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UNIVERSIDAD POLITÉCNICA DE MADRID
ESCUELA TÉCNICA SUPERIOR DE INGENIEROS AGRÓNOMOS
TESIS DOCTORAL
SISTEMA DE ALIMENTACIÓN PARA CONEJOS:
DEFINICIÓN DE LAS UNIDADES DE VALORACIÓN
NITROGENADA
("Rabbit feeding system: Comparison of different un i t s for nitrogen and
amino acid feedstuffs evaluation")
A n a I s a b e l G a r c í a R u i z
Ingeniero Agrónomo
Mayo, 2004
DEPARTAMENTO DE PRODUCCIÓN ANIMAL
ESCUELA TÉCNICA SUPERIOR D E INGENIEROS AGRÓNOMOS
Esta Tesis ha sido realizada como uno de los requisitos para la obtención del
grado de Doctor Ingeniero Agrónomo de la Escuela Técnica Superior de
Ingenieros Agrónomos de la Universidad Politécnica de Madrid.
El Doctorando
Ana I s a b u i z
Ingeniero Agrónomo
V°B°
Los d i r ec to re s de l a Tes i s
Rosa M^ C a r a b a ñ o Lúe
Dr. Ingeniero Agrónomo
fí A 0^4^ iMijsi^^
C a r l o s de B l a s Beo r l egu i
Dr. Ingeniero Agrónomo
^ mis cuatto abu£.lob
AGRADECIMIENTOS
(fi (Jis^OL ct^~ '€fl'íO-l>0-ñ.o u '^t.loi de. Lat pot todo &l iiampo qua me kan. dedicado datante
La liealLzación. de eita teiii peío tamhién., pot iu amiitadu iaiioi coni^oi. ^taciai a ¿oi doi pot
'ceaala'c/ne lot m^otet añoi de mi todavía "cotta " vida.
^t c^- ^eiúi ^illamide.pot. tet. mi masitíapeío ¡tohíe todo... pot ¿e l una t>aen.a amia,a.
^l S-avieí ^axcía u ^íu.'z.ia ^fícodemui pot tu auuda en. el laho'cato'cio u po'c todoí> loi
íuenoi momentoi que compatiimoi dentzo u fueía de nueifco deipacAo.
^l (/Ina ftpinoia po'c ¡tu valioia anuda en el Íaíoíatotí-O u tu dedicación en el man^o de
Lat con^at canuladat.
^t ^i.at.ia ^ilvit. u ^atlot ^¡{gd^iauez. l"^attu'cab")po'c tu apoup incondicional u tinc&'ca
amittad.
^l S.avie'c ^onzález po'c iui pacientei ta'cdei •Pcente al e^pecfcofotómetfco u tu animada
cont/cctación en lat comidat.
(/i 3.uan fptapo'c toda la auudap'cettada en lat navetupox "aquellot almue'czot",
^fl ^epe, B.avie'c ^{gd'd.gu.ez, ^olanda u ^{gta ^llatet po'c tu colabo'cación en diitintot
atp&ctot de eita tetit.
^ ^ntonio ^lotet u a Lot compañe'coi / at del "^ouífw and ^{ahbit ^isiewcck ^ntte."
po'c tu CU30U0 u aactencia en ¿a tecta final de eita tetit.
^ m.it mát intünat arntaai ^naela u fya, tin ellai lat co¿a¿ kahtian iido mucho mái
dificilei. ^ata votottat mi atatitud ei doble: ataciat poí bel co/no toi^, pot. h.abe'une b'zindado
detde hace ya m-uchah añat vueitta amittad u pot kaJie'zjne auudado tanto en lot momentot mát
dutoi.
^ toda mi familia, pe'co etpecialmente. a mit pad'cet, (flngel u ^íatibel u a mit
ketmanot, ^ngel y. ^wcia poi^que te lo meicecen y kan tábido ayuantatme pacientemente. ^
i/otottot cuatto: ^'caciatpo'c todo.
^pWca ^Intonio '^kacobo po'c tu eno'cme entiba, poí tu catino u po'c que'ce'c como quie'ce.
INDEX
General Index
INDEX
INDEX
RESUMEN
SUMMARY
1
5
11
CHAPTER I: Literature review and objectives. 16
Recent advances in nitrogen nutrition in rabbits. 16
1. Introduction 17
2 . Crude protein and total amino acids 17
3 . Digestible crude protein and amino acids 2 1
3 .1 . Faecal balance 21
3.2. Ileal balance 25
3.3. Apparent versus t rue digestibility 28
4. Objectives 3 0
5. References 3 0
CHAPTER II: Experiment 1 36
Effect of type of diet and caecotrophy on ileal endogenous nitrogen
and amino acidflotu in rabbits 36
1. Abstract 3 8
2 . Introduction 3 9
3 . Materials and Methods 4 0
3 .1 . Diets 40
3.2. Animáis and housing 42
3.3. Experimental procedure 43
3.4. Analytical methods 45
3.5. Statistical analysis 46
4. Resul ts 4 7
5. Discuss ion 5 4
General Index
6. Acknowledgements 57
7. References 57
CHAPTER IIJ: Experiment 2 63
Comparíson of different units for nitrogen and amino acid evaluation
in rabbit diets 63
1. Abstract 65
2. Introduction 66
3. Materials and Methods 67
3.1. Diets 67
3.2. Animáis and housing 71
3.3. Experimental procedure and calculations 71
3.4. Analytical methods 72
3.5. Statistical analysis 73
4. Results 74
5. Discussion 82
5.1. Apparent faecal versus ileal balance 82
5.2. Apparent versus true ileal digestibility 84
6. Acknowledgements 88
7. References 89
CHAPTER IV: GENERAL DISCUSSION AND CONCLUSIÓN 94
1. General discussion 95
2. Conclusión 96
ANNEXI I
1. Objetivo de la tesis III
2. Revisión Bibliográfica IV
2.1. Proteína bruta y aminoácidos totales IV
2.2. Proteína bruta digestible y aminoácidos digestibles VIII
General Index
3. Experimento 1. Efecto del tipo de alimentación y la cecotroña
sobre la estimación de la proteina y los aminoácidos de origen
endógeno en conejos. IX
4. Experimento 2. Comparación de diferentes unidades de valoración
nitrogenada de cuatro materias primas comúnmente empleadas en
piensos de conejos. XIV
4.1 Digestibilidad fecal versus ileal XV
4.2. Digestibilidad aparente versus verdadera XVII
5. Referencias bibliográfícas XVIII
RESUMEN
Resumen
RESUMEN
El objetivo general de esta tesis doctoral ha sido el tratar de establecer
las bases para un nuevo método de valoración de alimentos que permita
evaluar con mayor precisión el valor nitrogenado de piensos y materias
primas en conejos.
Los objetivos particulares fueron: I) el estudio de las pérdidas
nitrogenadas de origen endógeno en el conejo y II) la valoración de diferentes
materias primas comúnmente empleadas en piensos de conejos a través de
sus coeficientes de digestibilidad fecal aparente e ileal tanto aparente como
verdadero.
En el prim.er experimento, se estudió el efecto del tipo de alimientación
(sin nitrógeno o con nitrógeno) y de la cecotrofia, sobre la estimación de la
proteína y los aminoácidos de origen endógeno en el conejo. Para ello se
compararon los flujos ileales de proteína y aminoácidos de animales
alimentados por un lado, con piensos libres de nitrógeno (PF) y por otro, con
piensos cuya principal fuente de proteína era la caseína (C). Ambos piensos
se suministraron ad libitum a animales que realizaban la cecotrofia y a
animales a los que se les impedía realizarla. Para el experimento se
utilizaron animales fistulizados a nivel de íleon terminal con una cánula en T
de vidrio.
Los animales alimientados con el pienso PF mostraron un consumo de
pienso muy inferior (de prácticamente la mitad) al mostrado por los animales
alimentados con el pienso C. Por el contrario, los animales que consumían el
pienso PF tuvieron una mayor producción de cecotrofos (de
aproximadamente el doble) y con un mayor contenido en proteína. En los
animales alimentados con el pienso C se pudo establecer una relación lineal
entre el consumo total de MS (pienso + cecotrofos) y el ñujo ileal de nitrógeno
Resumen
endógeno. Esa misma relación no se observó en los animales que consumían
el pienso PF como consecuencia del bajo consumo observado.
La ingestión del pienso C condujo a flujos ileales de aminoácidos
(excepto para la glicina) un 27%, de media, más altos que el pienso PF. El
flujo ileal de glicina ñae un 20% más alto en aquellos animales que
consumían el pienso PF. Además, la composición en aminoácidos del
nitrógeno endógeno reveló que los animales que consumían el pienso PF
tenían una mayor proporción de arginina, fenilalanina y treonina. Las
diferencias cuantitativas entre los aminoácidos que componen las pérdidas
endógenas estimadas por estas dos vías, podrían deberse a variaciones en la
contribución relativa de las diferentes secreciones enzimáticas inducidas por
el pienso.
Dentro de los animales que consumían el pienso C, los que podían
realizar la cecotrofia mostraron mayores flujos ileales de fenilalanina que los
que no podían realizarla. Además, el análisis de aminoácidos del nitrógeno
endógeno reveló que los animales que realizaban la cecotrofia tenían una
mayor proporción de am.inoácidos esenciales tales como arginina, leucina,
lisina, fenilalanina y treonina. El efecto que la cecotrofia tuvo sobre la
estimación de los flujos nitrogenados de origen endógeno no fue tan
importante como se pensó en un principio debido probablemente al alto
coeficiente de digestibilidad del nitrógeno de los cecotrofos (79% estimado
mediante análisis in vitro).
Al comparar los valores de nitrógeno endógeno obtenidos en este
trabajo con los obtenidos en cerdos utilizando métodos similares se observó
que tanto con el pienso PF como con el C, las pérdidas de nitrógeno
endógeno eran de 2 a 3 veces superiores, respectivamente, en conejos que en
cerdos. Estas diferencias podrían explicarse principalmente por los mayores
contenidos en fibra de los piensos de conejos (27-33% vs. 3-8% de FND, en
conejos y cerdos, respectivamente).
Resumen
Debido al importante cambio en el comportamiento de los animales
que consumieron el pienso PF (baja ingestión de pienso y aumento de la
producción de cecotrofos) la utilización de piensos libres de nitrógeno, tiene
importantes limitaciones prácticas a la hora de determinar el nitrógeno
endógeno en conejos. Por este motivo, la utilización de piensos con proteína
altamente digestible resultó ser un método más adecuado para la
determinación del nitrógeno endógeno en conejos.
En el segundo experimento se evaluaron cuatro materias primas
diferentes a partir de sus coeficientes de digestibilidad fecal aparente, ileal
aparente e ileal verdadera de la proteína y los aminoácidos. Para la
determinación de los coeficientes de digestibilidad verdaderos se utilizaron
los resultados de nitrógeno y aminoácidos endógenos obtenidos en el primer
trabajo. Las materias primas escogidas fueron: heno de alfalfa, cebada,
salvado de trigo y harina de girasol. La selección de las materias primas se
hizo en base a su importancia en la formulación práctica de piensos
compuestos de conejos. En este experimento se utilizaron animales
fistulizados a nivel de íleon terminal con una cánula en T de vidrio. Los 4
piensos se formularon para ser isonutritivos. La principal fuente de pro teína
fue, en cada caso, el alimento que se quería valorar. La composición química
de estos piensos fue similar a la del pienso de caseína (C) del experimento
anterior.
Al comparar el flujo ileal de proteína (3.17 g/día, de media) y la
excreción total de pro teína (heces duras + cecotrofos) (3.74 g/día, de media)
se observó que había un balance positivo de proteína de 0.558 g/día (~ un
18% de lo que llegaba al íleon). Este balance positivo se podría explicar por
una entrada de urea al ciego procedente del catabolismo de los aminoácidos
en el hígado a través del torrente sanguíneo. Todos los alimentos mostraron
una digestibilidad fecal aparente de la proteína mayor que la ileal, al
Resumen
reciclarse un 36% del total de nitrógeno excretado mediante la cecotrofia (un
57% de media, del nitrógeno de los cecotrofos es de origen bacteriano).
Realizando balances similares al mostrado para la proteína pero con
los aminoácidos se observó que, esta proteína estaba enriquecida en lisina
(0.072 g/día; 63% del flujo ileal), metionina (0.026 g/día; 95% del flujo ileal)
y treonina (0.059 g/día; 40% del flujo ileal). Además este enriquecimiento, en
aminoácidos esenciales tendió a ser mayor en los piensos basados en alfalfa,
cebada y harina de girasol que en el basado en salvado. Esta tendencia
resultó paralela a los datos de nitrógeno microbiano obtenidos en este
trabajo (el pienso de salvado presentó la menor proporción de nitrógeno
microbiano en heces).
La actividad bacteriana en el ciego dio como resultado cambios
sustanciales en la composición de aminoácidos de la proteína. Como
consecuencia, pese a que un tercio aproximadamente de la lisina y la
metionina excretadas se reciclaron vía cecotrofia, la digestibilidad ileal de
ambos aminoácidos fue mayor que la digestibilidad fecal (2.8 y 5.5 unidades
porcentuales de media para los cuatro alimentos, respectivamente). Sin
embargo la treonina mostró una tendencia distinta, siendo la digestibilidad
ileal menor que la fecal. Esto se debió al ya de por sí elevado flujo ileal de
treonina (0.142 g/día de treonina vs. 0.118 y 0.028 g/día para lisina y
metionina, respectivamente) como consecuencia de su alta proporción en el
nitrógeno endógeno (5.53 g/16gN vs. 3.76 y 0.96 g/16gN para lisina y
metionina, respectivam.ente).
Debido a la gran cantidad de proteína y aminoácidos de origen
endógenos presentes en el conejo, los coeficientes de digestibilidad ileal
verdadera de proteína y aminoácidos de los cuatro alimentos fueron mayores
que sus correspondientes coeficientes de digestibilidad ileal aparente. Las
mayores diferencias entre estas dos unidades se obtuvieron en los alimentos
más bajos en proteína (cebada y salvado) mientras que esas diferencias
Resumen
fueron de menor magnitud en los ricos en proteína (harina de girasol). Esto
se debe a la proporción relativa que alcanza el nitrógeno endógeno respecto
al nitrógeno no digerido procedente del alimento.
Además, en este trabajo se ha visto que independientemente del valor
absoluto de digestibilidad alcanzado, el valor relativo que se establece entre
los alimentos no es constante, sino que varia dependiendo de la unidad
empleada en la valoración de estos alimentos y del aminoácido considerado.
La digestibilidad ileal y fecal aparente conducen a una subestimación
o sobreestimación, respectivamente, de la digestibilidad ileal verdadera de
los aminoácidos de los alimentos como consecuencia del elevado nitrógeno
endógeno y la importante actividad microbiana en el ciego de los conejos. Por
tanto, con el fin de obtener una valoración más precisa de los alimentos se
recomienda el uso de la digestibilidad ileal verdadera. Con el uso de esta
unidad de valoración en la formulación práctica se conseguiría un mejor
ajuste de los nutrientes del pienso a las necesidades del animal y una
reducción de la excreción de nitrógeno al medio. Sin embargo, llegados a este
punto hay que destacar la falta de datos y por tanto la necesidad de seguir
valorando más alimentos en estos términos.
10
SUMMARY
11
Summary
SUMMARY
The general aim of this doctoral thesis has been to establish the basis
for a new feed evaluation system. in rabbit increasing the accuracy of
feedstuff nitrogen evaluation. To achieve this general goal the nitrogen
endogenous losses in rabbits has been studied. Also, different feedstuffs
commonly used in rabbit feed formulation have been evaluated in terms of
protein and amino acids using different digestibility coefficients.
To reach these goals two different triáis were carried out. In the first
trial the effect of type of diet (casein-based or protein-free diet) and
caecotrophy on the ileal endogenous nitrogen and amino acid flow in rabbits
was studied. The ileal flow of nitrogen and amino acids of animáis fed with
casein based diet (C diet) or a protein-free diet (PF diet) were compared. Both
diets were given ad libitum access to animáis practicing caecotrophy but also
to animáis not allowed to practica it. For the experiment doe rabbits
surgically fitted with a glass T-cannula at ileum level were used.
Animáis fed PF diet had a very low feed DM intake (about halí).
Furthermore, soft faeces excretion was almost twice in animáis fed PF diet
than in animáis fed C diet. In animáis fed with the C diet, a linear
relationship was found between the ileal endogenous nitrogen flow
determined with this method and the total DM intake (feed + soft faeces).
However, this relationship was not observed in animáis fed the PF diet due
to their low feed DM intake.
The ingestión of C diet led to a higher (27 %, as average), endogenous
amino acid ileal flow than PF diet for all amino acids except for glycine. The
ileal flow of glycine was 20% higher in animáis fed the PF diet than in
animáis fed the C diet. Moreover, the amino acid composition of the ileal
endogenous losses revealed that animáis fed PF diet had higher proportion of
12
Summary
arginine, phenylalanine, threonine, than animáis fed C diet. Differences in
the amino acid pattern of the endogenous nitrogen losses depending on the
type of diet could be explained by differences in the relative proportion of the
endogenous nitrogen secretions.
Within animáis fed C diet, animáis practicing caecotrophy showed
higher ileal flow of phenylalanine than animáis not practicing it. Moreover,
the amino acid analyses of the endogenous nitrogen showed that when
caecotrophy was not prevented, higher proportions of arginine, leucine,
lysine, phenylalanine and threonine were found. The effect of caecotrophy on
the endogenous estimations was not as high as expected due to their high
nitrogen and amino acid digestibility coefficient (79% as average) performed
by in vitro analysis.
The comparison of the results obtained in this trial in rabbits not
practicing caecotrophy with the existing data in pigs, showed that the
endogenous ileal nitrogen flow for the PF and the C diet was between 2 and 3
times higher, respectively, in rabbits than in pigs. This difference might be
mainly related to the higher fibre content of rabbit than pig diets (27-33% vs.
3-8% of NDF, respectively).
The use of PF diets has limited practical application in rabbits to
estímate the nitrogen endogenous losses as the low feed DMI and the high
soft faeces DMI leads to abnormal feed/soft faeces intake ratio. For this
reason, the use of highly digestible containing protein diets resulted in a
better approach for the nitrogen endogenous losses estimations in rabbits.
In the second experiment, four different feedstuffs were evaluated
using different protein and amino acid digestibility coefficients (apparent
faecal digestibility and apparent and true ileal digestibility). The endogenous
losses obtained in the first experiment were used to determine the true ileal
digestibility coefficients. The feedstuffs chosen for this experiment were
13
Summary
alfalfa hay (AH), barley grain (BG), wheat bran (WB) and sunñower meal
(SM). For this experiment animáis were surgicaiiy fitted witti a giass T-
cannula at ileum ievei. Diets were formuiated in order to contain a maximai
nitrogen proportion from tiie feedstuffs studied in each diet. The
experimental diets were isonutritive and their chemical composition was
similar to the C diet.
A positiva balance of protein (0.57 g/day, as average, representing 18%
of ileal flow) was deduced when comparing average ileal flow of protein (3.17
g/day, as average) and total protein excreted in soft and hard faeces (3.74
g/day, as average). This balance has been explained since a proportion of
urea produced in the liver from amino acids catabolism is recycled into the
caecum through the blood. AU feedstuffs showed a higher CP digestibility at
faecal than ileal level, as soft faeces recycled as average a 36% of the total
protein excreted which was mainly of bacterial origin (67%, as average).
Making sim.ilar balances for the most limiting amino acids (using the
average valúes of the four feedstuffs), enrichment in lysine (0.072 g/day;
63% of the ileal flow), methionine (0.026 g/day; 95% of the ileal flow) and
threonine (0.059 g/day; 40% of the ileal flow) was observad. Furthermore,
the enrichment in these essential amino acids tended to be higher in diets
based on AH, BG and SM than in those based in WB. This result was
parallel to the lower proportion of microbial nitrogen on total nitrogen
excreted in hard faeces observed in diet based in WB with respect to the
other diets.
The bacterial activity in the caecum resulted therefore in substantial
changes in the amino acid composition of caecal crude protein. As a
consequence, although around one third of the total excretion of lysine and
methionine was recycled with soft faeces, ileal digestibilities of lysine and
methionine were higher (by respectively 2.8 and 5.5 percentage units, as
average of the four diets) than at faecal level. A different pattern was
14
Summary
observed for threonine digestibility, which tended to be lower a t ileal t h a n at
faecal level. This could be an effect of the part icularly high threonine
concentration in the ileal endogenous losses (5.53 g/16gN vs. 3.75 and 0.96
g/16gN for lysine and methionine, respectively). Moreover, the endogenous
ileal threonine has been observed to be highly digestible at the caecum in
pigs.
Due to the large endogenous losses found in the rabbits, higher true
than apparent ileal digestibilities of CP and amino acids were observed in all
the feedstuffs studied. Differences between these two units were of small
magnitude in the high-protein feedstuffs (SM) but much more pronounced in
the low-protein feedstuffs (BG and WB). This effect was consequence of the
relative proportion of endogenous to exogenous recoveries of protein and
amino acid in the ileal digesta.
Also in this experiment it was observed that the relative valué
established among feedstuffs for the CP and the most limiting essential
amino acids varied with the digestibility unit used and the amino acid
considered.
According to the results obtained, apparent ileal and faecal digestibility
lead to an underestimation or an overestimation, respectively, of the true
ileal digestibility of nitrogen and amino acids content of the feedstuffs due to
the high nitrogen endogenous losses impact and to the important caecal
bacteria activity of the rabbits. The use of true ileal digestibility is
recommended for a more precise evaluation of nitrogen and amino acids
content of the feedstuffs. By using true ileal digestibility a better approach to
the animal requirements and less nitrogen excretion to the environment
would be reached. However, at this stage insufficient data is available and
more feedstuffs should be evaluated to achieve its implementation in feed
formulation.
