Improved rabbit growth ferformance, faecal dry matter and energy digestibility, as a conse-

quence of a better digestibility of certain fractions of the fibre (NDF).

- Reduced linearly mortality, which is an agreement with the reduction of the frequency of de-

tection of the several potential pathogens mentioned and the general improvement of gut

health and sanitary status.

Partial substitution of boiled wheat by boiled rice and wheat bran (80:20) in order to study

the source of starch:

- Have no impact on growth performance, faecal digestibility and mucosal morphology.

- Impaired ileal starch digestibility and increased the ileal flow of this nutrient to the caecum.

- Altered intestinal microbiota al ileal and caecal level: decreasing the frequency of detection

of Campylobacter spp. (both ileum and caecum), Helicobacter spp. (at ileum) and Rumino-

coccus spp (at caeum) and increasing the Bacteroides spp. (at caecum). Despite all these

changes, mortality rate was not affected.

Total substitution of raw wheat by boiled wheat in order to study the effect of heat pro-

cessing of starch:

- Did not improve the growth performance, ileal or faecal digestibility and mucosa morpholo-

gy parameters. Only at ileal level slightly improved ileal digestibility of starch, but did not

modify the flow of this nutrient to the caecum.


172

- Alter caecum but not ileal microbiota, increasing frequency of detection of Campylobacter

spp. and Ruminococcus spp. with none effect on mortality.

Respect to the methodological aspect concerning to the length of the collection period to

determine nutrient digestibility in the post-weaning period.

- It would not be advisable to start digestibility trials before the 32 days of age and the number

of animals required to detect a significant difference among means would depend on the col-

lection period.

2. Implications:

As a consequence of the results obtained in the present thesis, it should be advisable

to consider the Neutral Detergent Soluble Fibre (NDSF) as a functional nutrient in rabbits

due to its positive effect reducing mortality in growing rabbits affecting by digestive diseas-

es (including ERE)-

In this thesis, sugar beet pulp (rich in pectins) was selected as the main raw material

to increase soluble fiber content. Diet including beet pulp had a great fermentability, induced

changes in intestinal microbiota, improved gut barrier function and reduced markly mortali-

ty. However, the beneficial effects appreciated might be not only attributed to the soluble

fraction of beet pulp as there was an important highly fermentable insoluble fibre which

might be also implicated on these effects. We must be aware of the physical and chemical

complexitiy of the soluble polysaccharides and their interaction with other components of

the diet which make difficult to define or isolate the single effect of SF components.

More experiments would be necessary to investigate the effect of the different frac-

tions (soluble and insoluble) of sugar beet pulp on growth performance and at the gastro-

intestinal tract level, as well to search for additional sources of soluble fiber.

At the results obtained in the present thesis it seems to be advisable guarantee around

a 13% of SF expressed as % of dry matter in post-weaning diets containing 33% of NDF and

17% of ADF as % of dry matter.

Respect to the use of highly digestible sources of starch, which do not escape to the

enzymatic digestion and could enter in the large intestine where it could be fermented and


173

promote the proliferation of pathogenic bacteria, it seems that neither the use of rice or heat

treatments has no advantage for rabbit growth performance and health.

On the opposite, the heat processing of wheat starch or its substitution by heat processed rice do not

seem an interesting alternative for practical rabbit diets.

EFFECT OF TYPE OF DIETARY CARBOHYDRATES ON DIGESTION...

Documents

Transcript of EFFECT OF TYPE OF DIETARY CARBOHYDRATES ON DIGESTION...