15
CHAPTER I: Literature review and objectives.
Recent advances in nitrogen nutrition in rabbits.
16
Chapter I: Literature review and objectives
1. Introduction
Rabbits need balancea diets that provide sufficient levéis of all
nutrients, including nitrogen, to meet the requirements of the different
productive processes. In order to achieve this target, practical diet
formulation needs a correct definition of requirements and the nutritional
content of the raw materials used.
Rabbits need nitrogen for maintenance, muscle growth, foetus growth,
milk production or hair production. These requirements differ in nitrogen
content but mainly in their amino acid pattern (Table 1). This is why the
nitrogen requiremrents are better defined as amino acids rather than in crude
protein. In European intensive rearing conditions, total nitrogen
requirements are met with alfalfa hay (30-50 %), oil seed meáis (30-40 %)
and cereals and their by-products (33 %). However, the ability of these feeds
to meet nitrogen or amino acid requirements varíes depending on the unit
used to define their nutritive valué.
Many efforts have been done to define the best unit to evalúate the
energy content of rabbit diets. However, small attention has been paid to
determine this unit in terms of nitrogen. A proper definition of this unit
allows increasing the accuracy of diet formulation, reducing the risks of
environmental poliution.
2. Crude protein and total amino acids
Crude protein is the more common unit used to express the nitrogen
requirements and the nutritive valué of feeds in rabbits. The main advantage
of this unit is the availability of information about the requirements and feed
composition. However, the use of this unit leads to diets with an excess of
protein with respect to requirements. Recently, Maertens et al. (1997) have
17
Chapter I: Literatura review and objectives
observed that it is possible to reduce the dietary crude protein contení from
17.0 to 15.7% without impairment of growing performance, by maintaining
the supply of lysine, sulphur amino acids and threonine above the
requirements. Within this interval, a reduction of faecal nitrogen excretion
(11%) was also observed. A strong relationship between dietary nitrogen level
and nitrogen excretion have been confirmed by Trocino et al. (2000), and a
predominant role was recognised to the last period of growth. These results
indicate that the use of total amino acid content instead of crude protein
content leads to a better evaluation of the nitrogen valué of a diet.
Otherwise, it is world wide accepted that animáis need an external
supply of essential amino acids to synthesise body proteins. There is
increasing information available on total amino acid composition of feeds.
Villamide et al. (1998) summarised the lysine, sulphur amino acids and
threonine content of the most common ingredients used in rabbit feed
formulation (cereals, cereal by-products, legume and oil seed meáis, an soma
forages and fibrous by-products). Additional information for the rest of
amino acids and feeds may be consultad in the publications of several
research centres in animal nutrition (a.g. NRC, 1998 (English); CVB, 2002
(Dutch); INRA, 2002 (French); FEDNA, 2003(Spanish)). However, valúes of
amino acid composition for other forages than alfalfa, or agricultural and
industrial fibrous by-products, frequently used in rabbit diets, are scarce.
There is also information available from studies dose/response about
the requirements of the most limiting amino acids in rabbit diets:
methionine, lysine, and threonine, both for growing (Colin, 1975;
Spreadbury, 1978; Berchiche and Lebas, 1994; Tabeada et al., 1994 and
1996, de Blas et al., 1998) and for lactating rabbits (Maertens and de Groóte,
1988, Taboada et al., 1994 and 1996, de Blas et al. 1998). The amino acid
recommendations have been summarised recently by de Blas and Mateos
(1998) (Tabla 1). There is not experimental support for the recommendations
in the rest of amino acids. An approach to balance the supply of amino acids
18
Chapter I: Literature review and objectives
is to use the concept of "ideal protein". This method was used in growing
rabbits by Moughan et al. (1988), and consists in supplying a dietary protein
with an amino acid pattern similar to that of the main protein synthesised.
In the case of growing rabbits this is the amino acid composition of whole
body and, for lactating rabbits the amino acid composition of milk. Tabla 2
shows the amino acid pattern of whole body and milk proteins expressed in
mg/gN and the relative content of each amino acid with respect to lysine.
Table 1. Nutrient requirement of intensively rearad rabbits as concentration/kg corrected to a dry matter content of 900 g/kg (de Blas and Mateos, 1998).
Nutrient Unit Breeding does Fattening rabbits Mixed feed
Digestible energy
Crude protein
Digestible protein
Lysine ^
Total
Digestible
Sulphurb.d
Total
Digestible
Threonine t'.e
Total
Digestible
MJ
g
g
g
g
g
g
g
g
11.1
184 (153-198)a
129 (114-139)
8.4 (100)^^
6.5 (100)
6.5 (77)
5.0 (75)
7.0 (83)
4.8 (73)
10.5
153 (145-162)
107 (102-113)
7.5 (100)
5.9 (100)
5.4 (72)
4.1 (70)
6.4 (85)
4.4 (75)
159
111
10.5
(154-162)
(108-113)
8.0
6.3
6.0
4.6
6.8
4.7
^Valúes in parenthesis indícate minimal and maximal valúes recommended. ''Total amino acid requireraents calculated for a contribution of synthetic amino acids of 0.15. '^Valúes in parenthesis indicate the relative valué respect to lysine. '^Methionine should provide a minimum of 35% of the total sulphur requirements. ''Maximal levéis of 5.2 and 7.6 g kg-i of digestible and total threonine, respectively, are recommended for breeding does.
19
Chapter I: Literature review and objectives
The present recommendat ions , relative to lysine, for total su lphur
amino acids and, especially, for total threonine (Table 1) are higher t h a n the
amino acid composition of whole body and milk (Table 2). These differences
show the limitations of the ideal protein, a s it a s s u m e s tha t metabolic
efficiency is similar for all the amino acids, and does not take into account
the main tenance requirements . There is not Information abou t amino acid
requi rements for main tenance in rabbi ts . However, in pigs, the main tenance
requirements , relative to lysine, are especially high in threonine and su lphur
amino acids (Williams, 1995).
Table 2. Amino growing rabbits) unpublished).
acid composition (mg/gN) of the whole body (53 day oíd and of the doe milk (Moughan et al, 1988; Nicodemus et al,
Amino acids
Lysine
Alanine
Arginine
Aspartic acid
Histidine
Isoleucine
Leucine
Methionine
Cystine
Glutamic acid
Glycine
Phenylalanine
Serine
Threonine
Tyrosine
Valine
Whole body
Absoluta valué
383
365
415
467
193
194
429
77
158
788
466
249
283
245
192
239
Relativa to lysine
100
74
108
121
50
51
112
20
41
205
121
65
74
64
50
62
Absoluta valué
451
228
328
451
159
304
567
150
175
1220
106
281
228
305
332
382
Milk
Ralative to lysine
100
50
73
100
35
67
125
33
39
270
23
62
50
67
73
85
20
Chapter I: Literature review and objectives
3. Digestible crude protein and amino acids
3.1. Faecal balance
From a biological point of view, neither crude protein ñor total amino
acids achieve to characterise accurately the nutritive valué of feeds. The
availability of an amino acid is the proportion of the total content that is
absorbed in the digestive tract and is available to cell for synthesis when it is
needed. Digestibility has been usad as synonymous of availability but this
concept only considers the disappearance of a nutrient (potentially absorbed)
in the digestive tract. Taking into account these differences, several units of
digestibility, measured at different segments of the digestive tract, have been
proposed in non-ruminant species. The most common procedure for
determining digestibility and to express requirements in rabbits is the faecal
balance (apparent faecal digestibility).
Digestible crude protein (DCP) requirements have been widely
investigated and summarised in several reviews (Maertens, 1992; Xiccato,
1996, Fraga 1998b, Xiccato, 1999). Thus, valuable Information is available
for optimising growth rate, milk production or meat quality. Practical
recommendations for rabbit diets are shown in Table 1. Requirements would
be better expressed in relation to dietaiy energy concentration. Valúes for
breeding does are around 20% higher than for growing rabbits (12 vs. 10 g
DCP MJ-i DE; Xiccato, 1996; De Blas et al., 1981).
Digestión implies obligatory losses of protein. These losses depend on
many factors. The main ones are relatad to chemical structure of proteins
and its accessibility to digestive enzymes. Accordingly, protein digestibility
varias widaly among tha ingrediants of a diat. According to Villamida at al.
(1998), crude protein digestibility in rabbits ranges from 15% (grape saad
meal) to 85% (soybean meal). The highest valúes of digestibility ara usually
21
Chapter I: Literature review and objectives
observed for storage proteins located in grains or seeds (cereals, legume and
oil seeds, and oil seed meáis) and the lowest for proteins protected by a cell
wall with a high degree of lignification (forages and fibrous by products) . In
fact, insoluble nitrogen in NDF or ADF solution seems to be one of the best
single predictor of protein digestibility (Martínez and Fernández, 1980;
Villamide and Fraga, 1998).
Table 3 shows crude and digestible protein contení of the main protein
sources used in rabbit diets. Crude protein overest imates the valué of
absorbable protein of all ingredients, b u t mainly in those with lower
digestibility, as wheat b ran or alfalfa hay. Although digestible protein
increases the accuracy of diet formulation, the u s e of th is un i t is restricted
due to the scarce Information of digestible content of raw materials . Alfalfa is
the best-evaluated ingredient, a s a total of 23 samples (14 dehydrated and 9
a s hay) have been studied.
Table 3. Crude and digestible protein content (g/kg) of some protein sources in rabbit's diets (Villamide et al, 1998).
Crude protein Digestible protein
Absolute Relative Absolute r-. i - i 1 1 1 Relative valué
valué valué valué Sunflower meal Wheat bran Bar ley grain
Alfalfa hay
320
160
120
180
100
50
33
56
270
107
90
117
100
39
33
43
However, for other dietary ingredients (sunflower meal, barley, and
oats), no more t h a n 4-6 ba tches have been evaluated, being the most
frequent si tuation tha t the referenced valué of c rude protein digestibility
comes j u s t from one batch.
Recently, Villamide and Fraga (1998) have proposed different
equat ions to predict the digestible protein content of feed ingredients
22
Chapter I: Literature review and objectives
combined in four groups: diy forages, cereals and cereal by-products,
protein concentrates and ñbrous by-products (Table 4).
Table 4. Prediction equations of digestible protein (g/kg DM) from crude protein content (g/kg DM). (Villaraide and Fraga, 1998).
^ '^" ' í^ 'a Intercept B N R^ RSD number^
1
2
3
4
-39.4 ± 11.1
-2.3 ±7 .3
-55.3 ±24.6
-19.2 ± 18.4
0.83 ±0.06
0.75 ±0.05
0.94 + 0.06
0.72 ±0.15
26
27
18
17
0.892
0.911
0.936
0.617
3.44
3.90
7.14
9.73 a 1: Dry forages; 2: Cereals and cereal by-products; 3: Protein concentrates; 4: Fibrous by-products.
The best single predictor was the crude protein content for all groups.
However, its ability to predict digestible protein was different for each group.
The worse relationship, in terms of precisión, was observed for fibrous by-
products. In this case, the inclusión of ADF content in the equation
improved the accuracy of prediction (R2= 0.895, RSD = 5.27).
An additional restriction to the use of faecal protein digestibility as
unit to evalúate nitrogen valué of feeds is that faecal digestibility of amino
acids differs among them and is different to that of crude protein. This could
be due to i) differences in relative endogenous losses, ii) the specificity of
proteases and peptidases involved in the enzymatic hydrolysis of proteins,
and iii) differences in the rates of amino acid absorption, as it has been
observed in other non-ruminant species as pigs, Differences in amino acid
composition of proteins contained in the vegetable structures (seed, leaves,
stalks, etc) could affect also its accessibility to digestive enzymes and then to
its digestibility. According to Low (1980), arginine and lysine would be
expected, among the essential amino acid, to appear first after enzymatic
hydrolysis, whereas threonine would be the last. Furthermore, the rates of
absorption are highest for arginine, methionine, isoleucine and leucine and
lowest for threonine and histidine (Li et al., 1993).
23
Chapter I: Literature review and objectives
Data obtained in rabbi ts for alfalfa hay agree with observations made
in pigs. In this way, Table 5 shows the apparen t crude protein and amino
acid faecal digestibility of three ba tches of alfalfa differing in NDF and
protein content (from 38.7 to 55.0% on DM, and from 17 to 22% on DM,
respectively). Although amino acids followed the same t rend t h a n crude
protein digestibility, the differences between extreme ba tches of alfalfa were
higher in lysine and threonine digestibility (17 and 13.5%, respectively) t h a n
for crude protein digestibility (10%). Fur thermore , lysine, methionine and
threonine digestibilities tended to be higher t han crude protein digestibility,
mainly in the alfalfa of better quality.
Table 5. Effect oftype of alfalfa hay on apparent faecal digestibility of protein and amino acids (96) (García et al, 1995b)
Crude protein
Lysine
Methionine
Threonine
A
74.4 a
81.4 a
79.8 a
77.4 a
Alfalfa hayí
B
71.5 b
76.0 b
72.5 b
72.2 b
C
57.4 c
57.2 c
72.5 b
57.0 c
SEM
1.0
2 .0
2 .0
2 .0
P
0.001
0.001
0.001
0.001
1 NDF content of alfalfa A, B and C were 38.7, 48.9 and 55.0% on DM, respectively.
^•^'^ Means in the same row not followed by a common letter differ (P < 0.05)
In the same way, Taboada et al. (1994 and 1996) and de Blas et al.
(1998) observad tha t digestibility of synthetic amino acids is higher (from
0.93 to 1.0) t h a n the corresponding amino acids contained in raw mater ials
(from 0.54 to 0.80 a s average). These resul ts show t h a t the use of faecal
digestible amino acids could improve the character isat ion of nitrogen valué
of feeds, bu t also the definition of requirements . Some discrepancies in total
amino acid requirements reported in the l i terature could be partially
explained by differences of amino acid digestibility in the diets used to
determine them. Considering a diet with the same supply of digestible
threonine (4.8 g /kg, Table 1), total dietary threonine content should vary
24
Chapter I: Literature review and objectives
froxn 7 .5 to 5 .6 if t h e d i e t a r y i n c l u s i ó n of s y n t h e t i c t h r e o n i n e a c c o u n t s for O
to 3 0 % of t h e to ta l s u p p l y , respec t ive ly . O n t h e c o n t r a r y , a d ie t f o r m u l a t e d
a c c o r d i n g to to t a l t h r e o n i n e r e c o m m e n d a t i o n s (7 g / k g , T a b l e 2), m a y s u p p l y
a n e x c e s s or a déficit of t h r e o n i n e d e p e n d i n g if d i e t a i y t h r e o n i n e d iges t ib i l i ty
is h i g h e r o r lower t h a n 0 . 6 8 6 (Table 1). Acco rd ing to De B l a s e t a l . (1998) ,
b o t h s i t u a t i o n s will l ead to a r e d u c t i o n in l a c t a t i o n a n d g r o w t h p e r f o r m a n c e .
A p p a r e n t faecal d iges t ib le lys ine , s u l p h u r a m i n o a c i d s a n d t h r e o n i n e
r e q u i r e m e n t s h a v e b e e n d e t e r m i n e d in l a c t a t i n g d o e s a n d g rowing r a b b i t s
(Table 1). R e c o m m e n d a t i o n s for t h e o t h e r a m i n o a c i d s m i g h t b e c a l c u l a t e d
from t h e idea l p r o t e i n p a t t e r n . T h e m a i n p r o b l e m to u s e t h i s u n i t in d ie t
f o r m u l a t i o n is t h e l ack of d a t a of d iges t ib le a m i n o ac id c o n t e n t of feeds . At
t h i s po in t , it c o u l d be c lea r t h a t a m a i n object ive for t h e f u t u r e w o u l d b e to
get more information on faecal digestible amino acid content of feeds.
However, there are some doubts about the goodness of this unit for
characterisation of avaiiable amino acids. In other non-ruminant species
(pigs and poultty), research in this área for the last 30 years has led to
propose ileal true digestibility, rather than faecal, as the best unit for
nitrogen evaluation. Accordingly, it should be established, as a first step, the
best unit of digestibility allowing the most accurate estimation of avaiiable
amino acid content of the feeds in rabbits.
3.2. Real balance
The main reason to choose ileal digestibility as the best correct term to
evalúate feeds in non-ruminant species is that ileum is the last segment of
the digestive tract where amino acids can be absorbed. As it has been shown
in several experiments in pigs (Zebrowska, 1973; Just , et al., 1981), most of
the nitrogen that disappears from the large intestine is not retained,
indicating that compounds absorbed are not used for protein synthesis.
25
Chapter I: Literature review and objectives
From this point of view, it is possible that faecal digestibility were not a
so incorrect term to evalúate the nitrogen valué of feeds in rabbits due to
caecotrophy. Rabbits can use for protein synthesis part of the amino acids
that disappear after the ileum through ingestión and digestión of soft faeces.
The main effect of soft faeces reingestion is related to protein
reutilization. Caecal microorganism can use for their protein synthesis, the
undigested nitrogen of the digesta and the endogenous nitrogen but also,
urea recycled into the caecum through the blood. Ammonia is the main end
product of nitrogen catabolism in the caecum and the main source of
nitrogen for microbial protein synthesis. When protein intake exceeds the
requirements for body protein synthesis, the excess is catabolised and
produce urea as end product being partially recycled to the caecum.
Forsythe and Parker (1985) estimated that 25% of the total caecal ammonia
comes from the hydrolysis of the urea. In this sense, Emaldi et al. (1979)
found that the main enzymatic activities of the caecal microorganism were in
decreasing order: ammonia-user, ureolytic, proteolytic and cellulolytic.
Different authors have related an increment in the caecal ammonia
concentration with an increase in the dietary digestible crude protein
content (Carabaño et al., 1988, 1989, 1997; Fraga et al., 1991; Motta-
Ferreira et al., 1995; García et al., 1995a, 1995, 2000). There are many data
on the chemical composition of soft faeces suggesting that it is similar to
that of the caecal contents (Ganuza et al., 1999). When comparing the
protein concentration of soft faeces (CPSF) with that of caecal contents (CPcc)
of rabbits fed 31 different diets obtained employing the same methodology,
the following regression equation was obtained (Fraga, 1998a):
CPsF (g/kg DM] = 100.88 + 6.89 (+ 0.8) CPcc R2 = 0.712 P < 0.001
26
Chapter I: Literature review and objectives
García et al. (1995a) obtained that the proportion of microbial nitrogen
respect to total nitrogen content in soft faeces varied from 0-31 to 0-68
depending on the amount and type of fibre in the diet.
Yoshida et al. (1968) when comparing germ-free versus conventional
rabbits reportad a lower digestibility of protein by the conventional rabbits
resulting from the protein synthesised by microbes. Soft faeces are
commonly richer than hard faeces in total amino acid content but especially
in some essential amino acids such as lysine, methionine and threonine In
Table 6 the amino acid composition of hard and soft faeces is shown.
Table 6. Amino acid composition (g 16/g nitrogen) of soft faeces (SF) and hard faeces (HF) in rabbits.
Essential amino acids Cystine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Tyrosine Valine Non essential amino acids Alanine Arginine Aspartic acid Glutamic acid Glycine Proline Serine
Proto, SF
-
1.30 3.87 5.69 4.74 1.58 3.59 5.26 3.33 5.05
5.21 3.15 9.15 10.25 4.47 3.84 3.89
1976 HF
-
1.71 4.10 6.20 4.56 1.13 4.19 4.94 2.80 4.38
5.19 3.57 8.66 10.25 5.19 5.21 4.40
Nicodemus, 1999 SF
1.41 1.35 3.94 5.61 5.05 1.70 3.41 5.35
-
5.35
5.87 2.72 9.35 11.53 4.74
-
4.31
27
Chapter I: Literatura review and objectives
The bacterial activity in the caecum resultad in substantial changas in
the amino acid composition of its cruda protein. Yoshida et al. (1971) found
an increased content of five essential amino acids (lysine, mathionine, valina
leucine and isoleucine) when comparing normal with germ-free rabbits.
According to this, soft faeces contribution to the total intake of lysine,
sulphur amino acids and threonine is higher than the contribution of crude
protein (Nicodemus et al., 1999) (figure 1).
Figure 1. Relative contribution of soft faeces to total intake of crude protein
(CP) and amino acids (%).
CP=15
MET+CYS=17
LYSINE=18
^ ^ ^ ^ ^ K < ¿ > " ' ' THRE0NINE=21
1 0 1 2 1 4 1 6 1 8 20 22
3.3. Apparent versus true digestibilitij
As it has baan mantionad bafore, ileal or faecal flows contain, in
addition to the undigested residue of tha fead, a variable and quantitativaly
important residue of bacterial and endogenous nitrogen origin. The classical
definition of endogenous nitrogen comes from Mitchell (1924). According to
his definition, endogenous nitrogen is the nitrogen found in chyme or faeces
when a nitrogen-free diet has been fed. This endogenous protein are derived
from the gastrointestinal tract and comprise protein, peptides, amino acids
and other N-containing substances from saliva, hile, pancreatic, gastric and
intestinal secretions, bacteria and sloughed epithelial cells. The excretion of
28
Chapter I: Literature review and objectives
endogenous or non-dietary protein leads to a variable under-estimation of
the proportion of dietaiy protein and amino acids absorbed by the animal.
For this reason, in other non-ruminants, the ileal apparent digestibility has
been substituted by the ileal true digestibility, as the latter one takes into
account the endogenous losses of the animal.
The endogenous protein losses are widely variable and seem to be
inñuenced primarily by dietary dry matter intake and secondarily by the
composition of the diet. Endogenous losses have been divided into basal
endogenous losses (non-specific), directly related to the diy matter intake
(DMI), and specific endogenous losses, induced by specific characteristics of
the feed.
Different methods as the N-free diet method, the use of diets with
highly digestible protein sources (casein or •wheat gluten), the regression
method, the enzymatically hydrolysed casein (EHC)/ultra filtration method,
the homoarginine method, the isotopic dilution method, etc. have been
described to determine the endogenous crude protein or amino acid fraction
in the ileal digesta of the pigs (Souffrant, 1991; Boisen and Moughan, 1995;
Nyachoti et al., 1997). Depending on the nature of the diets and the
experimental approach used, results of measurements on endogenous crude
protein provide Information on the flow of basal endogenous CP, or on the
total (basal plus specific) flow of endogenous crude protein.
In the pigs, the basal endogenous crude protein losses when
experimental N-free diets are ingested are 10-15 g/kg DMI. However, when
protein-containing diets are ingested under more practica! conditions, these
losses increase to 20 g/kg DMI. Moreover, when vegetable feeds are ingested,
there is often an extra loss induced generally by the presence of fibre or
antinutritional factors, resulting in a total endogenous protein loss in the
range of 20 to 40 g/kg DMI.
29
Chapter I; Literature review and objectives
4. Objectives
The global aim of this doctoral thesis has been to establish the basis
for a new feeding system in rabbits increasing the accuracy of the feedstuff
nitrogen evaluation for further use in practical feed formulation.
To reach this general aim the foliowing particular goals were defined:
1. To determine the endogenous nitrogen losses, which have been
never determined before in rabbits.
2. To evalúate in terms of protein and amino acids the nutritiva valué
of different feedstuffs commonly included in rabbit feed formulation by using
different protein and amino acid digestibility units.
To achieve the se particular goals two experiments were carried out.
These two experiments are presented in Chapter II and III, respectively.
In the first experiment, the effect of type of diet (protein-containing diet
or protein-free diet) and caecotrophy on the endogenous nitrogen and amino
acid losses estimations has been studied.
In the second experiment, four different feedstuffs were evaluated
using different protein and amino acid digestibility coefficients (apparent
faecal digestibility and apparent and true ileal digestibility). The endogenous
losses obtained in the first experiment were used to determine the true ileal
digestibility coefficients. The feedstuffs chosen for this experiment were
alfalfa hay (AH), barley grain (BG), wheat bran (WB) and sunflower meal
(SM).
30
Chapter I: Literature review and objectives
5. References
Berchiche, M. and Lebas, F. 1994. Supplementation en methionine d'un
aliment á base de feverole: effet sur la croissance, le rendement á
l'abattage, et la composition de la carcasse chez le lapin. World Rabbit
Science 2: 135-140
Boisen, S. and Moughan, P.J. 1996. Dietary influences on endogenous ileal
protein and amino acid loss in the pig- a review. Acta Agriculturae.
Scandinavica 45: 154-164.
Carabaño, R., Fraga, M.J., Santoma, G. and De Blas, J.C. 1988. Effect of
diet on composition of cecal contents and on excretion and
composition of soft faeces and hard faeces of rabbits. Journal of
Animal Science 66: 901-910.
Carabaño, R.M., Fraga, M.J. and De Blas, J.C. 1989. Effect of protein source
in fibrous diets on performance and digestive parameters of fattening
rabbits. Journal of Applied Rabbit Research 12: 201-204.
Carabaño, R.M., Motta Ferreira, W., De Blas, J.C. and Fraga, M.J. 1997.
Substitution of sugarbeet pulp for alfalfa hay in diets for growing
rabbits. Animal Feed Science and Technology 65: 249-256.
Colin, M. 1975. Effets sur la croissance du lapin de la supplementation en L-
lysine et en DL-méthionine de régimes végétaux simplifiés. Annales de
Zootechnie 24: 465-474.
CVB. 2002. Veevoedertabel. Central Veevoederbureau. Lelystad. The
Netherlands
De Blas, J . C , Pérez, E., Fraga, M.J., Rodríguez, J.M. and Galvez, J.F. 1981.
Effect of diet on feed intake and growth of rabbits from weaning to
slaughter at different ages and weights. Journal of Animal Science 52:
1225-1232.
De Blas, J.C. and Mateos, G.G. 1998. Feed formulation. In The Nutrition of
the Rabbit (ed. J.C. de Blas and J. Wiseman), pp. 241-253.
Commonwealth Agricultural Bureau, Wallingford, UK.
31
Chapter I: Literature review and objectives
De Blas, J.C., Taboada, E., Nicodemus, N., Campos, R., Piquer, J. and
Méndez, J. 1998. Performance response of lactating and growing
rabbits to dietaiy threonine contení. Animal Feed Science and
Technology 70: 151-160.
Emaldi, O., Crociani, F. and Matteuzi, D. 1979. A note on the total viable
counts and selective enumeration of anaerobio bacteria in the caecal
content, soft and hard faeces of rabbit. Journal of Applied
Bacteriology. 46: 169-172.
Forsythe, S.J. and Parker, D.S. 1985. Ammonia nitrogen turnover in the
rabbit caecum and exchange with plasma urea. British Journal of
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Fraga, M.J., Pérez de Ayala, P., Carabaño, R. and De Blas, J.C. 1991. Effect
of fibre on the rate of passage and on the contribution of soft faeces to
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1566-1574.
Fraga, M.J. 1998a. Protein digestión. In The Nutrition of the Rabbit (ed. J.C.
de Blas and J. Wiseman), pp. 39-53. Commonwealth Agricultural
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Ganuza, J. M., Balcells, J., Pérez, J.F., Fondevila, M. and Parker, D.S.
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García, J., De Blas, J.C., Carabaño, R. and García, P. 1995a. Effect of type of
lucerne hay on caecal fermentation and nitrogen contribution through
caecotrophy in rabbits. Reproduction Nutrition Development 35: 267-
275.
32
Chapter I: Literature review and objectives
García, J., Perez-Alba, L., Alvarez, C , Rocha, R., Ramos, M. and De Blas,
J.C. 1995b. Prediction of nutritive valué of lucerne hay in diets for
growing rabbits. Animal Feed Science and Technology 54: 33-44.
García, J., Carabaño, R., Pérez-Alba, L. and De Blas, J.C. 1995. Effect of
fibre source on neutral detergent fibre digestión and caecal traits in
rabbits. In Proceedings of the sixth world rabbit congress (ed. F.
Lebas), pp. 175-179. Toulouse, France.
García, J., Perez-Alba, L., Carabaño, R. and De Blas, J . C , 2000. Effect of
fíber source on cecal fermentation and nitrogen recycled through
cecotrophy in rabbits. Journal of Animal Science 78: 638-646.
INRA. 2002. L'alimentation des Animaux Monogastriques: Poro, Lapin,
Volailles. Institute de la Recherche Agronomique. París.
Just , A., Jorgensen, H. and Fernandez, J.A. 1981. The digestive capacity of
the caecum-colon and the valué of the nitrogen absorbed from the
hindgut for protein synthesis in pigs. British Journal of Nutrition 46:
209-219
Li, S., Sauer, W.C. and Kan, M.Z. 1993. The effect of dietary protein level on
amino acid digestibility in early-weaned pigs. Journal of Animal
Physiology and Animal Nutrition 70: 26-37.
Low, A.G. 1980. Nutrient absorption in pigs. Journal of Science of Food
Agriculture 31: 1087-1130.
Maertens, L. and De Groóte, G. 1988. The effect of dietary protein-energy
ratio and lysine content on the breeding results of does. Archives
Geflügelk. 52: 89-95.
Maertens, L. 1992. Rabbit nutrition and feeding: a review of some recent
developments. Journal Applied Rabbit Research 15: 889-913.
Maertens, L., Luzi, F. and De Groóte, G. 1997. Effect of dietary protein and
amino acids on the performance, carcass composition and N-excretion
of growing rabbits. Annales de Zootechnie 46: 255-268.
Martínez, J. and Fernández, J. 1980. Composición, digestibilidad, valor
nutritivo y relaciones entre ambos de diversos piensos para conejos.
2nd World Rabbit Congress. Barcelona. 1: 214-223.
33
Chapter I: Literature review and objectives
Mitchell, H.H. 1924. A method of determining the biological valué of protein.
Journal of Biological Chemistry 58: 873-882.
Motta Ferreira, W., Fraga, M.J. and Carabaño, R. 1995. Inclusión of grape
pomace in substitution for lucerne hay in diets for growing rabbits.
Animal Science 53: 157-174.
Moughan, P.J., Schulze, W.E. and Smith, W.C. 1988. Amino acid
requirements of growing meat rabbit. 1. The amino acid comiposition of
rabbit whole-body tissue-a theoretical estímate of ideal amino acid
balance. Animal Production 55: 153-152.
Nicodemus, N., Mateos, J., De Blas, J.C., Carabaño, R. and Fraga, M.J.
1999. Effect of diet on amino acid composition of soft faeces and the
contribution of soft faeces to total amino acid intake, trough
caecotrophy in lactating doe rabbits. Animal Science 69: 157-170.
NRC. 1998. Nutrients requirements of swine. National Research Council.
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Nyachoti, C.M., de Lange, C.F.M., McBride, B.W. and Schulze, H. 1997.
Significance of endogenous gut nitrogen losses in the nutrition of
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Proto, V. 1976. Fisiologia della nutrizione del coniglio con particolare
riguardo alia ciecotrofia. Rivista di Coniglicoltura n.7: 15-33.
Souffrant, W.E. 1991. Endogenous nitrogen losses during digestión in pigs.
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sulphur-containing amino acids. British Journal of Nutrition 39: 601-
613.
Taboada, E., Méndez, J., Mateos, G.G. and De Blas, J.C. 1994. The
responso of highly productive rabbits to dietary lysine content.
Livestock Production Science 40: 329-337.
34
Chapter I: Literature review and objectives
Tabeada, E., Méndez, J. and De Blas, J.C. 1995. The response of highly
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191-203.
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different protein levéis: effects on growth performance, meat quality
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and protein digestibility of feed ingredients for rabbits from chemical
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evaluation. In The Nutrition of the Rabbit (ed. J.C. de Blas and J.
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Williams, P.E.V. 1995. Digestible amino acids for non-ruminant animáis:
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Yoshida, T., Pleasants, J.R., Reddy, B. and Wostman, B.S. 1971. Amino acid
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Zebrowska, T. 1973. Digestión and absorption of nitrogenous compounds in
the large intestine of pigs. Roczniki Nauk Rolnizych B95: 85-90.
35
CHAPTER II; Experiment 1
Effect of type of diet and caecotrophy on ileal endogenous
nitrogen and amino acid flow in rabbits
(Animal Science, IN PRESS)
36
Chapter II: Experiment 1
Effect of type of diet (casein-based or protein-free diet) a n d caecotrophy on
ileal endogenous nitrogen and amino acid flow in rabbits^
A.I. García*, J .C. de Blas, and R. Carabano
Departamento de Producción Animal, E. T. S. Ingenieros Agrónomos,
Universidad Politécnica 28040, Madrid, Spain
t Financial suppor t was provided by the Spanish Comisión Interminesterial
de Ciencia y Tecnología (Project AGF199-1109)
* Present address : Nutreco Poultry and Rabbit Research Centre, Casarrubios
del Monte, 45950, Toledo, Spain.
37
Chapter II: Experiment 1
1. Abstract
Twenty-eight New Zealand White x Californian doe rabbits weighing
3791 ± 14Ig were surgically fitted with a glass T-cannula at ileum level.
Animáis were given ad libitum access to the casein-based diet (C) and to the
protein-free diet (PF). The only difference in the ingredient composition of the
diets was the substitution of 15% of maize starch by casein in the C diet.
Animáis were randomly allotted to each experimental diet. Seven rabbits per
diet were not allowed to practice caecotrophy. The C diet resulted in higher
feed DMI (111.5 vs. 51.5 g/day); feed nitrogen intake (3.12 vs. 0.20 g/day),
ileal ñow of DM (55.0 vs. 23.8 g/day) and ileal flow of nitrogen (0.72 vs. 0.24
g/day) but lower soft faeces DMI (21.7 vs. 41.4 g/day) than the PF diet. A
linear relationship between ileal endogenous nitrogen flow and total DM
intake was found for animáis fed the C but not for the PF diet. The ingestión
of C diet led to a higher (27 %, as average), endogenous amino acid ileal flow
than PF diet for all amino acids except for glycine. The ileal flow of glycine
was 20% higher in animáis fed the PF diet than in animáis fed the C diet.
The C diet resulted in higher content of nitrogen, threonine and proline in
soft faeces than the PF diet. On the opposite, the ingestión of the PF diet led
to a higher content of lysine and methionine in soft faeces than the C diet.
Within the C-diet, animáis practicing caecotrophy showed higher ileal flow of
DM (73.4 vs. 56.0 g/day) and phenylalanine (0.834 vs. 0.677 g/kg DMI) than
animáis not practicing it. Caecotrophy led to higher endogenous proportion
of some of the most important limiting essential amino acids in rabbits as
arginine, lysine, phenylalanine and threonine. In rabbits, the use of PF diets
has limited practical application to estimate the nitrogen endogenous losses
as the low feed DMI and the high soft faeces DMI leads to abnormal feed/soft
faeces intake ratio. Moreover, due to the important effect of caecotrophy in
the amino acid endogenous composition, it is important to correct the total
amino acid content of the ileal chyme by the undigested amino acid residue
coming from the soft faeces intake. This correction could be made, despite
38
Chapter II: Experiment 1
the methodological complexity implicit, avoiding caecotrophy by fitting
animáis with a wooden collar as in the present trial.
Keyívords: Rabbits, Casein-based diet, Protein-free diets, Endogenous
nitrogen, Amino Acids
2. Introduction
The most common procedure for nitrogen feedstuff evaluation and to
express nitrogen requirement in rabbits is the faecal balance (Fraga, 1998).
In other non-ruminant species (pigs and poultry), ileal digestibility has been
proposed, rather than faecal balance, as the best unit for nitrogen evaluation
of raw materials (Tanksley and Knabe, 1984; Sauer and Ozimek, 1986). The
main reason to choose ileal digestibility for nitrogen evaluation is that ileum
is the last segment of the digestive tract where amino acids can be absorbed
and used for protein synthesis (Zebrowska, 1973). Otherwise, standardised
digestibility has been proposed rather than apparent digestibility because
the first one takes into account the endogenous nitrogen losses of the
animal. This residue of endogenous origin leads to an under-estimation of
the amount of dietary amino acids absorbed. In single-stomached animáis,
several methods have been developed and applied to quantify these
endogenous losses being feeding protein-free diets or diets containing highly
digestible protein (casein or wheat gluten) the most common procedures
used. Each method or approach has its own advantages and limitations as it
is reviewed by Jansman et al. (2002). Several works, have shown that the
estimation of endogenous protein excretion determined using a protein-free
diet is lower than that found when a protein-containing diet is given, and
suggest that the protein-free method underestimates endogenous protein
excretion (Souffrant et al, 1997; Donkoh and Moughan, 1999). Rabbits can
use part of the amino acids not absorbed after the ileum for the protein
synthesis, through ingestión and digestión of soft faeces (caecotrophy). This
39
Chapter II: Experiment 1
phenomenon may contribute from 15 to 38% to total protein and amino acid
intake (Carabaño et al, 1988; Fraga et al, 1991; Nicodemus et al, 1999).
Therefore, in rabbits practicing caecotrophy, the contents of ileal flow are
constituted not only of undigested residuos from dietary protein and
producís of endogenous origin (digestiva enzymes, mucoproteins,
desquamated cells, urea and amino acids produced by cellular breakdown)
but also of undigested residues coming from the ingestión of soft faeces.
The present study was undertaken to compare the ileal endogenous
nitrogen flow determined either using casein or a protein-free diet and also
to study the effect of caecotrophy on the ileal endogenous flow of nitrogen
and amino acids in rabbits fitted with simple T-cannula at the terminal
ileum.
3. Materials and Methods
3.1. Diets
A casein-based (C) and a protein-free (PF) diet were formulated.
Ingredient composition and chemical analysis of diets are shown in Table 1
and 2, respectively. The only difference in the ingredient composition was the
substitution of 16% of maize starch by casein in the C diet to meet the
protein requirements of growing rabbits (de Blas and Mateos, 1998). Dietaiy
fibre content is particularly important in rabbit nutrition due to their high
requirements of fibre. Part of these fibre requirements of rabbits is related to
the effect of the large size fibre partióles on the passage rate of digesta
through the gut (Gidenne et al., 1998; de Blas and Mateos, 1998). A
deficiency in the fibre supply leads to high incidence of digestible problems
and changes in the digestive physiology of the rabbits as prolonged retention
timie in the caecum (Lebas et al., 1998). In this context, low-protein fibrous
sources as sunflower hulls, wheat straw, pectin and cellulose were included
40
Chapter II: Experiment 1
in the diet formulation. Sunflower hull and wheat s t raw were included in the
diets to reach a correct t rans i t time and ra te of passage of the digesta
th rough the intest inal t ract whereas pectin and cellulose were added to
ensu re a m i n i m u m fermentable subs t ra te en t rance into the caecum for the
main tenance of microbial metabolism. Both diets also included 2 g /kg of
fibre mordan ted with yt terbium according to the procedure of Uden et al.
(1980). Ytterbium was used as indigestible marker . The marked fibre was
produced from a mixture of sunflower hul ls and wheat s t raw (1:1) washed
with detergent in a n automat ic washing machine . Diets were pelleted and
animáis were given ad libitum access to feed and water dur ing all the
experiment.
Table 1. Ingredient composition ofthe experimental diets
í tem C diet PF diet
Ingredient, (g/kg)
Purified casein 150 Maize s ta rch 300 460
Lard 15 15
Sunflower hul ls 170 170
Wheat s traw 170 170
Purified cellulose 70 70 Pectinst 60 60
Sodium chloride 5.0 5.0
Calcium carbonate 10 10
Dicalcium phospha te 10 10
Sepiolite* 23 23 Vi tamin/minera l premix§ 5 5
Sunflower hu l l /whea t s traw + Yb# 2 2 + Provided by Sanofi Bio-Industries Ibérica t Provided by TOLSA, S.A. § Provided by Trouw Nutrition España S.A. (Madrid, Spain): Mineral and vitamin composition (mg/kg of feed): Mg, 290; Na, 329; S, 275; Co, 0.7; Cu, 10; Fe, 76; Mn, 20; Zn, 59.2; I, 1.25; choline, 250; riboflavin, 2; niacin, 20; vitamin Be, 1; vitamin K, 1; vi tamin E, 20 lU/kg diet; thiamine, 1; vitamin A, 8,375 lU/kg, and vitamin D3, 750 lU/kg. * Sunflower hull and w^heat stravi? (1:1) marked with Ytterbium.
41
Table 2.
í tem
Chemical composition ofthe experimental diets fe/kg
C diet
Chapter II: Experiraent 1
DM basis)
PF diet
Nitrogen
Starch
NDF
ADF
ADL
NDF-Nt
GE (MJ/kg DM) DE (MJ/kg DM)
Arginine
Cystine
Histidine
Isoleucine Leucine
Lysine
Methionine
Phenylalanine Threonine
Valine
Alanine
Aspartic acid
Glutamic acid Glycine
Proline
Serine
Tyrosine
28
245
276
179
50
1.3 17.8 8.99
5.9
1.4
6.1
10.1
16.6
13.4
4.3
8.5 7.8
13.1
6.2
14.7
38.3
4.2
20.4
10.0
8.1
3.8
380
271
180
47
1.3
16.6
9.40
1.70
0.46
0.52
0.86
1.44
0.96
0.38
0.81
0.84
1.34
1.01
2.01
3.15
1.24
0.93
0.95
0.60
t NDF-N = nitrogen bound to the NDF
3.2. Anim.als and housing
Twenty eight New Zealand White x Californian doe rabbi ts weighing
3791 ± 141 g were surgically fitted with a glass T-cannula a t ileum, 10 to 15
cm before the ileo-caeco-colic junct ion, according to the procedure described
by Gidenne et al. (1988), and following the principies for care of an imáis in
experimentation (Spanish Royal Decree 2 2 3 / 8 8 , 1988). After a 5 weeks
42
Chapter II: Experiment 1
recovery period rabbits reached their previous level of DMI and were ready to
begin the trial. Animáis were housed individually in wired metabolism cages
measuring 405 x 510 x 320 mm and allowing a sepárate complete collection
of uriñe and faeces. A cycle of 12-h of light and 12-h of dark was used
throughout the experiment. The light was switched on at 07:30. Heating and
forced ventilation systems allowed the building temperature to be
maintained between 15 and 24°C.
3.3. Experimental procedure
Animáis were randomly allotted to each experimental diet (14 rabbits
per diet). Seven rabbits in each diet were not allowed to practice caecotrophy
by fitting them with a wooden collar (150 g and 25 cm diameter).
Experimental procedure was different for animáis fed the C and the PF diet
in order to avoid or minimize any detrimental effect of a prolonged intake of
the PF diet (Hodgkinson et al., 2000a). The total length of the study for
animáis practising caecotrophy and fed C and PF diets was 20 and 12 days,
respectively, including adaptation, ileal collection and soft faeces collection
period (Figure 1). A total of 4 ileal samples from each rabbit were collected
under gravity for 1-h during 4 consecutive days (one per day) for the C diet
and during 2 consecutive days (two per day) for the PF diet. A reduction of
the length of the ileal sample collection period (to minimize any detrimental
effect of the PF diet) by intensifying the frequency of collection was justified,
as it has no effect on the ileal chyme composition (Blas et al, 2000). Two of
the four-ileal samples were collected during the soft faeces intake period at
11:00 and mixed together. The other two-ileal samples were collected during
the hard faeces excretion period at 20:00 and mixed together. Both mixed
sa:m.ples were stored at -20°C, freeze-dried and then pooled in 1:1
proportion. Soft faeces collection was determined individually on days 3 and
6 after the ileal collection period, according to the procedure described by
Carabaño et al. (1988). Rabbits not allowed to practice caecotrophy in each
43
Chapter II: Experiment 1
diet followed the same procedure described above for ileal sampling b u t
wearing a wooden collar from two days before s tar t ing the ileal collection
period (Figure 1).
Figure 1 .•Experimentalprocedure ofthe casein-based diet (C-diet) andprotein-
free diet (PF-diet) allotuing or not caecotrophy
C-Dict
Practicing caecotrophy
0(1
Not practicing caecotrophy
PF-Diet
Practicing caecotrophy
Od
Adaptat ion Period
Ileal Collection
ll:O0h 20:00h
lOd l i d 14d
Adaptat ion Period
Ileal Collection
l l i O O h 2 Q : 0 0 h
Ileal Collection
Adaptat ion Period ' ii;o();
4d Sd 6d
Soft Facces Collection
Soft Faeces Collection
17d 20d
12d
Not practicing caecotrophy
Od
Ileal Collection
Adaptat ion Period
2d 4d 5d 6d
Ileal flows of nitrogen and amino acids were determined by the dilution
technique of a marker (mordant ytterbium). The indigestible fraction of
nitrogen contained in the fibre sources (NDF-N) of the experimental diets
const i tute a source of exogenous losses at the end of the ileum which was
taken into account according to Schulze et al. (1994). The a m o u n t of
endogenous ileal nitrogen was calculated as foliows:
44
Chapter II: Experiment 1
a) Animáis practicing caecotrophy
TNF (g/d) = [total intake of ytterbium (feed + soft faeces) / ileal
ytterbium concentration] x ileal nitrogen
where TNF = total nitrogen floAV
NDF-NF (g/d) = [total intake of ytterbium (feed + soft faeces) / ileal
ytterbium concentration] x ileal NDF-N
where N D F - N F = nitrogen bound to NDF flow
ENF (g/d) = TNF - NDF-NF
where ENF = endogenous nitrogen flow
b) Animáis not practicing caecotrophy
TNF (g/d) = [feed intake of ytterbium / ileal ytterbium concentration] x
ileal nitrogen
NDF-NF (g/d) = [feed intake of ytterbium / ileal ytterbium
concentration] x ileal NDF-N
ENF (g/d) = T N F - N D F - N F
Endogenous ileal amino acid flows were calculated substituting the
ileal nitrogen and NDF-N by their respective amino acids concentration.
3.4. Analytical methods
AU Chemical analyses were conducted in duplícate. Procedures of the
AOAC (1995) were used to determine DM (930.15), and N (954.01) of diets,
45
Chapter II: Experiment 1
ileal content and soft faeces and also to determine the starch content of the
diets according to the alpha-amyloglucosidase method (995.11). Neutral-
detergent fibre, acid-detergent ñbre and acid-detergent lignin were
determinad according to the sequential method of Van Soest et al. (1991).
Nitrogen bound to neutral detergent fibre (NDF-N) was determinad as
described by Licitra et al. (1996). Gross energy of diets was determined by
adiabatic calorimetry. Amino acids were analysed only in ten rabbits per diet
(five practicing caecotrophy and five not). Amino acids were determined
following acid hydrolysis using a Beckman System 5300HPA amino acid
analyser (Fullerton, CA, USA). Samples (150 mg) were hydrolysed by reflux
in 50 mi of 5 mol/1 HCl with 10 g/1 added phenol for 24-h at 120°C. For the
determination of sulphur amino acids (methionine and cystine), samples
were oxidized with performic acid at 0°C for 16-h and then, neutralized with
1 g of sodium meta-bisulphite before analysis. Tryptophan, being destroyed
during acid hydrolysis, was not determined. Ytterbium content of diets, ileal
digesta and soft faeces were analysed by atomic absorption spectrometiy
(Smith Hieftje 22, Thermo Jarrel Ash, MA, USA) using predosed samples of
faeces to prepare common matrix standards. Previously, samples were ashed
(600°C) and then digested by boiling with a solution of 1.5 mol/1 HNO3 and
KC1(3.81 g/1).
3.5. Statistical analysis
Data were analysed as a completely randomised assay with type of diet
and caecotrophy as main effects. The GLM procedure of the Statistical
Analysis Systems Institute (1990), Versión 6, 4^ Edition (SAS Inst. INC.,
Cary, NC) was used to perform this analysis. Contrasts were used for mean
comparisons. Data of endogenous nitrogen flow were related to total DMI
using the REG procedure. Comparison of regression equations was done
following the procedure outlined by Snedecor and Cochran (1989).
46
Chapter II: Experiment 1
4. Results
Two of the rabbits fed the PF diet and practicing caecotrophy died
during the adaptation period. Two of the rabbits in each diet wearing the
wooden collar to prevent caecotrophy were discarded for bad adaptation to
the collar. The rest of rabbits remained in good health throughout the
experiment and no pathological changes in the ileum or the gastro-intestinal
tract were observed in post-slaughter examination.
The effect of the diet and caecotrophy on DMI, nitrogen intake and ileal
flow of DM and nitrogen is shown in Table 3. Feeding PF diet led to a very
lew feed DM intake (about half) than that observed in diet C. Furthermore,
soft faeces excretion was almost twice (41.4 vs. 21.7 g DM/day), so that the
proportion of soft faeces on total DM intake (sot faeces + feed intake) was
much higher (37.0 vs. 17.2 %) in animáis fed PF than C diet. These facts
difficult the interpretation of the results, as could affect the
representativeness of ileal samples, which were coUected in a proportion 1:1
at 11:00 and 20:00 h assuming a normal circadian pattern of soft faeces and
feed intake. As a consequence, the effect of type of diet on all the traits
studied was restricted to animáis were caecotrophy was prevented, whereas
the effect of caecotrophy was only analysed in animáis fed C diet. Prevention
of caecotrophy in animáis fed diet C implied a decrease of ileal ñow of DM (by
23.7 %, P < 0.01), although no differences were detected in the ileal flow of
nitrogen. Furthermore, in animáis where caecotrophy were prevented,
feeding diet C led to higher (P < 0.001) ileal flow of DM and nitrogen (by 135
and 200 %, respectively) than that observed in anim.als fed PF-diet.
47
Chapter II: Experiment 1
Table 3. Effect oftype ofdiet and caecotrophy on DM intake, nitrogen intake and üealflow ofDM and nitrogen (g/day)
í tem
Feed DMI
Soft faeces DMI
Total DMI (feed + SF)
Feed nitrogen intake
Soft faeces nitrogen intake
Total nitrogen intake
Ileal flow of DM
Ileal flow of nitrogen
(feed + SF)
C diet Practicing
caecotrophy (n = 7)
104.1
21.7
125.8
2.91
0.47
3.39
73.4
0.85
Not practicing caecotrophy
(n = 5)
111.5
-
111.5
3.12
-
3.12
56 .0
0.72
PF diet Practicing
caecotrophy (n=5)
70.6
41.4
112.0
0.27
0.62
0.89
74.0
0.64
Not practicing caecotrophy
(n=5)
51.5
-
51.5
0.20
-
0.20
23.8
0.24
SEM
5.891
2.810
6.202
0.116
0.054
0.127
3.407
0.051
Contrastt
1
•k-k-k
-
•krkk
•k-k-k
-
•k:kk
•kkk
rkkk
2
NS
-
NS
NS
-
NS
**
NS
t 1 = Effect of diet (animáis fed C-diet and not practicing caecotrophy vs. animáis fed PF-diet and not practicing caecotrophy)
2 = Effect of caecotrophy (animáis fed C-diet and practicing caecotrophy vs. animáis fed C-diet and not practicing caecotrophy)
48
Chapter II: Experiment 1
A linear relationship between endogenous nitrogen ñow (ENF) and total
DM intake was found (Figure 2) for animáis fed the C diet, either practicing
caecotrophy (ENFI) or not (ENF2). Comparison of the regression equat ions
showed tha t the slopes and the intercepts differed significantly (P < 0.05).
The regressions equat ions obtained were:
E N F I (mg/day kgO- S) = -189 (±118; P = 0.17) + 10.7 (±2.44; P = 0.007)
Total DMI (g/day kgO.75) R2 = o.79
ENF2 (mg/day kgO-75) = 153 (±24.4; P = 0.007) + 2.83 (+0.55; P = 0.014)
Total DMI (g/day kgO.75) R2 = Q.QO
Figure 2. Linear regression relationships between the endogenous nitrogen flow (mg/day kg^'^^j and total DMI (g/day kg^'^^} of animáis fed the casein-based diet practicing (•; ENFI) or not (A; ENFQ) caecotrophy.
1 c
1
450 -
400
350 -
• / ^ •
• ^ ^
•
300
250 1
200 35 40 45 50
Total DMI
55 60
No significant effect of DMI on the endogenous nitrogen ñow was found
for animáis fed the PF diet (P = 0.556 and P = 0.339 for animáis practicing
and not caecotrophy, respectively). The endogenous nitrogen ñow of an imáis
fed the PF diet practicing or not caecotrophy was, a s average, 253.9 and
49
Chapter II: Experiment 1
84.8 mg/day kgO^s, respectively, for an average daily feed intake of 44.4 and
19.4 g/day k^'^^, respectively.
Endogenous flow of amino acids in animáis consuming C or PF diet,
and practicing or not caecotropliy is shown in Table 4. Due to the effect of
DMI on tile basal endogenous losses, the endogenous amino acid ileal flows
are expressed as g/l<:g DMI. The ingestión of C diet led to a higher (27 %, as
average), endogenous amino acid ileal flow than PF diet for all amino acids
except for glycine. The ileal ñow of glycine was 20% higher (P < 0.05) in
animáis fed the PF diet than in animáis fed the C diet. No effect of diet (P >
0.05) was detected for arginine, phenylalanine, threonine and tyrosine.
Prevention of caecotrophy only affects (P < 0.05) to the ileal flow of
phenylalanine, which was a 19% higher (P < 0.05) in animáis practicing
caecotrophy than in animáis not practicing it.
The effect of diet and caecotrophy on the amino acid profile (g/ 16gN) of
the endogenous ileal protein is presented in Table 5. No effect of type of diet
was observed in the ileal endogenous proportion of cystine, histidine,
leucine, lysine, methionine, valine, alanine, proline and tyrosine. Animáis fed
diet C had 23, 32 and 32% higher (P < 0.001) proportion of isoleucine,
glutamic acid and serine, respectively, in the nitrogen endogenous losses
than animáis fed PF-diet. However, higher proportion of ileal endogenous
arginine, phenylalanine, threonine, aspartic acid and glycine (16, 23, 13, 7
and 42% respectively, P < 0.05) were observed in animáis fed the PF than the
C-diet. Within the C-diet, animáis practicing caecotrophy showed higher
endogenous ileal proportion of arginine, leucine, lysine, phenylalanine,
threonine, alanine, aspartic acid, proline and tyrosine (12, 7, 14, 20, 11, 14,
7, 11 and 16%, respectively, P< 0.05) than animáis not practicing it.
50
Chapter II: Experiment 1
Table 4. Effect oftype ofdiet and caecotrophy on endogenous ileal amino acidflows (g/kg DMI)
ítem Arginine Cystine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Valine Alanine Aspartic acid Glutamic acid Glycine Proline Serine Tyrosine
Practicing caecotrophy (n=
1.848 1.241 0.611 1.485 1.904 1.501 0.383 0.834 2.207 2.251 1.421 3.005 5.053 2.067 2.159 2.574 0.826
Cdiet
Not practicing =5) caecotrophy (n=5)
1.654 1.273 0.640 1.512 1.804 1.310 0.352 0.677 1.999 2.145 1.237 2.826 5.097 2.453 1.958 2.672 0.701
Practicing caecotrophy (n=
1.474 0.999 0.486 0.924 1.341 1.272 0.334 0.674 1.608 1.652 1.269 2.343 2.941 1.933 1.160 1.498 0.691
PF diet
=5) Not practicing
caecotrophy (n=5) 1.415 1.007 0.481 0.837 1.333 0.896 0.255 0.630 1.657 1.599 0.972 2.176 2.512 3.066 1.432 1.304 0.583
SEM -
0.113 0.080 0.039 0.077 0.110 0.080 0.022 0.050 0.133 0.098 0.082 0.179 0.247 0.212 0.120 0.128 0.048
Contrastt
1
NS *
**
•í;'k-Jc
**
**
**
NS
NS **
*
*
***
*
**
***
NS
2
NS
NS
NS
NS
NS
NS
NS *
NS
NS
NS
NS
NS
NS
NS
NS
NS
t 1 = Effect of diet (animáis fed C-diet and not
2 = Effect of caecotrophy (animáis fed C-diet
practicing caecotrophy vs. animáis fed PF-diet and not practicing caecotrophy)
and practicing caecotrophy vs. animáis fed C-diet and not practicing caecotrophy)
51
Table 5. Effect oftype ofdiet and caecotrophy on endogenous arrdno acid composition (g/ 16gN)
C diet PF diet
Practicing Not practicing Practicing Not practicing ítem caecotrophy (n=5) caecotrophy (n=5) caecotrophy (n=5) caecotrophy (n=5)
SEM
Chapter II: Experiment 1
Contrast t
1 2
Arginine
Cystine
Histidine
Isoleucine
Leucine
Lysine
Methionine
Phenylalanine
Threonine
Valine
Alanine
Aspartic acid
Glutamic acid
Glycine
Proline
Serine
Tyrosine
4.63
3.11
1.53
3.72
4.77
3.76
0.96
2.09
5.53
5.64
3.56
7.53
12.66
5.18
5.41
6.45
2.07
4 .08
3.14
1.58
3.73
4 .45
3.23
0.87
1.67
4 .93
5.29
3.05
6.97
12.57
6.05
4 .83
6.59
1.73
4 .31
2.92
1.42
2.70
3.92
3.72
0.98
1.97
4.70
4 .83
3.71
6 .85
8.60
5.65
3.38
4 .38
2.02
4 .85
3.45
1.65
2.87
4.57
3.07
0.87
2.16
5.68
5.48
3.33
7.46
8.61
10.51
4 .91
4.47
2.00
0.16
0.15
0.05
0.07
0.10
0.10
0.05
0.07
0.16
0.13
0.11
0.16
0.12
0.60
0.10
0.10
0.10
**
NS
NS •k'k'k
NS
NS
NS •k-k-k
•k-k
NS
NS •k
ickk
•kkk
NS •k-k-k
NS
NS
NS
NS
NS
NS **
*
NS
NS **
NS *
t 1 = Effect of diet (animáis fed C-diet and not practicing caecotrophy vs. animáis fed PF-diet and not practicing caecotrophy)
2 = Effect of caecotrophy (animáis fed C-diet and practicing caecotrophy vs. animáis fed C-diet and not practicing caecotrophy)
52
Chapter II: Experiment 1
The effect of the diet on nitrogen and amino acid concentra t ion of soft
faeces (SF) is shown in Table 6. Diet C led to a 32, 11 and 10% higher (P <
0.05) content in the soft faeces of nitrogen, threonine and proline t h a n PF
diet, respectively. On the contrary, the ingestión of diet PF led to 10 and 13%
higher content (P < 0.05) of lysine and methionine t h a n diet C, respectively.
No effect of diet was detected for the rest of amino acids analysed.
Table 5. Effect oftype of diet on nitrogen (g/kg DM) and amino acid composition (g/16gN) of soft faeces
í tem C diet (n-5)
PF diet (n=5)
SEM Significance
Nitrogen
Amino acid
Arginine
Cystine
Histidine
Isoleucine
Leucine
Lysine
Methionine
Phenylalanine
Threonine
Valine
Alanine
Aspartic acid
Glutamic acid
Glycine
Proline
Serine
Tyrosine
22.1
4.42
2.28
1.38
3.47
5.01
4.11
1.16
2.49
5.60
5.33
4.18
8.20
9.81
4.38
3.68
4.28
2.44
15.0
4.33
2.40
1.37
3.37
4.72
4.58
1.34
2.37
4.97
5.22
4.42
8.22
9.83
4.71
3.32
4.03
2.47
1.41
0.22
0.16
0.05
0.09
0.11
0.13
0.04
0.09
0.17
0.12
0.13
0.21
0.23
0.13
0.08
0.13
0.08
**
NS
NS
NS
NS
NS *
*
NS *
NS
NS
NS
NS
NS *
NS
NS
53
Chapter II: Experiment 1
5. Discussion
In the present work, feeding the rabbits with PF diet led to a significant
decrease (54%) of feed DMI respect to the C diet. As a consequence, the DE
intake of animáis fed PF diet was 246 kJ /day kgO-75 which is below the
maintenance energy requirements of rabbit does (400 kJ /day kgO- s Parigi
Bini and Xicatto, 1998). Furthermore, a significant increase of daily soft
faeces intake was also observed in animáis fed the PF diet. Caecotrophy
normally represents from 9 to 18% of total DMI (feed intake + soft faeces)
(Carabaño and Piquer, 1998) while for the PF-diet caecotrophy represents in
the present work 37% of the total DMI. This fact suggests an adaptative
behaviour in order to compénsate a deficient nutrient intake. In the same
way, Hornicke and Bjornhag (1980) also observed an increase on the soft
faeces intake to compénsate the deñcient nutrient absorption induced by a
pancreatic duct ligation.
The endogenous ileal nitrogen losses in single-stomached animáis are
widely variable and are influenced primarily by dietary DMI (Butts et al,
1993a; Hess and Séve, 1999) and secondarily by diet composition (Boisen
and Moughan, 1996). In the present study, the endogenous ileal nitrogen
flow of the C diet varied according to DMI. The lack of response to the DMI
for PF diet could be explained by the low feed intake but also by the low
variation range of total DMI showed by the rabbits fed this diet. Boisen and
Moughan (1996) showed in a review that basal ileal nitrogen losses (directly
related to the DMI) varied from 1.6 to 2.4 g/kg DMI when experimental N-
free diets were fed. However, when protein-containing diets were given, these
losses increased to 3.2 g/kg DMI. Moreover, when vegetable feeds were
ingested, there were often extra losses induced generally owing to the
presence of fibre or antinutritional factors, resulting in total endogenous
nitrogen losses in the range of 3.2 to 6.4 g/kg DMI. The endogenous nitrogen
54
Chapter II: Experiment 1
and the amino acid flows obtained in the present s tudy, with the C diet were
also higher t h a n those obtained with the PF diet with the exception of
glycine. The resul ts obtained agree with severa! works carried out in other
single-stomached an imáis (Butts et al., 1993b; Donkoh et al., 1995;
Hodgkinson et al., 2000b), which suggest a s t imulatory effect of dietary
protein and pept ides on the endogenous protein secretion. Lower proteolytic
enzyme activities in the páncreas and intestine and an increased ra te of
breakdown and reabsorpt ion of secreted enzymes have been reportad in
animáis fed a protein-free diet (Schneeman, 1982). Differences in the
quant i ty or in the amino acid composition of the components of the
endogenous nitrogen losses could explain the differences observed in our
s tudy in the amino acid pat tern of the endogenous nitrogen losses with the
type of diet. In this way, the C diet, could increase the pancreat ic and
intestinal endogenous secretions leading to variat ions in the relative
contribution of the different enzymatic secret ions const i tut ing the
endogenous losses. The increased recovery of endogenous glycine in ileal
digesta of the PF diet could probably resul t from a relatively large
contribution of bile respect to other components of the endogenous protein
(De Lange et al, 1989). The higher endogenous ileal flow of nitrogen and
amino acids of animáis fed the C diet could be also explained by dietaiy
amino acids or peptides from the undigested casein of the C diet.
Hodgkinson et al. (2000b) found a significant effect of dietary peptide
concentrat ion on the endogenous ileal flows of nitrogen and all the amino
acids of growing pigs, with an increase in the endogenous ileal amino acid
flow with increasing dietary enzyme-hydrolysed casein concentrat ion. In the
present trial, no correlation between the amino acid composition of the
casein and the ileal chyme of animáis fed the C diet and not practicing
caecotrophy were found. In this context, Hagemeister and Erbersdobler
(1985) showed tha t the ileal t rue digestibility valué of casein in Gottinger
minia ture pigs was 99 .5% and when the endogenous losses in pigs were
est imated by feeding them diets containing highly digestible protein s u c h a s
55
Chapter II: Experiment 1
casein, a true digestibility of 99% for CP and amino acids was considered
(Jansman et al, 2002).
The soft faeces contribution to the total nitrogen intake of rabbits was
14 and 70% for the C and the PF diets, respectively. Nitrogen concentration
of soft faeces was low for both diets compared to literature data (Spreadbury,
1978; Carabaño et al, 1988; Nicodemus et al, 1999). The type of fibre
included in the diets could explain this ñnding. García et al (2000) found
that rabbits fed diets based on sunflower huUs and barley straw produced
soft faeces with lower nitrogen contení than animáis fed diets based on other
fibre sources with higher peptic constituents, higher proportions of fine
dietary particles (< 0.315 mm) and lower proportion of ADL and NDF. As it is
shown in Table 5, caecotrophy led to higher endogenous proportion of some
of the most important limiting essential amino acids in rabbits as arginine,
lysine, phenylalanine and threonine. This is an expected result as soft faeces
are enriched respect to the hard faeces in essential amino acid such as
lysine, sulphur amino acids and threonine (Carabaño and Piquer, 1998).
However, the effect of caecotrophy on the endogenous estimations was not as
high as it was thought due to their high nitrogen and amino acid digestibility
coefficient (79% as average) performed by in vitro analysis (García, A.I. et al,
unpublished results).
To the authors ' knowledge, estim.ates of the endogenous ileal nitrogen
and amino acids flows have never been reported before in rabbits. The
comparison of the results obtained in this trial in rabbits not practicing
caecotrophy and those reviewed by Jansman et al (2002) in pigs, showed
that the daily endogenous ileal nitrogen flow (g/kg DMI) for the PF and the C
diet was, respectively, 2.7 and 3.2 times higher in rabbits. Similarly, the ileal
endogenous amino acid flows were, as average, 2.7 and 3.0 times higher,
respectively, than those reported by the same authors. The difference
between the present study in rabbits and that reported in pigs may be a
species difference, as there is no evidence that the endogenous amino acid
56
Chapter II: Experiment 1
flows are similar in the rabbit as in the pig but also may be due to the higher
fibra content of rabbit diets than pig diets (27-33% vs. 3-8% of NDF, for
rabbits and pigs diets, respectively) and by differences in the methodology
used to obtain the endogenous losses.
In conclusión, in rabbits, the use of PF diets has limited practical
application to estímate the nitrogen endogenous losses as the low feed DMI
and the high soft faeces DMI leads to abnormal feed/soft faeces intake ratio.
Moreover, due to the important effect of caecotrophy in the amino acid
endogenous composition, it is important to correct the total amino acid
content of the ileal chyme by the undigested amino acid residue coming from
the soft faeces intake. This correction could be made, despite the
methodological complexity implicit, avoiding caecotrophy by fitting animáis
with a wooden collar as in the present trial. Data obtained in this work could
be used for further calculation of the true ileal digestibility valúes of feed
ingredients for rabbits.
6. Acknowledgements
The authors are grateful to A. Espinosa for her technical assistance in
the laboratoiy and in the ileal collection and to Trouw Nutrition for the
analyses of the amino acids. Financial support was provided by CYCIT,
Project AGF199-1109.
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57
Chapter II: Experiment 1
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Boisen, S. and Moughan, P.J. 1995. Dietary inñuences on endogenous ileal
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But t s , C. A., Moughan, P.J., Smith, W . C , Reynolds, G.W. and Garrick, D.J.
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composition of soft faeces and hard faeces of rabbi ts . J o u r n a l of
Animal Science 56: 901-910.
Carabaño, R. and Piquer, J . 1998. The Digestive System of the Rabbit. In
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Chapter II: Experiment 1
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Donkoh, A., Moughan, P.J. and Morel, P.C.H. 1995. Comparison of methods
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359-366.
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fibre source on cecal fermentation and nitrogen recycled through
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Wallingford, UK.
Hagemeister, H. and Erbersdobler, H. 1985. Chemical labelling of dietary
protein by transformation of lysine to homoarginine: a new technique
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59
Chapter II: Experiment 1
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Press, Lancaster.
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of pigs. Animal Peed Science and Technology 98: 49-60.
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Corring and A. Rerat), 2e seminaire internat ional Jouy-en-Josas -
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Schulze, H., Van Leeuwen, P., Verstegen, M.W.A., Huisman , J . , Souffrant,
W.B. and Ahrens, F. 1994. Effect of level of dietary neu t ra l detergent
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Souffrant, W. B., Février, C , Laplace, J .P . and Hennig, U. 1997. Comparison
of methods to est ímate ileal endogenous nitrogen and amino acids in
piglets. Proceedings of the Yll^ internat ional symposium on digestive
physiology in pigs (ed. J .P . Laplace, C. Fevrier and A. Barbeau), EAAP
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61
Chapter II: Experiment 1
Zebrowska, T. 1973. Digestión and absorption of nitrogenous compounds in
the large intestino of pigs. Roczniki Nauk Rolnizych B95: 85-90
62
CHAPTER III; Experíment 2
Comparison of different units for nitrogen and amino acid
evaluation in rabbit diets
(Animal Science, submitted)
63
Chapter III: Experiment 2
Comparison of different units for nitrogen and amino acids evaluation in
rabbit dietst
A.I. García*, J.C. de Blas, and R. Carabaño§
Departamento de Producción Animal, E.T.S. Ingenieros Agrónomos,
Universidad Politécnica 28040, Madrid, Spain
t Financial support was provided by the Spanish Comisión Interminesterial
de Ciencia y Tecnología (Project AGF199-1109)
* Present address: Nutreco Poultry and Rabbit Research Centre, Casarrubios
del Monte, 45950, Toledo, Spain.
§To whom correspondence should be addressed
64
Chapter III: Experiment 2
1. Abstract
Twenty New Zealand White x Californian doe rabbi ts weighing 3841 ±
117 g were surgically fitted with a glass T-cannula a t i leum level. Animáis
were given ad libitum access to feed and water dur ing all the experiment.
Four diets were formulated in order to contain a maximal nitrogen
proportion from alfalfa hay (AH), barley grain (BG), wheat b ran (WB) and
sunflower meal (SM) in each diet. True ileal digestibility of crude protein and
amino acid of AH, BG, WB and SM was determined and compared with those
digestibility valúes obtained by us ing simplified ba lances (apparent ileal and
faecal digestibility). The endogenous losses were determined feeding rabbi ts
with diets containing casein with an a s sumed 100% digestibility. All feedstuff
showed higher CP digestibility at faecal t h a n ileal level. However, amino acid
digestibilities were higher or lower at faecal or ileal level depending on the
amino acid and the feedstuff studied. The WB feedstuff, showed higher faecal
t h a n ileal digestibility valúes for most of the amino acids. Contrary, for the
AH, BG and SM feedstuff higher amino acid AID t h a n AFD valúes were
observed. Making a balance us ing the average valué of the four feedstuffs,
between the ileal flow of protein (3.17 g/day, as average) and the total
protein excreted in soft and hard faeces (3.74 g /day, a s average) an overflow
of protein of 0.568 g/day, as average, was observed (18% of the ileal flow).
However, the effect of this overflow on the faecal digestibility valué was lesser
appreciable t h a n expected due to the soft faeces excretion and re-ingestion
(36% of the total protein excreted). Making similar ba lances for the most
limiting amino acids enr ichment in lysine, methionine a n d threonine were
observed. An impor tant activity of the gut microflora was observed and from
0.40 to 0.49 and from. 0.62 to 0.69 of the total nitrogen in ha rd faeces and
soft faeces, respectively, was of bacterial origin. Due to the large endogenous
losses, higher t rue ileal digestibility (TID) t h a n appa ren t ileal digestibility
(AID) of CP and amino acids were observed in all the feedstuffs s tudied.
DiíTerences between TID and AID were of small magni tude in the high-
65
Chapter III: Experiment 2
protein feedstuffs (SM) bu t m u c h more pronounced in the low-protein
feedstuffs (BG and WB). The relative valué establ ished among feedstuffs for
the CP and the main essential amino acids changed depending on the
digestibility un i t used and the amino acid. In conclusión, the u s e of AID and
AFD lead to an underes t imat ion or an overestimation, respectively, of the
ileal utilization of nitrogen and amino acids contení of the feedstuffs. The u s e
of TID is recommended for a more precise nitrogen feedstuffs evaluation.
Keywords: Rabbits, Faecal and Ileal digestibility, Apparent and True
digestibility, Amino Acid.
2. Introduction
Estimations of disappearance of nutrients (potentially absorbed) in the
digestive tract are necessary to evalúate more precisely the nutritive valué of
feeds. Several units of digestibility, measured at different segments of the
digestive tract, have been proposed. Nowadays, the most common procedure
for nitrogen feedstuff evaluation is, in rabbits, the faecal balance (Fraga,
1998). In other single-stomached species (pigs and poultry), ileal balance
was proposed, rather than faecal, as the best unit for nitrogen evaluation
(Tanksley and Knabe, 1984; Sauer and Ozimek, 1986) as faeces are
contaminated with endogenous microbial nitrogen and ileum is the last
segment of the digestive tract where am.ino acids can be absorbed and used
for protein synthesis (Zebrowska, 1973). Similar results have been reported
in rabbits when caecotrophy is prevented (Belenguer et al., 2003). In pigs,
standardised and true ileal digestibilities have been proposed rather than
apparent ileal digestibility as the first ones are corrected by the ileal
endogenous nitrogen flow. The endogenous losses lead to an
underestimation of the amount of amino acids absorbed v hen apparent
units are used. Differences between standardised and true ileal digestibility
are found in the methodology used for the endogenous losses estimation. In
the standardised digestibility unit's endogenous flo'ws are determined by
66
Chapter III: Experiment 2
feeding animáis with a protein free diet. When other methodology is used,
the digestibility unit is commonly better known as true ileal digestibility. In a
previous work (García et al., 2004), the endogenous losses of the rabbits
were determined feeding animáis with diets containing casein with an
assumed 100% digestibility. This residue of endogenous origin is
characteristically higher in rabbits than in other single-stomached species.
Furthermore, rabbits are able to use microbial protein synthesized after the
ileum (in the caecum) through caecotrophy, which should be taken into
account in the total digestibility balance.
The present study was undertaken to determine the true ileal
digestibility of four different raw materials commonly used in rabbit
formulation and to compare these results with those obtained by using
simplified balances (apparent ileal digestibility and faecal digestibility) in
order to establish, the best digestibility unit allowing the most accurate
estimation of available amino acid content of the feeds in rabbits.
3. Materials and Methods
3.1. Diets
In European rearing conditions the total nitrogen requirements are
met with alfalfa (30-50%), oil seed meáis (30-40%) and cereals and their by-
products (33%). For this reason alfalfa hay (AH), sunflower meal (SM), barley
grain (BG) and wheat bran (WB) were the feedstuffs selected for the present
study. Four diets were formulated in order to get a maximal proportion of the
protein from the feedstuffs studied on the total dietary protein content.
Purified casein, maize starch, lard, sunflower hulls, wheat straw, purified
cellulose, pectins, vitamins and minarais were added to make diets
isonutritive (CP, starch and fibre content and type of fibre). Diet composition
was also similar to the casein-based diet used in a previous study to
determine the endogenous nitrogen and amino acid flow (García et al, 1999
67
Chapter III: Experiment 2
and García et al., 2004). The experimental diets meet or exceed the actual
requirements of adult rabbits (de Blas and Mateos, 1998). Chemical
composition of the feedstuffs studied is shown in Table 1.
Table 1. Chemical composition of feedstuffs (g/kg dry m.atter)
Diets
DM
CP
Starch
NDF
ADF ADL
Essential Amino Acids
Arginine
Cystine Histidine
Isoleucine
Leucine
Lysine
Methionine Phenylalanine
Threonine
Valine
Non essential Amino Acids
Alanine Aspartic acid
Glutamic acid
Glycine
Proline
Serine
Tyrosine
Alfalfa Hay 876
202
10.2
438
285 70.7
10.87
4.32 3.24
8.91
14.36
8.46 2.79
8.80
8.28
12.20
9.78
19.82
19.51
10.02 6.67
0.763
5.54
Barley Grain 873
121
602
223 54.0 12.7
7.32
2.86 2.67
4.71
8.03
4.20
1.95 5.68
4 .05
6.85
4.70
7.78
27.89
4 .73 13.57
0.486
3.49
Wheat Bran 864
160
216
400 115
36.4
11.42
2.95
4 .56
6.64
11.22
6.85
3.15
7.13
5.87
8.72
8.21 10.67
37.42
8.19 11.82
0.810
5.22
Sunflower Meal 887
393
14.5
344
211 84.9
30.81
6.04
9.29
17.13
23.62
14.22 8.61
16.66
14.24
20.40
16.63
37.30
61.27
21.78 15.84
1.591
9.48
Ingredient composition and chemical analysis of alfalfa hay (AH),
barley grain (BG), wheat bran (WB) and sunflower meal (SM) diets are shown
in Table 2 and 3, respectively. Diets also included 2 g/kg of fibre mordanted
with ytterbium according to the procedure described by Uden et al. (1980).
Ytterbium was used as indigestible marker. The marked fibre comes from a 68
Chapter III: Experiment 2
mixture of sunflower hulls and wheat straw (1:1) washed with detergent in
an automatic washing machine. Diets were pelleted and animáis were given
ad libitum access to feed and water during all the experiment.
Table 2. Ingredient composition of experimental diets (g/kg)
Diets
Alfalfa hay
Barley grain
Wheat Bran
Sunflower meal
Puriñed casein
Maize s tarch
Lard
Sunflower hul ls
Wheat s t raw
Puriñed cellulose
Pectinst
Sodium chloride
Calcium carbonate
Dicalcium phospha te
Sepiolite^^
Vi tamin/minera l premix§
Sunflower h u ü / w h e a t s traw + Yb*
Alfalfa Hay
300
-
-
-
110
250
15
100
70
60
40
5
10
10
23
5
2
Barley Grain
-
470
-
-
110
-
15
165
95
30
60
5
10
10
23
5
2
Wheat Bran
-
-
300
-
120
190
15
140
80
40
60
5
10
10
23
5
2
Sunflower Meal
-
-
-
330
40
250
15
50
140
70
50
5
10
10
23
5
2
t- Provided by Sanofi Bio-Industries Ibérica *• Provided by TOLSA, S.A. § Provided by Trouw Nutrition España S.A. (Madrid, Spain): Mineral a n d vitamin composition (mg/kg of feed): Mg, 290; Na, 329; S, 275; Co, 0.7; Cu, 10; Fe, 76; Mn, 20; Zn, 59.2; I, 1.25; choline, 250; riboflavin, 2; niacin, 20; vitamin Be, 1; vitamin K, 1; vitamin E, 20 lU/kg diet; thiamine, 1; vitamin A, 8,375 lU/kg, and vitamin D3, 750 lU/kg. * Sunflower hull and wheat straw (1:1) marked with Ytterbium.
69
Chapter III: Experiment 2
Table 3. Chemical composition of expeñmental diets (g/kg dry matter)
Diets
DM
CP
Starch
NDF
ADF
ADL
NDF-CPt
GE ( k J / k g DM)
Essential Amino Acids
Arginine
Cystine
Histidine
Isoleucine
Leucine
Lysine
Methionine
Phenylalanine
Threonine
Valine
Non essential Amino Acids
Alanine
Aspartic acid
Glutamic acid
Glycine
Proline
Serine
Tyrosine
Alfalfa Hay
911
175
253
262
156
44.4
17.9
173
7.70
2.01
5.83
9.44
15.32
11.39
4.06
8.53
7.55
12.28
7.14
15.03
32.23
5.35
16.21
8.83
6.74
Barley Grain
903
175
250
299
160
44.8
11.9
180
9.01
2.85
5.77
9.31
15.41
11.35
5.04
8.37
7.29
12.41
6.49
13.43
39.15
5.24
17.19
9.06
6.25
Wheat Bran
911
179
262
2 8 3
154
41 .8
10.4
180
8.63
1.80
5.55
8.96
14.90
11.57
3.84
7.52
7.15
11.97
6.75
13.86
35 .85
5.24
15.90
9.28
6.54
Sunflower Meal
909
177
231
261
152
42.8
12.8
172
12.69
2.67
4.84
7.98
11.79
7.85
3.54
7.27
6.53
9.99
5.93
15.59
32.38
8.15
10.89
7.57
4.66
t NDF-CP: c m d e protein bound to the NDF
70
Chapter III: Experiment 2
3.2. Animáis and housing
Twenty New Zealand White x Californian doe rabbits weighing 3841 +
117 g were surgically fitted with a glass T-cannula at ileum, 10 to 15 cm
before the ileo-caeco-colic junction, according to the procedure described by
Gidenne et al. (1988), and foUowing the principies for care of animáis in
experimentation (Spanish Royal Decree 223/88, 1988). After a 6 weeks
recovery period rabbits reached their previous level of DMI and were ready to
begin the trial. Animáis were housed individually in wired metabolism cages
measuring 405 x 510 x 320 mm and allowing a sepárate complete collection
of uriñe and faeces. A cycle of 12-h of light and 12-h of dark was used
throughout the experiment. The light was switched on at 07:30. Heating and
forced ventilation systems allowed the building temperatura to be maintained
between 15 and 24°C.
3.3. Experimental procedure and calculations
After the surgical recovery period five animáis per diet were randomly
allotted to each experimental diet. Rabbits remained in apparent good health
throughout the experiment and no pathological changes in the ileum or the
gastro-intestinal tract were observed in post-slaughter examination.
Following 10 days of feed adaptation period, feed intake and hard faeces
excretion were recorded for each rabbit over a 4 days period. After the hard
faeces collection period a total of four ileal samples from each rabbit were
collected under gravity for 1-h during 4 consecutive days (one per day). Two
of the four-ileal samples were collected during the soft faeces intake period at
11:00 and mixed together. The other two-ileal samples were collected during
the hard faeces excretion period at 20:00 and mixed together. Both mixed
samples were stored at -20°C, freeze-dried and then pooled in 1:1
proportion. Soft faeces intake was determined individually 3 and 5 days after
71
Chapter III; Experiment 2
the ileal-sampling period, according to the procedure described by Carabaño
etal. (1988).
The different digestibility units of the feedstuffs were estimated taking
into account several factors. Each diet contained two protein sources:
Purified casein and the feedstuff studied. Casein was supposed to be 100%
digested at ileal level. Due to the particular importance of dietary fibre in
rabbit nutr i t ion, low-protein fibrous sources (wheat s t raw and sunñower
huU) were included in the diets. To subt rac t the undigested residue of
protein coming from these fibrous sources, the ileal flow and the faeces
excretion were corrected according to the procedure described by Schulze et
al. (1994), by the intake of CP and amino acids bound to the NDF fraction of
the wheat s t raw and sunñower hull . Moreover, the indigestible res idue of
protein and amino acids coming from the soft faeces intake was also
subt rac ted from the ileal flo^v determining the in vitro digestibility of CP and
amino acids of the soft faeces. The mean valué of the in vitro soft faeces
digestibility used was est imated in a total n u m b e r of four samples (one soft
faeces mixed sample per diet).
For the t rue digestibility un i t s the ileal endogenous losses (lEL) were
est imated according to the following equation (García et al., 2004) taking into
account the inclusión level of the feedstuff in each experimental diet:
lEL (mg/dav kgO-751 = - 189 (+ 118; P = 0.17) + 10.7 (± 2.44; P = 0.007)
Total DMI (g/day kgO-75)
3.4. Analytical methods
All Chemical analyses were conducted in duplícate. Procedures of the
Association of Official Analytical Chemists (1995) were used to determine DM
(930.15), and N (954.01) of diets, hard faeces, ileal contení and soft faeces
72
Chapter III: Experiment 2
and also to determine the starch contení of the diets and feedstuffs
according to the alpha-amyloglucosidase method (996.11). Neutral-detergent
fibre, acid-detergent fibre and acid-detergent lignin were determined
according to the sequential method of Van Soest et al. (1991). Nitrogen
bound to neutral detergent fibre (NDF-N) was determined as described by
Licitra et al. (1995). Gross energy of diets was determined by adiabatic
calorimeter. Amino acids were determined following acid hydrolysis using a
Beckman System 6300HPA amino acid analyser (Fullerton, CA, USA).
Samples (150 mg) were hydrolysed by reflux in 50 mi of 6 mol/1 HCl with 10
g/1 added phenol for 24-h at 120°C. For the determination of sulphur amino
acids (methionine and cystine), samples were oxidized with performic acid at
0°C for 16-h and then, neutralized with 1 g of sodium meta-bisulphite before
analysis. Tryptophan, being destroyed during acid hydrolysis, was not
determined. Ytterbium content of diets, ileal digesta and soft faeces were
analysed by atomic absorption spectrometry (Smith Hieftje 22, Thermo Jarrel
Ash, MA, USA) using predosed samples of faeces to prepare common matrix
standards. Previously, samples were ashed (600°C) and then digested by
boiling with a solution of 1.5 mol/1 HNO3 and KCl (3.81 g/1). In vitro
digestibility of soft faeces was determined by the en2ymatic in vitro method
developed by Ramos et al. (1992) based on the method for pigs proposed by
Boisen (1991). The microbial nitrogen content in soft faeces and hard faeces
was calculated from the purine nitrogen concentration using the total purine
analysis method of Zinn and Owens (1980) as modified by Ushida et al.
(1985). A purine:bacterial nitrogen ratio of 0.89 mg RNA/mg of total nitrogen
was previously determined on a bacterial preparation isolated from caecal
contents of 20 does (García et al., 1995).
3.5. Statistical analysis
Data were analysed as a completely randomised design with the diet as
main effect by using the GLM procedure of the Statistical Analysis Systems
73
Chapter III: Experiment 2
Inst i tute (1990), Versión 6, 4^ Edition (SAS Inst. INC., Cary, NC). Means of
digestibility coefficients were compared us ing a protected t-test.
4. Results
In Table 4, DMI of diets and soft faeces (g DM/day) and total (N) and
microbial (Nm) nitrogen concentrat ion and the ratio Nm/N of soft faeces and
hard faeces are presented. A significant effect of diet was observed for feed
and soft faeces intake. An average feed DMI valué of 140 g DM/day was
obtained for AH, BG and SM. However, animáis fed the WB-based diet
reached a 15% lower valué. The highest SF intake was obtained for the SM-
based diet (30 g DM/day) while the lowest was reached in animáis fed the
BG and WB-based diet (20 g DM/day, a s average). No effect of diet was
detected on the nitrogen soft faeces composition result ing in an average
valué of 27.9 and 18.9 g /kg DM for N and Nm, respectively, and 0.57 for
Nm/N. For nitrogen hard faeces composition significant differences among
diets were detected for the ratio Nm/N. The highest ratio was found in the
barley grain and sunflower meal based diets and the lowest in the wheat
b ran diet. However, no effect of diet was observed in the N and Nm of the
ha rd faeces (18.6 and 8.4 g /kg DM, a s average, respectively).
The amiino acid composition of soft faeces from an imáis fed with alfalfa
hay, barley grain, wheat b ran and sunflower meal based diets (g/16gN) and
the mean valúes of the in vitro amino acid digestibility of the soft faeces are
presented in Table 5. Significant differences among diets on the amino acid
composition of the soft faces were detected for lysine, methionine, threonine,
alanine, glutamic acid and tyrosine. The highest in vitro digestibility
coefficient was obtained for tyrosine and cystine while the lowest was
determined for glycine and proline.
74
Chapter III: Experiment 2
Table 4. DMI of diets and soft faeces and total and microbial nitrogen contení of soft and hará faeces of alfalfa hay (AH), harley grain (BG), ivheat bran (WB) and sunflower meal (SM) based diets.
ítem AH BG WB SM P , L
Feed Intake, g DM / day
Soft Faeces Intake, g DM / day
Soft Faeces composition, g/kg DM
Total Nitrogen content (N)
Microbial Nitrogen content (Nm)
Ratio Nm / N
Hard Faeces composition, g/kg DM
Total Nitrogen content (N)
Microbial Nitrogen content (Nm)
Ratio Nm / N
»• ^ Means in the same row with different letters differ (P < 0.05)
133.1 ab
24.6 ab
27.2
18.9
0.592
19.0
8.19
0.430 ab
146.5 a
19.1 b
32.1
22.3
0.688
18.8
9.27
0.488 a
119.9 b
21.4 b
26.5
16.6
0.624
19.0
7.62
0.404 b
138.9 ab
29.9 a
26.0
17.8
0.687
17.6
8.42
0.482 a
*
**
NS
NS
NS
NS
NS
*
6.10
1.74
1.84
1.67
0.025
0.85
0.60
0.024
75
Chapter III: Experiment 2
Table 5. Amino acid (AA) composition ofsoft fasces (g/ 16gN) of alfalfa hay (AH), barley grain (BG), ivheat bran (WB), and sunflower meal (SM) and mean valué ofthe in vitro amino acid digestibility ofsoftfaeces
AH BG WB SM SEM In vitro s.e. (n = 5) Digestibility (n = 4)
Essential AA Arginine Cystine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Valine Non essential AA Alanine Aspartic acid Glutamic acid Glycine Proline Serine Tyrosine
4.05 2.39 1.46 4.01 5.34
4.94 b 1.40 b 2.77
5.31 b 5.71
4.59 b 8.98
10.0 b 4.48 3.84 4.34 2.79
4.14 2.39 1.48 4.57 6.03
5.67 a 1.67 a 3.15
6.07 a 6.27
5.44 a 10.12 11.9 a 4.87 3.80 4.61 3.16
4.18 2.33 1.47 4.09 5.68
4.79 b 1.44 b 2.94
5.94 a 5.99
5.06 ab 9.56
10.3 b 4.66 3.93 4.50 3.03
4.54 2.46 1.52 4.27 5.83
5.02 b 1.56 ab
3.08 5.51 ab
5.99
5.01 ab 9.85
11.0 ab 5.04 4.22 4.39 2.67
NS NS
NS
NS
NS *
*
NS *
NS
*
NS *
NS
NS
NS **
0.177 0.101 0.049 0.137 0.212 0.193 0.062 0.107 0.189 0.156
0.155 0.335 0.342 0.148 0.213 0.150 0.090
0.607 0.770 0.614 0.705 0.698 0.726 0.750 0.705 0.713 0.698
0.641 0.728 0.703 0.549 0.576 0.647 0.784
0.048 0.010 0.045 0.041 0.040 0.041 0.032 0.035 0.038 0.042
0.051 0.035 0.038 0.050 0.056 0.042 0.023
a. b Means in the same row with different le t ters differ (P < 0.05)
76
Chapter III: Experiment 2
Apparent faecal and ileal digestibility of crude protein (CP) a n d amino
acids of the experimental diets (AFDdiet and AIDdiet, respectively) are
presented in Table 6. There were not significant differences in the AFDdiet of
CP among diets (0.751, as average). However, the BG and WB based diets
showed the highest AIDdiet of CP whereas the SM had the lowest valué. The
AFDdiet of the amino acids were usual ly highest in the BG diet and lowest in
the WB and SM diets. Only the AFDdiet of glycine was higher for SM t h a n for
BG diet (0.757 vs. 0.674, respectively). For the AH and WB diets the highest
AFDdiet was obtained for proline and glutamic acid, respectively, and for BG
and SM diets the AFDdiet of m.ethionine was found a s the highest valué. The
lowest AFDdiet was obtained for cystine in all the experimental diets. There
were not significant differences among diets in the AIDdiet of alanine, aspar t ic
acid and glutamic acid. As in the faecal balance, the AIDdiet of the amino
acids were usual ly higher in the BG diet and lower in the WB and SM diets.
Only the AIDdiet of arginine and glycine was higher for the SM t h a n for the
BG diet (0.760 vs. 0.684 and 0.552 vs. 0 .493, respectively). The highest and
the lowest AIDdiet valué were obtained, in all diets, for methionine and
cystine, respectively.
Apparent faecal digestibility of CP and amino acids of the feedstuffs
(AFD) are presented in Table 7. The SM showed the highest AFD of CP while
the WB had the lowest valúes (0.852 vs. 0 .551 , respectively). The AFD of the
amino acids were usual ly highest in SM and lowest in WB. Only the AFD of
histidine and glutamic acid was lower for AH t h a n for WB (0.532 vs. 0.679,
0.607 vs. 0.754, respectively). The highest AFD was obtained for
phenylalanine, proline, glutamic acid and methionine for the AH, BG, WB
and SM feedstuff, respectively, whereas the lowest AFD was obtained for
serine, threonine, cystine and tyrosine for the AH, BG, WB and SM,
respectively.
77
Chapter III: Experiment 2
Table 6. Apparent Faecal and Real
ítem
CP
Essential AA
Arginine
Cystine
Histidine
Isoleucine
Leucine
Lysine
Methionine
Phenylalanine
Threonine
Valine
Non essential AA
Alanine
Aspartic acid
Glutamic acid
Glycine
Proline
Serine
Tyrosine
Digestibility of crude protein
Apparent Faecal Digestibility (AFDdkt)
Alfalfa Hay
0.760
0.744 b
0.345 c
0.886 b
0.840 b
0.862 b
0.838 b
0.892 b
0.873 a
0.713 be
0.817 a
0.767 a
0,804 a
0.867 b
0.644 b
0.896 a
0.763 b
0.838 b
Barley Grain
0.751
0.820 a
0.658 a
0.906 a
0.865 a
0.881 a
0.874 a
0.917 a
0.890 a
0.764 a
0.840 a
0.765 a
0.789 a
0.902 a
0.674 b
0.893 a
0.816 a
0.875 a
Wheat Bran
0.739
0.742 b
0.261 d
0.861 c
0.803 c
0.837 c
0.841 b
0.850 c
0.842 b
0.740 ab
0.776 b
0.739 b
0.767 b
0.867 b
0.591c
0.845 c
0.772 b
0.844 b
Sunflower Meal
0.766
0.837 a
0.590 b
0.864 c
0.817 c
0.810 d
0.789 c
0.879 b
0.854 b
0.694 c
0.770 b
0.745 ab
0.799 ab
0.871 b
0.757 ab
0.870 b
0.781 ab
0.761 c
P
NS
***
***
***
***
***
***
***
* A
•k
•k-k-k
•k
*
***
•k-kk
•krk-k
•k
•k-kk
(CP) and
SEM (n = 5)
0.0072
0.0057
0.0122
O.0029
0.0048
0.0040
0.0051
0.0039
0.0046
0.0088
0.0056
0.0060
0.0054
0.0027
0.0104
0.0035
0.0086
0.0052
amino acids (AA) of the experimental diets
Apparent Ileal Di:
Alfalfa Hay
0.635 be
0.656 b
0.291 b
0.814 a
0.742 b
0.798 a
0.778 b
0.848 ab
0.836 a
0.600 a
0.720 a
0.702
0.697
0.757
0.497 b
0.809 a
0.579 b
0.801a
Barley Grain
0.654 ab
0.684 b
0.426 a
0.802 a b
0.776 a
0.809 a
0.806 a
0.877 a
0.819 b
0.608 a
0.739 a
0.694
0.688
0.793
0.493 b
0.822 a
0.641 a
0.765 a
gestibility (AIDdiet)
Wheat Bran
0.666 a
0.667 b
0.190 c
0.773 b
0.728 b
0.780 b
0.787 b
0.817 c
0.800 c
0.623 a
0.718 a
0.658
0.665
0.768
0.468 c
0.785 b
0.605 ab
0.770 a
Sunflower Meal
0.615 c
0.760 a
0.381 a
0.766 b
0.737 b
0.746 c
0.720 c
0.834 be
0.804 be
0.541 b
0.672 b
0.693
0.706
0.768
0.562 a
0.717 c
0.555 b
0.724 b
P
**
**
**
*
*
**
A * *
*
**
*
*
NS
NS
NS k-k-k
•k-k-k
k
k
SEM (n=5)
0.0072
0.0092
0.0170
0.0090
0.0066
0.0048
0.0036
0.0076
0.0039
0.0093
0.0089
0.0102
0.0084
0.0059
0.0059
0.0046
0.0106
0.0102 a, b, c,íi Means in the same row with different letters differ (P < 0.05)
78
Chapter III; Experiment 2
Tab le 7. Apparent Faecal Digestibüity (AA) of the feedstuffs
í tem
CP
ESvSential AA
Arginine
Cystine
Histidine
Isoleucine
Leu cine
Lysine
Methionine
Phenylalanine
Threonine
Valine
Non essential AA
Alanine
Aspartic acid
Glutamic acid
Glycine
Proline
Serine
Tyrosine
of crude protein (CP) and
Apparent Faecal Digestibüity (AFD)
Alfalfa Hay
0 . 6 9 1 b
0.586 c
0 .501 c
0.632 d
0.668 b
0.719 b
0.550 be
0.696 b
0 .763 b
0.503 b
0.564 b
0.680 b
0.698 b
0.607 d
0.651 b
0.626 b
0.466 c
0.542 b
Barley Grain
0.683 b
0.771 b
0.699 b
0.779 b
0.682 b
0.732 b
0.597 b
0.735 b
0.812 b
0.533 b
0 . 5 7 1 b
0.608 be
0.576 c
0.830 b
0.592 c
0.852 a
0.621 b
0.705 a
Wheat Bran
0 . 5 6 1 c
0.654 e
0.354 d
0.679 c
0 .468 c
0.571 c
0.480 c
0.602 c
0 .668 e
0.525 b
0.394 c
0.607 c
0.451 d
0.754 c
0.508 d
0.602 b
0.514 c
0.568 b
Sunflower Meal
0 .852 a
0.884 a
0 .778 a
0.882 a
0 .851 a
0.836 a
0.796 a
0 .921 a
0.894 a
0 .771 a
0.817 a
0.810 a
0.864 a
0.892 a
0 .845 a
0.916 a
0.848 a
0.750 a
amino
P
•k-k-k
•k-k-k
***
* • * • *
•k-kk
•k'k-k
•k-k
•k-k-k
•k'k-k
k-k
k'k-k
•k-k
•k-k-k
***
•k-kk
•k-k-k
•k-k
-k'k-k
acids
SEM (n = 5)
0 .0173
0.0152
0 .0153
0.0124
0.0241
0.0130
0.0255
0.0134
0 .0141
0.0314
0 .0155
0.0201
0.0171
0.0062
0.0156
0.0191
0.0256
0.0141
a, b, c, d Means in the same row with different letters differ (P < 0.05)
79
Chapter III: Experiment 2
Apparent and t rue ileal digestibility of CP and amino acids of the
feedstuffs are presentad in Table 8 (AID and TID). The highest and the lowest
AID were obtained for the SM and WB feedstuff, respectively. For the TID of
CP, the SM reached the highest valué, the BG showed a n intermedíate valué
and the AH and the WB feedstuff had the lowest valúes. Similar to the ileal
CP digestibility, the AID and the TID of the amino acids vi^ere usual ly highest
in the SM feedstuff and lowest in the WB. The highest AID was obtained for
phenylalanine and proline for the AH and the BG feedstuff, respectively, and
for methionine for the WB and the SM feedstuff. The lowest AID was
obtained for serine and threonine for the AH and the BG feedstuff,
respectively, and for cystine in the WB and the SM feedstuff. In te rms of
TFD, the best-digested amino acid was phenylalanine, proline, methionine
and glutamic acid for the AH, BG, WB and SM feedstuff, respectively.
However, the 'worst digested amino acid expressed in the same uni t -was
serine, glycine, aspart ic acid and glycine for the AH, BG, WB and SM
feedstuff, respectively.
80
Chapter III: Experiment 2
Table 8. Apparent and True Real Digestibüity of crudeprotein
í tem
CP
Essential AA
Arginine
Cystine
Histidine
Isoleucine
Leucine
Lysine
Methionine
Phenylalanine
Threonine
Valine
Non essential AA
Alanine
Aspartic acid
Glutamic acid
Glycine
Proline
Serine
Tyrosine
Apparent Ileal Digestibüity (AID)
Alfalfa Hay
0.591 b
0.736 b
0 .601 ab
0.613 b
0.649 b
0.745 b
0.594 b
0.744 be
0.812 b
0.562 b
0.659 b
0.685 b
0.669 b
0.594 c
0.623 b
0.558 b
0.421 c
0.707 b
Bar ley Grain
0.619 b
0.662 be
0.538 b
0.582 b
0.705 b
0.719 b
0.616 b
0.803 b
0.739 c
0.457 c
0.641 b
0.604 c
0.589 c
0.785 b
0.464 c
0.856 a
0.583 b
0.573 be
Wheat Bran
0.529 c
0.623 c
0.216 c
0.501 b
0.435 c
0.551 c
0.472 c
0.690 c
0.648 d
0.439 c
0.526 c
0.465 d
0.326 d
0.678 c
0.449 c
0.570 b
0.371 c
0.499 c
Sunflower Meal
0.807
0.888
0.661
0.856
0.887
0.863
0.845
0.938
0.915
0.738
0.834
0.830
0.850
0.908
0.730
0.791
0.806
0.837
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
P
•k
•k*
•k-k-k
**
•k-kk
**
kkk
kkk
k-k
kk
k
**
***
***
**
***
**
*
(CP) and
SEM (n = 5)
0.0146
0.0238
0.0265
0.O372
0.O194
0.0218
0.0244
0.O176
0.O152
0.0260
0.0253
0.O205
0.OO74
0.0312
0.0276
0.O255
0.O398
0.O392
amino acids (AA) of the feedstuffs
True Ileal Digestibüity (TID)
Alfalfa Hay
0.742 b
0.855 b
0.831 a
0.714 b
0.770 c
0.839 b
0 .717 c
0.842 be
0 .878 b
0.752 b
0 .793 b
0.760 b
0.776 b
0.797 be
0 .763 a
0.801 b
0 .683 c
0 .781 b
Barley Grain
0.796 ab
0.783 be
0.769 a
0.697 b
0.862 b
0.833 b
0.792 b
0.898 b
0.808 e
0.725 b
0.802 b
0.722 b
0.778 b
0.854 b
0.669 b
0.923 a
0.828 b
0.684 be
Wheat Bran
0.698 b
0.751 e
0.556 b
0.608 b
0.619 d
0.686 e
0.652 d
0.791 e
0.739 d
0.744 b
0.729 b
0.574 c
0.554 e
0.749 c
0.637 b
0.705 c
0.584 d
0.635 e
Sunflower Meal
0.861 a
0.929 a
0.815 a
0.899 a
0.949 a
0.919 a
0.915 a
0.967 a
0.949 a
0.846 a
0 . 9 1 1 a
0.864 a
0.906 a
0.982 a
0.790 a
0.893 a
0.942 a
0.905 a
P
*
**
***
**
itírA
•k^k-k
^•k-k
**
***
**
*
***
***
***
i *
**
***
**
SEM ( n = 5 )
O.0321
0.0189
0 .0208
0.0290
0.0157
O.0159
0.0115
0.0121
0.0130
0.0106
0.0182
0.0204
0.0078
0.0124
0.0152
0 .0183
0.0185
0.0245
a, b, c, d Means in the same row with different letters differ (P < 0.05)
Chapter III: Experiment 2
5. Discussion
5.1. Apparent faecal versus ileal balance
Most of the caecal bacteria sho'w proteolytic activity (Emaldi et
al.,1979) allowing to deaminate the ileal protein and to use the carbón
skeleton as an energy source for microbial growth and to produce ammonia
from amino nitrogen. Ammonia can be absorbed through the caecal wall and
excretad in the urina as urea. However, a positive balance of protein (0.568
g/day, as average, representing 18% of ileal flow) can be deduced when
comparing average ileal flow of protein (3.17 g/day, as average) and the total
protein excretad in soft and hard faecas (3.74 g/day, as average). This
balance might be explained since a proportion of uraa produced in the livar
from amino acids catabolism is racyclad into the caecum through the blood.
Forsythe and Parker (1985) estimated that 25% of the total caecal ammonia,
which is the main nitrogen source for the microbial growth, comes from the
hydrolysis of blood urea.
Otherwise, all feedstuffs showed a higher CP digestibility at faecal than
ileal level (see Tables 7 and 8), as soft faeces recycled as average a 36% of the
total protein excreted which was mainly of bacterial origin (67%, as average,
see Table 4). Because of this recycling, the magnitude of difference between
ileal and hard faeces crude protein flow was small in our study (9.5%, as
average).
Making similar balances for the most limiting amino acids (using the
average valúes of the four feedstuffs), an enrichment in lysine (0.072 g/day;
63% of the ileal flow), methionine (0.026 g/day; 95% of the ileal flow) and
threonine (0.059 g/day; 40% of the ileal flow) was observed when comparing
total excretion in hard faeces and soft faeces with respect to ileal apparent
flow. An important microbial net synthesis for methionine and lysine has
82
Chapter III: Experiment 2
been also reported in pigs (Sauer et al, 1982 and 1991). Fur thermore , the
enr ichment in these essential amino acids tended to be higher in diets based
on AH, BG and SM t h a n in those based in WB. This resul t is parallel to the
lower proportion of microbial nitrogen on total nitrogen excreted in ha rd
faeces observad in diet based in WB with respect to the other diets (Table 4).
The bacterial activity in the caecum resul ted therefore in subs tant ia l
changas in the amino acid composition of caecal crude protain. In the same
way, when normal rabbi ts were compared with germ-free rabbi ts , an
increased contant of five essential amino acids (lysine, methionina, valine,
leucine and isoleucine) was found reflecting the importance of the caecal
microbial protein synthesis (Yoshida et al., 1971). As a consequence,
a l though a round one third of the total excretion of lysine and methionine
was recycled with soft faeces, ileal digestibilities of lysine and methionine
were higher (by respectively 2.8 and 5.5 percentage un i t s , a s average of the
four diets) t h a n at faecal level, which is the opposite t rend to tha t found for
crude protein. A different pat tern was observed for threonine digestibility,
which tended to be lower at ileal t h a n at faecal level, a l though its
concentrat ion in the microbial protein is also relevant. Threonine
concentrat ion is particularly high in ileal endogenous losses (5.53 g/16gN vs.
3.75 and 0.96 g/15gN for lysine and methionine, respectively; García et al.,
2004), which implies highest ileal flow of threonine (0.142 g /day vs. 0.118
and 0.028 g /day for lysine and methionine, respectively). Endogenous ileal
threonine might be highly digestible at the caecum, which would explain the
negativa balance found between ileal and faecal level. Threonine h a s also
been shown as one of the amino acids which usual ly d isappear at a large
extent in the large intestine of pigs, leading to higher faecal t h a n ileal
digestibility valúes (Sauer et al, 1982; Mosenthin et al, 1994).
Faecal c rude protein digestibility is the mos t available un i t for the
nitrogen evaluation of feedstuffs. The lack of da t a abou t the AID of crude
protein and amino acids, in rabbi ts , makes difficult the comparison between
83
Chapter III: Experiment 2
the different digestibility coefficients obtained for each feedstuff in the
present work and the existing literatura data. When the protein AFD of the
different feedstuffs (Table 7) was compared with an average valué compiled
by Villamide et al. (1998), similar digestibility coefficients for AH (0.69 vs.
0.54), BG (0.68 vs. 0.67) and SM (0.85 vs. 0.80) were obtained. However,
substantially lower valué for WB (0.56 vs. 0.73) was obtained in the present
work. The protein digestibility valué of different wheat brans obtained from
several works in rabbits and reviewed by Blas et al. (2000) ranged from 0.65
to 0.88. A possible explanation for this wide variation was found in
differences on the endosperm/bran ratio present in this wheat by-product as
a consequence of its manufacturing procedure. In spite of the few available
AID valúes in rabbits, wide variations have been observed on the ratio
AID/AFD among different research works (Gidenne, 1992; Merino and
Carabaño, 1992; Motta-Ferreira et al, 1996; García et al, 1996) based on
similar diets formulation and feedstuff ingredients. In these works, from 0.59
to 1.13 of the faecal digested protein in rabbits was digested before the
caecum. This wide variation emphasizes the importance of evaluating the
ileal digestibility of feedstuffs in rabbits, despite the methodological
complexity that is implicit in this technique.
5.2. Apparent versus trae ileal digestibility
As it was expected, the TID of CP and amino acids of the feedstuffs
were higher than their corresponding AID valúes due to the large
endogenous losses.
The largest differences between TID and AID were found in all
feedstuffs for cystine, threonine and proline (24, 22 and 21 percentage units
of difference, as average, respectively) indicating their relatively high
proportion in the endogenous protein. Depending on the feedstuff studied
larges differences were also observed for aspartic acid, glutamic acid, glycine
and serine. The lowest differences between TID and AID were obtained for
84
Chapter III: Experiment 2
phenylalanine and methionine (6.5 and 8.1 percentage un i t s of difference, as
average, respectively). In pigs, it h a s been observad tha t differences between
TID and AID are of small magni tude in high-protein feedstuffs b u t m u c h
more pronounced in low-protein feedstuffs (Mosenthin et al., 2000). Similar
resul ts were observed in the present work. The effect of t ransforming the
appa ren t into the t rue digestibility valúes were of small magni tude in the SM
(7.1 percentage un i t s of difference between TID and AID, as average for all
the amino acids), a s a consequence of the lower proportion of endogenous to
exogenous recoveries of protein and amino acid in the ileal digesta. This
effect was m u c h more pronounced in the low-protein feedstuffs studied (15
and 17 percentage un i t s of difference between TID and AID, as average for all
the amino acids for BG and WB, respectively).
Due to the lack of da ta in rabbi ts , FEDNA tables rules for feed
formulation (2003), INRA tables (2002) and CVB tables (2002) were used to
compare the s tandardised ileal digestibility of pigs (SID) with the TID of
rabbi ts obtained in the present work for CP and the mos t limiting amino
acids. The SID are the preferred approach for feed formulation in pigs a s it is
not affected by the level of intake or amino acid content of the experimental
diet allowing accurate comparisons between different feedstuffs (Mariscal
Landin, 1997). As average, a n 1 1 % higher TID t h a n SID valué have been
obtained from this comparison. The highest differences between these two
un i t s were found for AH feedstuff (26% higher TID t h a n SID valúes, as
average). For the AH feedstuff, tables show a n average SID valué of 0 .523,
0.582, 0.742 and 0.634 for CP, lysine, methionine and threonine,
respectively. The higher TID than SID valué of the AH could indícate a better
util isation of th is feedstuff by rabbi ts t h a n by pigs because of the
caecotrophy recycled of CP and amino acids. But, par t of this difference may
also be a t t r ibuted to differences in the chemical composition of the AH
studied and to the non-specific endogenous losses determinat ion. In th is
way, Boisen and Moughan (1996) reported tha t the non-specific endogenous
recoveries differ depending on the method used for their est imation (protein-
85
Chapter III: Experiment 2
free diets or protein-containing diets) but also depending on dietary factors
such as the contení of fibre or antinutritional factors. In this way, the alfalfa
SID valué for pigs could be underestimated as the high fibre content of AH
may led to a higher endogenous recoveries than those estimated with a
common low fibre-containing diet. Moreover, it should be mentioned that as
AH is not common feedstuffs for pig nutrition the available data to establish
the SID valué are fewer than for the BG, WB and SM feedstuffs.
For practical application on feed formulation more important than the
absolute valué of the digestibility coefficient, independently of the unit used,
are the relativa valué established am.ong different feedstuffs. In the present
work, the relative valué of AH, BG and WB respect to SM changed for the CP
and for the most limiting essential amino acids depending on the digestibility
unit used (see Figure 1 to 4). This effect was more pronounced for threonine
than for CP, lysine or methionine. In this sense it is known that threonine
content is high in the endogenous losses.
Figure 1. Relative valué of alfalfa hay, barley grain and tuheat bran respect to the sunflower medí (valué = 100) for the apparent and trae, faecal and ileal digestibility of crude protein
100
90
80 . /
70
60
50
. /
D Alfalfa Hay H Barley Grain S Wheat Bran
^
\¿L VL
m
Apparent Faecal Apparent Ileal Trae Ileal
86
Chapter III: Experiment 2
Figure 2. Relative valué of alfalfa hay, barley grain and wheat bran résped to the sunfloiver meál (valué = 100) for the apparent and true, faecal and ileal digestibüity of lysine
D Alfalfa Hay ü Barley Grain
100/"
Apparent Faecal Apparent Ileal
El Wheat Bran
True Ileal
Figure 3. Relative valué of alfalfa hay, barley grain and wheat bran respect to the sunflower meál (valué = 100) for the apparent and trae, faecal and ileal digestibüity of methionine
n Alfalfa Hay lü Barley Grain n Wheat Bran
lOOi
90
80
70-
60
.4
50
^ ^
y—y' Apparent Faecal Apparent Ileal True Ileal
87
Chapter III: Experiment 2
Figure 4. Relative valué of alfalfa hay, barley grain and wheat bran respect to the sunfloiuer meal (valué = 100) for the apparent and true, faecal and ileal digestibüity ofthreonine
D Alfalfa Hay • Barley Grain
100-r
90-
80-
70-
60
50
yZ
/ " a
H Wheat Bran
Apparent Faecal Apparent Ileal íí^IZ
True Ileal
In conclusión, AID and AFD lead to an underestimation or an
overestimation, respectively, of TID of nitrogen and amino acids contení of
the feedstuffs due to the high nitrogen endogenous losses impact and to the
important caecal bacteria activity. The use of TID is recommended for a more
precise evaluation of nitrogen and amino acids content of the feedstuffs. By
using TID a better approach to the animal requirements and less nitrogen
excretion to the environment would be reached. However, at this stage
insufficient data is available and more feedstuffs should be evaluated to
achieve its implementation in feed formulation.
6. Acknowledgements
The authors are grateful to A. Espinosa for her technical assistance in
the laboratoiy and in the ileal collection and to Trouw Nutrition Spain for the
Chapter III: Experiment 2
analyses of the amino acids. Financial suppor t was provided by CYCIT,
ProjectAGF 199-1109.
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Chapter III: Experiment 2
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spectrophotometry. Influence of sample treatment and preservation.
Reproduction Nutñtion Development 25: 1037-1046.
Van Soest, J. P., Robertson, J.B. and Lewis, B.A. 1991. Methods for dietary
fibre, neutral detergent fibre and non-starch polysaccharides in
relation to animal nutrition. Journal ofDairy Science 74: 3583-3597.
Villamide, M.J., Maertens, L., De Blas, J.C. and Pérez, J.M. 1998. Peed
evaluation. In The Nutrition of the Rabbit (ed. J.C. de Blas and J.
Wiseman), pp. 89-101. Commonwealth Agricultural Bureau,
Wallingford, UK.
Yoshida, T., Pleasants, J.R., Reddy, B. and Wostm.an, B.S. 1971. Amino acid
composition of caecal contents and faeces in germ. free and
conventional rabbits. Journal of Nutrition 101: 1423-1429.
92
Chapter III: Experiment 2
Zebrowska, T. 1973. Digestión and absorption of n i t rogenous compounds in
the large intestina of pigs. Roczniki Nauk Rolnizych B95: 85-90.
Zinn, R. and Owens, F. 1980. Rapid procedure for quantifying nucleic acid
contení of digesta. In Protein Requirements for Cattle. Protein
Symposium, Oklahoma State Univ., Stillwater. pp 26-30.
93
CHAPTER IV: GENERAL DISCUSSION AND CONCLUSIÓN
94
Chapter IV: General Discussion and Conclusión
1. General discussion
The objectives outlined at the beginning of this doctoral thesis have
been finally reached through the experimental triáis developed. The bases for
new nitrogen feed evaluation system establishment in rabbit have been
defined. The nitrogen endogenous losses of the rabbits have been determined
by tvi o different methods (casein-based or protein-free diet). Furthermore,
four different feedstuffs commonly used in rabbit feed formulation have been
evaluated according to the nev ^ nitrogen evaluation system proposed.
Rabbits fed the protein-free diet presented a feed intake below the
energy requirements of adult does but a soft faeces intake abnormally higher
according to the animáis feed intake. These facts suggest that this
methodology (commonly used in pigs for the basal endogenous losses
determination) could have limited practical application in rabbits. In this
sense, the use of a casein-based diet (highly digestible containing-protein
diets, with an assumed 100% digestibility at ileum level) resulted in a better
approach for the nitrogen endogenous losses estimations than the use of
protein-free diets. Accordingly, the true ileal digestibility coefficient of crude
protein and amino acids of alfalfa hay, barley, wheat bran and sunflower
meal were determined using the nitrogen endogenous losses obtained by the
casein-based diet.
Nitrogen and amino acid digestión of soft faeces was high (79%).
However, caecotrophy had a signiñcant effect on the nitrogen endogenous
losses leading to a higher ileal flow of the most limiting amino acids. For this
reason, the undigested nitrogen residues coming from soft faeces at ileum
level were subtracted from the total nitrogen found in the ileal digesta.
The true ileal digestibility coefficients have resulted of relevant
importance in rabbits due to their high nitrogen endogenous losses
95
Chapter IV: General Discussion and Conclusión
estimations, which were between twice and three times higher than in pigs.
For practical application on feed formulation more important than the
abvsolute valué of the digestibility coefficient, independently of the unit used,
is the relative valué established among different feedstuffs. However, in this
doctoral thesis, it has been demonstrated that this relative valué among
feedstuffs changed for the crude protein and for the most limiting essential
amino acids depending on the digestibility unit used.
From these results it can be concluded that apparent ileal and faecal
digestibility lead to an underestimation or an overestimation, respectively, of
the true ileal digestibility of nitrogen and amino acids content of the
feedstuffs due to the high nitrogen endogenous losses impact and to the
important caecal bacteria activity of the rabbits. Therefore, the use of true
ileal digestibility is recommended for a more precise evaluation of nitrogen
and amino acids content of the feedstuffs. By using true ileal digestibility a
better approach to the animal requirements and less nitrogen excretion to
the environment would be reached. However, at this stage insufficient data is
available and more feedstuffs should be evaluated to achieve its
implementation in feed formulation.
2. Conclusión
1. Feeding rabbits with a casein-based diet results in a better method
for the nitrogen endogenous losses estimations than the use of a protein-free
diet.
2. The nitrogen endogenous losses estimated feeding animáis with a
casein-based diet are related to the total DM intake (feed + soft faeces).
3. The nitrogen and amino acid content of the soft faeces results in a
highly digestible source of nitrogen with an estimated digestibility of 0.79.
96
Chapter IV: General Discussion and Conclusión
4. Caecotrophy leads to changes in the amino acids composition of the
nitrogen endogenous losses. Within the most limiting essential amino acids,
caecotrophy mainly affects lysine and threonine content of the ileal
endogenous losses.
5. The nitrogen endogenous losses in rabbits are significantly higher
than the endogenous losses determined in pigs. This could be mainly
explained by the higher fibre content of the rabbit diets.
6. The nitrogen endogenous losses in rabbits practicing caecotrophy
have the following amino acid composition (g/16gN): arginine 4.63; cystine
3.11; histidine 1.53; isoleucine 3.72; leucine 4.77; lysine 3.75; methionine
0.96; phenylalanine 2.09; threonine 5.53; valine 5.54; alanine 3.55; aspartic
acid 7.53; glutamic acid 12.66; glycine 5.18; proline 5.41; serine 6.45;
tyrosine 2.07. Threonine is one of the highest contained limiting amino acid
in the nitrogen endogenous losses of the rabbit.
7. The bacterial activity in the caecum results in substantial changes
in the amino acid composition of its crude protein content leading to a
variable amino acid enrichment in the excreta depending on the feedstuff.
This fact leads to variable differences between the apparent faecal and ileal
digestibility depending on the effect of each feedstuff on the microbial protein
synthesis.
8. Differences between the apparent and true ileal digestibility are of
sraall magnitude in the high-protein feedstuffs but much more pronounced
in the low-protein feedstuffs. This is consequence of the relative proportion
of the endogenous to exogenous recoveries of protein and amino acid in the
ileal digesta.
97
Chapter IV: General Discussion and Conclusión
9. Due to the different effect of feedstuffs on the caecal microbial
activity and the nitrogen endogenous losses, the relative valué established
among them change depending on the digestibility unit used and the amino
acid considered. For this reason, true ileal digestibility is consider as the
most precise digestibility unit to define the nutritive valué of the nitrogen
feedstuff contení in rabbits.
98
ANNEX I
Annexl
DEPARTAMENTO DE PRODUCCIÓN ANIMAL
ESCUELA TÉCNICA SUPERIOR DE INGENIEROS AGRÓNOMOS
El amplio resumen en lengua castellana presentado a continuación ha sido
realizado como uno de los requisitos necesarios para la presentación de la
Tesis Doctoral en inglés y poder optar así al título de Doctorado Europeo.
El Doctorando
Ana I s a b e l G a r c í a Ruiz
Ingeniero Agrónomo
V°B°
Los d i r ec to res de la Tes i s
R o s a M^ C a r a b a ñ o Luengo
Dr. Ingeniero Agrónomo
i 1 ñ
C a r l o s de B l a s B e o r l e g u i
Dr. Ingeniero Agrónomo
II
Annexl
1. Objetivo de la tesis
En nutrición de conejos, son muy pocos los trabajos dirigidos a
definir la unidad que mejor estime el valor nitrogenado de los alimentos más
frecuentemente empleados en los piensos de conejos. Hasta el momento, la
digestibilidad fecal aparente es la unidad más precisa que se emplea con
este fin. Sin embargo, en otras especies monogástricas (porcino y aves), el
concepto de unidad de valoración nitrogenada ha ido evolucionando
progresivamente a lo largo del tiempo. En el caso del porcino, por ejemplo, se
pasó de medir la digestibilidad a nivel fecal (mediante recogida natural de
heces) a medirla a nivel ileal (recogida de la digesta al final del intestino
delgado). Posteriormente, y ya siempre a nivel ileal se pasó del concepto de
digestibilidad aparente al de digestibilidad verdadera y más adelante al de
digestibilidad real proporcionando, en cada paso, una mejor caracterización
del valor de las proteínas de los alimentos. En el caso del conejo, la correcta
definición de la unidad de valoración nitrogenada que mejor caracterice el
valor de los alimentos permitiría: I) incrementar la precisión en la
formulación práctica de piensos ajustándose así mejor a las necesidades de
los animales y, II) disminuir la excreción de nitrógeno al medio y por tanto la
contaminación ambiental.
Por todo ello, el objetivo general de esta tesis doctoral ha sido el
tratar de establecer las bases para un nuevo método de valoración de
alimentos que permita evaluar con mayor precisión el valor nitrogenado de
piensos y materias primas en conejos.
Los objetivos particulares fueron: I) el estudio de las pérdidas
nitrogenadas de origen endógeno en el conejo y II) la valoración de diferentes
materias primas frecuentemente empleadas en piensos de conejos a través
de sus coeficientes de digestibilidad fecal aparente e ileal tanto aparente
como verdadero.
III
Annexl
Para ello se realizaron dos experimentos. En el primer
experimento, se estudió el efecto del tipo de alimentación (sin nitrógeno o
con nitrógeno) y de la cecotrofia, sobre la estimación de la proteína y los
aminoácidos de origen endógeno en el conejo. En el segundo experimento se
evaluaron cuatro materias primas diferentes a partir de sus coeficientes de
digestibilidad fecal aparente, ileal aparente e ileal verdadera de la pro teína y
los aminoácidos. Para la determinación de los coeficientes de digestibilidad
verdaderos se utilizaron los resultados obtenidos en el primer trabajo. Las
materias primas escogidas fueron: harina de girasol, cebada, heno de alfalfa
y salvado de trigo. La selección de las materias primas se hizo en base a su
importancia en la form.ulación práctica de piensos compuestos de conejos.
2. Revisión Bibliográfíca
2.1. Proteína bruta y aminoácidos totales
La pro teína bruta es la unidad más comúnmente empleada en la
práctica para expresar las necesidades y el valor nutritivo de los alimentos
en el conejo. La principal ventaja de esta unidad es la cantidad de datos e
información que hay disponible sobre necesidades y sobre composición de
alimentos. Sin embargo, el uso de esta unidad conduce a piensos con un
exceso de proteína respecto a las necesidades. Recientemente, Maertens et
al. (1997) observó que se podía reducir el contenido en pro teína bruta del
pienso desde un 17.0 hasta un 15.7 % sin perjudicar los rendimientos de
aniraales en cebo, suministrándoles lisina, aminoácidos azufrados y treonina
por encima de las necesidades. Además, al reducir el nivel de proteína en el
pienso se observó que se reducía en un 11% la excreción de nitrógeno fecal.
Del mismo modo, Trocino et al. (2000) confirmaron la estrecha relación
existente entre el nivel de proteína de los piensos y la excreción de nitrógeno
en heces que es especialmente importante en la fase final del cebo. Estos
resultados indican por tanto, que basar la formulación de piensos en la
composición de aminoácidos de las materias primas en lugar de en su
IV
Annex I
contenido en proteína bruta podría conducir a una mejor evaluación del
valor nitrogenado del mismo.
Poco a poco, la inform.ación sobre el contenido total en
aminoácidos de los alimentos ha ido aumentando. En el trabajo realizado
por Villamide et al. (1998) se recogió la composición en lisina, aminoácidos
azufrados y treonina de los alimentos que con más frecuencia se emplean en
la formulación de piensos compuestos de conejos (cereales, subproductos de
cereales, leguminosas, harinas de semillas oleaginosas, algunos forrajes y
otros subproductos fibrosos). Información adicional sobre el resto de
aminoácidos y sobre otros alimentos se puede obtener de diversas
publicaciones procedentes de distintos centros de investigación de nutrición
animal (pe. NRC, 1998 (Inglés); CVB, 2002 (Holandés); INRA, 2002 (Francés);
FEDNA, 2003 (Español)). Sin embargo, cuando se busca información sobre
la composición en aminoácidos de forrajes distintos a la alfalfa o de
subproductos fibrosos, ya sean agrícolas o industriales, comúnmente
utilizados en la alimentación del conejo, la información de que se dispone es
escasa.
En cuanto a las necesidades de aminoácidos limitantes
(metionina, lisina, y treonina), hay información disponible procedente de
estudios dosis-respuesta tanto para conejos en cebo (Colin, 1975;
Spreadbury, 1978; Berchiche y Lebas, 1994; Tabeada et al., 1994 y 1996; de
Blas et al., 1998) como en lactación (Maertens y de Groóte, 1988; Taboada et
al., 1994 y 1996; de Blas et al., 1998). Sin embargo, no existen trabajos que
apoyen o justifiquen las recomendaciones dadas para el resto de
aminoácidos. Las recomendaciones para los tres aminoácidos que
normalmente aparecen com.o limitantes en piensos de conejos han sido
resumidas recientemente por de Blas y Mateos (1998) (Tabla 1).
Otro concepto que se utiliza habitualmente con el fin de
proporcionar a los animales un aporte equilibrado de aminoácidos, es el de
proteína ideal. Este método fue utilizado por Moughan et al. (1988) en
V
AnnexI
conejos en crecimiento, y consistió en complementar la proteína del pienso
con un perfil de aminoácidos sim.ilar al de las proteínas normalmente
sintetizadas por el animal. Es decir, en el caso de estar trabajando con
animales en crecimiento, el perfil de aminoácidos a aplicar debería ser el del
cuerpo completo del animal mientras que para conejos en lactación habría
que utilizar el correspondiente a la leche de las conejas. En la Tabla 2 se
muestra la composición en aminoácidos tanto del cuerpo completo de los
conejos como el de la leche de la coneja, expresados en mg/gN y en valor
relativo respecto a la lisina (valor absoluto del aminoácido/valor absoluto de
lisina).
Tabla 1. Necesidades nutricionales para conejos en condiciones de explotación
intensiva expresadas como concentración/kg y corregidas por un contenido en
MS de 900 g/kg (de Blas y Mateos, 1998)
Nutriente
Energía digestible
Proteína bruta
Proteína bruta
Lisina
Total
Digestible
Azufrados ^-^
Total
Digestible
Treonina b.e
Total
Digestible
Unidad
MJ
g
g
g
g
g
g
g
g
Conejas
11.1
184 (153-198)^
129 (114-139)
8.4 (100)^
6.6 (100)
6.5 (77)
5.0 (76)
7.0 (83)
4.8 (73)
Conejos en cebo
10.5
153 (145-162)
107 (102-113)
7.5 (100)
5.9 (100)
5.4 (72)
4.1 (70)
6.4 (85)
4.4 (75)
Piensos
10.5
159 (154-162)
111 (108-113)
8.0
6.3
6.0
4.6
6.8
4.7 ^Valores mínimos y máximos recomendados
ti Aminoácidos totales calculados para un aporte en aminoácidos sintéticos de 0.15
=Valor relativo a lisina
• La metionina debe proporcionar como mínimo el 35% de las recomendaciones totales en
aminoácidos azufrados
^Niveles máximos de 7.5 y 5.2 g/kg de treonina total y digestible, respectivamente, son
recomendadas para conejas reproductoras.
VI
Annex I
Tabla 2. Composición en aminoácidos (mg/gN) del cuerpo completo (conejos en
crecimiento de 53 días de edad) y de la leche de conejas (Moughan et al. ,
1988; Nicodemus et al, datos sin publicar).
Aminoácidos
Cistina
Histidina
Isoleucina
Leucina
Lisina
Metionina
Fenilalanina
Treonina
Tirosina
Valina
Alanina
Arginina
Ac. Aspártico
Ac. Glutámico
Glicina
Serina
Valor
Cuerpo
Relativo a absoluto lisina
158
193
194
429
383
77
249
245
192
239
365
415
467
788
456
283
41
50
51
112
100
20
65
64
50
62
74
108
121
205
121
74
Valor absoluto
175
159
304
567
451
150
281
305
332
382
228
328
4 5 1
1220
106
228
Leche
Relativo a lisina
39
35
67
125
100
33
62
67
73
85
50
73
100
270
23
50
Las actuales recomendaciones, expresadas como valor relativo
respecto a lisina, de aminoácidos azufrados y especialmente de treonina
(tabla 1) son superiores a la composición en aminoácidos de la proteína del
cuerpo com.pleto del conejo y de la leche de la coneja (tabla 2). Estas
diferencias ponen de manifiesto las limitaciones que presenta la utilización
del concepto de proteína ideal, al asumir que la eficacia de utilización
metabólica es la misma para todos los aminoácidos y al no tener en cuenta
las necesidades de mantenimiento de los animales para cada uno de estos
aminoácidos.
VII
Annex I
Respecto a este último punto, destacar que no existe información
acerca de las necesidades de mantenimiento en lo referente a aminoácidos
en conejos. Sin embargo, en cerdos y aves, se ha observado que las
necesidades de mantenimiento expresadas como valor relativo respecto a
lisina, son especialmente altas en treonina y en el caso de cerdos también en
aminoácidos azufrados.
2.2. Proteína bruta digestible y aminoácidos digestibles
El contenido total de nutrientes de un pienso o alimento determinados
por análisis químicos no implica que biológicamente los animales puedan
disponer de ellos para su utilización. Por ello, desde un punto de vista
biológico, y pese a ser de lo que más información existe, ni el contenido en
proteína bruta ni el contenido total en aminoácidos de un alimento consigue
caracterizar de forma precisa y correcta su valor nutritivo. Los aminoácidos
pueden quedar bajo formas no disponibles para el animal por hidrólisis
incompletas de la proteína, por no poder las enzimas proteolíticas acceder a
la proteína, por inhibición de enzimas (pe. inhibidores de tripsina) o por
inhibición de la absorción de aminoácidos. Otros factores que pueden afectar
negativamente a la disponibilidad de los aminoácidos son los tratamientos
térmicos de piensos durante el procesado, la presencia de una pared celular
indigestible que impida la entrada de proteasas a la célula y la presencia de
inhibidores de proteína presentes habitualmente en materias primas de
origen vegetal. Por todo esto, resulta importante evaluar los piensos o
alimentos como fuentes de aminoácidos biológicamente disponibles. A este
respecto, es común el uso del concepto de "disponibilidad" como sinónim.o de
"digestibilidad". Sin embargo, la definición estricta de ambos términos es
distinta. Digestible significa que un nutriente ha sido absorbido (ha
desaparecido del tracto digestivo) mientras que, disponible implica que ese
nutriente queda además disponible en la célula para su utilización en el
metabolismo tisular. Hasta ahora, dado que no se ha desarrollado la
metodología y tecnología necesaria para realizar de forma rutinaria la
medición de los aminoácidos disponibles a nivel celular, el término de
VIH
Annexl
"digestibilidad" parece ser el más adecuado para describir el valor
nitrogenado de un pienso. Dentro de este concepto la unidad más empleada
para la valoración nitrogenada de alimentos en conejos es la digestibilidad
fecal aparente. Sin embargo, hay dudas acerca de la bondad o precisión de
esta unidad a la hora de caracterizar un alimento. En otras especies
monogástricas (aves y porcino) la investigación en este campo derivó en la
definición de otra serie de unidades de valoración como fueron en primer
lugar, la digestibilidad ileal aparente, y posteriormente la digestibilidad ileal
verdadera o estandarizada y más tarde la digestibilidad ileal real. El que no
hubiera absorción de aminoácidos dirigidos a la síntesis de proteína corporal
a partir del íleon puso de manifiesto la importancia de definir el valor
nitrogenado de los alimentos a partir de balances ileales y no de balances
fecales. Además, dentro de los balances ileales, las unidades de
digestibilidad verdadera y real tienen en cuenta que junto al residuo no
digerido del alimento encontrado al final del intestino delgado hay también
un residuo de nitrógeno endógeno que da lugar a una subestimación del
valor nitrogenado de los alimentos. Son muchos los procedimientos descritos
en cerdos para la determinación del nitrógeno endógeno. Dependiendo de la
naturaleza o composición de los piensos y del procedimiento experimental
aplicado en la determinación de las pérdidas endógenas los resultados
obtenidos podrían variar.
3. Experimento 1. Efecto del tipo de alimentación y la cecotrofia sobre
la estimación de la proteína y los aminoácidos de origen endógeno en
conejos.
La estimación del nitrógeno endógeno y su composición en
aminoácidos resulta imprescindible para la determinación posterior de
coeficientes de digestibilidad más precisos que el obtenido mediante
balances fecales. En animales monogástricos como cerdos y pollos ya se han
propuesto unidades de digestibilidad medidas a nivel ileal que tienen en
cuenta las pérdidas endógenas. Estas pérdidas de nitrógeno de origen
IX
Annexl
endógeno proceden de enzimas digestivas, mucoprote ínas , descamaciones
celulares, urea , etc. En general, las pérdidas endógenas conducen a u n a
subest imación de la cant idad de los aminoácidos absorbidos. Los métodos
m á s comú.nmente empleados para determinar es tas pérdidas endógenas son
el empleo de piensos libres de nitrógeno o de piensos que contengan fuentes
de proteína m u y digestibles a nivel ileal como caseína o gluten de trigo. Tal y
como expuso J a n s m a n et al. (2002), cada uno de estos métodos tiene s u s
propias ventajas y limitaciones. En diversos trabajos realizados en porcino se
h a observado que el nitrógeno endógeno est imado mediante piensos libres de
nitrógeno es menor que el obtenido por otros métodos sugiriendo u n a
subest imación del mismo (Souffrant et al., 1997; Donkoh y Moughan, 1999).
Los conejos pueden u s a r par te de los aminoácidos no absorbidos después
del íleon p a r a la síntesis de proteína a través de la ingestión y posterior
digestión de las heces b landas o cecotrofos. Este fenómeno, conocido como
cecotrofia, puede consti tuir entre u n 15 y u n 3 8 % del total de la pro te ína y
los aminoácidos ingeridos (Carabaño et al., 1988; Fraga et al., 1991). Por
tanto , el contenido en nitrógeno y aminoácidos del flujo ileal de u n conejo
es ta rá formado no sólo por res iduos no digeridos procedentes del pienso y
res iduos de origen endógeno sino también por el residuo no digerido
procedente de la ingestión de cecotrofos.
El presente experimento se h a llevado a cabo, p a r a definir el método
m á s indicado pa ra la determinación de las pérdidas endógenas en conejos.
Para ello se compararon los flujos ileales de nitrógeno de animales
a l imentados por u n lado, con piensos libres de nitrógeno y por otro, con
piensos cuya principal fuente de proteína era la caseína. Además se estudió
el efecto de la cecotrofia sobre la est imación del nitrógeno endógeno
utilizando para su determinación animales que prac t icaban cecotrofia y
animales a los que se les impedía realizarla.
Un total de 28 animales de raza Neozelandés Blanco x Californiano,
con u n peso de medio de 3791 ± 141 g, fueron ñstul izados en íleon terminal
(a u n a dis tancia entre 10 y 15 cm de la válvula ileo-cecal) con u n a cánu la en
X
Annex I
T de vidrio de acuerdo al procedimiento descrito por Gidenne et al. (1988).
Los animales tuvieron acceso directo y sin restricciones al pienso PF (libre de
nitrógeno) o al C (pienso cuya única fuente de nitrógeno era caseína). La
única diferencia entre los piensos fue su composición en materias primas al
sustituirse un 16% de almidón de maíz por caseína en el pienso C. Los
animales fueron distribuidos al azar a cada uno de los piensos
experimentales (14 animales por pienso). Siete animales por pienso llevaron
puesto un collar cervical de madera durante toda la prueba que les impedía
realizar la cecotrofia.
La ingestión del pienso libre de nitrógeno (PF) condujo a unos niveles
de ingestión de pienso y de excreción de cecotrofos de la mitad y el doble,
respectivamente, que los animales que ingirieron el pienso de caseína (C).
Por tanto, la ingestión de cecotrofos respecto a la ingestión total de MS fue
muy superior en aquellos animales que consumieron el pienso PF (37.0 vs.
17.2%, para el pienso PF y C, respectivamente). Este hecho dificultó
considerablemente la interpretación de los resultados, al poder estar
afectando la representatividad de las muestras de contenido ileal. Estas
muestras, fueron recogidas en una proporción de 1:1 a las 11:00 y las 20:00
h asumiendo un ciclo normal de excreción de cecotrofos e ingestión de
pienso. Por tanto, para evitar confusiones en la interpretación de los
resultados, el estudio del efecto del tipo de pienso se restringió a aquellos
aníimales que no realizaban la cecotrofia y el estudio del efecto de la
cecotrofia se restringió a aquellos animales que consumían el pienso C.
En cuanto al efecto que el tipo de pienso tuvo sobre la estimación de
las pérdidas endógenas cabe destacar que: I) el pienso C dio lugar a
mayores ingestiones de MS (111.5 vs. 51.5 g/dia), de nitrógeno (3.12 vs.
0.20 g/día), y mayores flujos ileales de MS ( 55.0 vs. 23.8 g/día) y nitrógeno
(0.72 vs. 0.24 g/día) que el pienso PF. II) La ingestión de cecotrofos tanto de
MS como de nitrógeno fue superior en animales que consumieron el pienso
PF que el pienso C (41.4 vs. 21.7 y 0.52 vs. 0.47 g/día, respectivamente).
Resultados parecidos se obtuvieron en animales a los que se les había
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Annex I
realizado una ligadura en el conducto pancreático (Hornicke y Bjórnhag,
1980). Esta ligadura daba lugar a una menor absorción de nutrientes que
los animales trataron de compensar con una mayor ingestión de cecotrofos.
III) En animales que consumieron el pienso C se obtuvo una relación lineal
entre el consumo total de MS y el flujo ileal de nitrógeno endógeno
determinado. Esa misma relación no se observó en los animales que
consumían el pienso PF. La falta de respuesta observada en estos animales
pudo deberse, de nuevo, al bajo consumo de MS observado y a la falta de la
variación necesaria para establecer un rango amplio de consumos que pueda
correlacionarse con los flujos de nitrógeno endógeno. IV) La ingestión del
pienso C condujo a valores de flujo total de aminoácidos un 27% superiores
de media que el pienso PF. A este respecto, Boisen y Moughan (1995)
muestran en su revisión que las pérdidas endógenas básales estimadas con
piensos libres de nitrógeno varían de 1.5 a 2.4 g/kg MSI. Sin embargo,
cuando se utilizan piensos con proteina altamente digestible, estas pérdidas
son de 3.2 g/kg MSI (entre un 50 y un 37% superiores que las estimadas
mediante piensos libres de nitrógeno). Además, estos mismos autores
muestran que si los piensos son vegetales, la presencia de fibra o de factores
anti-nutricionales conducen a unas pérdidas endógenas que podrían variar
entre 3.2 y 6.4 g/kg MSI. Por otro lado, el tipo de pienso también tuvo un
efecto significativo sobre la composición en aminoácidos de las pérdidas
endógenas en el presente trabajo. Estas diferencias podrían explicarse por
variaciones en la contribución relativa de las diferentes secreciones
enzimáticas inducidas por el pienso. V) El pienso C condujo a un
enriquecimiento de los cecotrofos en nitrógeno, treonina y prolina mientras
que el pienso PF dio lugar a cecotrofos con raayor contenido en lisina y
metionina que el C.
La prevención de la cecotrofia en los animales que consumieron el
pienso C no tuvo ningún efecto negativo sobre la ingestión de MS y nitrógeno
total ni sobre el flujo ileal de N pero sí sobre el flujo ileal de MS (73.4 vs. 56.0
g/día). La cecotrofia tuvo escaso efecto sobre el flujo ileal de los aminoácidos
(g/kg MSI). Así, los animales que no podían realizar la cecotrofia tan sólo
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Annexl
mostraron diferencias respecto a los que sí podían realizarla en el flujo ileal
de fenilalanina que resultó ser un 19% menor en el primer caso. En cuanto
al efecto de la cecotrofia sobre la composición en aminoácidos del N
endógeno, los animales que podían practicarla presentaron una mayor
proporción aminoácidos esenciales como arginina, leucina, lisina,
fenilalanina y treonina. Este resultado tal y como se esperaba, es
consecuencia del enriquecimiento de los cecotrofos respecto a las heces
duras en aminoácidos esenciales procedentes en gran parte de la síntesis
microbiana en ciego (Carabaño y Piquer, 1998). Sin embargo, el hecho de
que el coeficiente de digestibilidad del nitrógeno de los cecotrofos
(determinado por métodos in vitro) fuera alto (79%) hizo que su efecto sobre
el flujo de aminoácidos endógenos (g/kg MSI) no fuera tan importante como
se pensó inicialmente.
Debido a la falta de datos referentes a las pérdidas endógenas en
conejos, los resultados obtenidos en este trabajo se compararon con los
presentados por Jansman et al. (2002) en cerdos. De esta comparación se
extrae que tanto con el pienso C como con el PF, las pérdidas de nitrógeno
endógeno obtenidas fueron 3.2 y 2.7 veces superiores en conejos. Estas
diferencias podrían deberse a diferencias entre especies animales puesto que
no hay ningún dato previo de estos resultados en conejos, pero también
podrían explicarse por los mayores contenidos en fibra de los piensos de
conejos (27-33% vs. 3-8% en conejos y cerdos, respectivamente) y por
diferencias metodológicas.
Debido al bajo consumo de MS que presentaron los animales
alimentados con el pienso PF, la ingestión de energía digestible (ingirieron
246 kJ /d ía y kgO-75) fue un 45% inferior a las necesidades de mantenimiento
de conejas adultas (400 kJ /d ía y kgO-75) (Parigi Bini y Xiccato, 1998). Este
hecho, junto al incremento en la ingestión de cecotrofos, indica que la
utilización de los piensos libres de nitrógeno podría tener importantes
limitaciones prácticas pudiendo resultar más precisa la utilización de
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Annex I
piensos con proteína altamente digestible para la determinar de las pérdidas
endógenas en conejos.
4. Experimento 2. Comparación de diferentes unidades de valoración
nitrogenada de cuatro materias primas comúnmente empleadas en
piensos de conejos.
Tal y como se ha indicado en la introducción general, para poder
evaluar con mayor precisión el valor nutritivo de los alimentos es necesario
estimar la desaparición de nutrientes a lo largo del tracto digestivo. Para ello,
se han propuesto varias unidades de digestibilidad medidas en distintos
tramos del aparato digestivo. En la actualidad, el procedimiento más
comúnmente empleado en conejos es el de los balances fecales (Fraga,
1998). Sin embargo, en otras especies monogástricas como pollos o cerdos,
los balances ileales se han propuesto como unidades de valoración
nitrogenada más precisas (Tanksley y Knave, 1984; Sauer and Ozimek,
1986). El íleon es el último segmento del intestino donde los aminoácidos
pueden ser absorbidos y utilizados para la síntesis de proteínas corporales
(Zebrowska, 1973). Lo mismo ocurre en conejos a los que se les impide
realizar la cecotrofia (Belenguer et al., 2003). Dentro del concepto del
balance de nutrientes medido a nivel ileal, la digestibilidad ileal
estandarizada e ileal verdadera, han sido propuestas como mejores unidades
de valoración al corregir por el efecto del nitrógeno endógeno determinado
mediante piensos libres de nitrógeno o piensos proteicos con proteína
altamente digestible, respectivamente.
En el presente trabajo se ha determinado la digestibilidad ileal
verdadera de 4 alimentos diferentes comúnmente empleados en piensos de
conejos. Además se comparan esos valores con los obtenidos por balances
más sencillos (digestibilidad ileal aparente y fecal). El objetivo de esta prueba
fue establecer la unidad que m.ejor define el valor nitrogenado de estos
alimentos. Las materias primas escogidas fueron: heno de alfalfa, cebada,
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Annex I
salvado de trigo y harina de girasol. La elección de estas materias primas se
hizo en base a su importancia en la formulación práctica de piensos
compuestos de conejos.
Un total de 20 conejas de raza Neozelandés Blanco x Californiano, con
un peso de medio de 3841 ± 117 g, fueron canuladas a nivel de íleon
terminal (a unos 10 ó 15 cm de la válvula ileo-cecal) con una cánula en T de
vidrio de acuerdo al procedimiento descrito por Gidenne et al. (1988). Los
animales tuvieron acceso directo y sin restricciones al pienso y al agua. Se
formularon 4 piensos isonutritivos cuya principal fuente de proteína era, en
cada caso, el alimento que se quería valorar: heno de alfalfa, cebada, salvado
de trigo y harina de girasol. La composición química de estos piensos fue
similar a la del pienso de caseína del experimento anterior. Los coeficientes
de digestibilidad ileal verdadera fueron determinados a partir de los datos de
nitrógeno endógeno obtenidos en el experimento anterior con el pienso de
caseína (C). Los animales fueron distribuidos al azar a cada uno de los
piensos experimentales a razón de 5 animales por pienso.
4.1 Digestibilidad fecal versus ileal
La actividad proteolítica mostrada por las bacterias cecales (Emaldi et
al., 1979) permite la utilización de la cadena carbonada y del nitrógeno
procedente de la desaminación de la proteína ileal para el crecimiento
bacteriano y la producción de amoniaco. Este amoniaco puede ser absorbido
a través de la pared cecal y excretado en la orina en forma de urea. Sin
embargo, al comparar el flujo ileal de proteína (3.17 g/día, de media) y la
excreción total de proteína (heces duras + cecotrofos) (3.74 g/día, de media)
se observó que había un balance positivo de proteína de 0.568 g/día (« un
18% de lo que llegaba al íleon). Este balance positivo se podría explicar por
una entrada de urea al ciego procedente del catabolismo de los aminoácidos
en el hígado a través del torrente sanguíneo. Forsythe and Parker (1985)
estimaron que un 25% del total de amoniaco en ciego (principal fuente de
nitrógeno para los microorganismos) procedía de la hidrólisis de urea.
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Annex I
Todos los alimentos mostraron una digestibilidad fecal aparente de la
proteína mayor que la ileal (0.697 vs. 0.535 de media para la digestibilidad
fecal e ileal, respectivamente) al reciclarse un 35% del total de nitrógeno
excretado mediante la cecotrofia (un 67% de media, del nitrógeno de los
cecotrofos era de origen bacteriano). Sin embargo, la digestibilidad de los
aminoácidos fue mayor o menor a nivel fecal o ileal dependiendo de la
materia prima y del aminoácido estudiado. Por tanto, el uso de proteína
digestible o de aminoácidos digestibles podría cambiar la caracterización del
valor nitrogenado de los alimentos.
Realizando balances similares al mostrado para la proteína pero con
los aminoácidos se observó que, ese balance positivo de proteína estaba
enriquecido en lisina (0.072 g/día; 63% del flujo ileal), metionina (0.025
g/día; 95% del flujo ileal) y treonina (0.059 g/día; 40% del flujo ileal).
Además este enriquecimiento, en aminoácidos esenciales tendió a ser m.ayor
en los piensos basados en alfalfa, cebada y harina de girasol que en el
basado en salvado. Esta tendencia resultó paralela a los datos de nitrógeno
microbiano observados (dependiendo de la composición del pienso, entre un
40 y un 49% y entre 62 y 69% del nitrógeno total en heces y cecotrofos,
respectivamente, era de origen microbiano) al ser el pienso de salvado el de
más bajo contenido en nitrógeno bacteriano.
La actividad bacteriana en el ciego dio como resultado cambios
sustanciales en la composición de aminoácidos de la proteína. Como
consecuencia, pese a que un tercio aproximadamente de la lisina y la
metionina excretadas se reciclaron vía cecotrofia, la digestibilidad ileal de
ambos fue mayor que la digestibilidad fecal. Sin embargo la treonina mostró
una tendencia distinta, siendo la digestibilidad ileal menor que la fecal. Esto
se debió al ya de por sí elevado flujo ileal de treonina (0.142 g/día de
treonina vs. 0.118 y 0.028 g/día para lisina y metionina, respectivamente)
como consecuencia de su alta proporción en el nitrógeno endógeno (5.53
g/ 15gN vs. 3.76 y 0.96 g/ 16gN para lisina y metionina, respectivamente).
XVI
Annex I
4.2. Digestibüidad aparente versus verdadera
Tal y como se esperaba, los coeficientes de digestibüidad ileal
verdadera de proteína y aminoácidos de los alimentos fueron mayores que
los coeficientes de digestibüidad ileal aparente como consecuencia de las
importantes pérdidas nitrogenadas endógenas obtenidas para los conejos
(0.536 vs. 0.774 de media, para la digestibüidad ileal aparente y verdadera
de la proteína, respectivamente). En cuanto a los aminoácidos, en todos los
alimentos se observaron importantes diferencias entre estas dos unidades
para cistina, treonina y prolina. Estos resultados se corresponden con los
altos niveles encontrados para estos aminoácidos en las pérdidas endógenas.
Y dependiendo del alimento, también se observaron diferencias importantes
para el ácido aspártico, ácido glutámico, glicina y serina. Además, en cuanto
a los alimentos, las mayores diferencias entre estas dos unidades se
obtuvieron para los alimentos de m.enor contenido en proteína bruta, es
decir, para la cebada y el salvado. Por el contrario, las menores diferencias
entre estas dos unidades se obtuvieron para la harina de girasol. Estos
resultados son consecuencia, al igual que ocurre en cerdos, de la diferente
proporción que implica el nitrógeno endógeno respecto al nitrógeno no
digerido procedente del alimento.
Al no existir datos previos de digestibüidad verdadera en conejos, los
resultados obtenidos en este trabajo se compararon con la digestibüidad ileal
estandarizada de cerdos existente en la literatura. De esta comparación se
obtuvo que la digestibüidad ileal verdadera de la proteína y los aminoácidos
de estos cuatro alimentos en conejos fue de media un 11% superior que la
ileal estandarizada de los cerdos. Las mayores diferencias en esta
comparación se obtuvieron para el heno de alfalfa.
En la formulación práctica, el valor relativo que se establece entre los
alimentos es más importante que el valor absoluto de digestibüidad
alcanzado. En el presente trabajo se ha visto que la relación que se establece
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Armex I
entre los alimentos estudiados no es constante, sino que varia dependiendo
de la unidad empleada en la valoración tanto para la proteína como para los
aminoácidos. Las variaciones observadas en el valor relativo que se establece
ente los alimentos para cada uno de los coeficientes de digestibilidad fueron
más importantes en el caso de la treonina que en la proteína bruta, la lisina
o la metionina.
La digestibilidad ileal y fecal aparente conducen a una subestimación
o sobreestimación, respectivamente, de la digestibilidad ileal verdadera de
los aminoácidos de los alimentos como consecuencia del elevado nitrógeno
endógeno y la importante actividad microbiana en el ciego de los conejos. Por
tanto, con el fin de obtener una valoración más precisa de los alimentos se
recomienda el uso de la digestibilidad ileal verdadera. Con el uso de esta
unidad de valoración en la formulación práctica se conseguiría un mejor
ajuste de los nutrientes del pienso a las necesidades del animal y una
reducción de la excreción de nitrógeno al medio. Sin embargo, llegados a este
punto hay que destacar la falta de datos y por tanto la necesidad de seguir
valorando más alimentos en estos términos,
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