Rabbit feeding system : assesment of different digestible units of nitrogen and amino acids in feedstuffs for rabbits = Sistema de alimentación para conejos: definición de las unidades de valoración nitrogenada

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(1)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 u n i t s for nitrogen a n d. amino acid feedstuffs evaluation"). Ana Isabel García Ruiz. Ingeniero Agrónomo. Mayo, 2 0 0 4.

(2) 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 Isab. uiz. Ingeniero Agrónomo. V°B° Los d i r e c t o r e s d e l a T e s i s. fíiMijsi^^ A 0^4^ R o s a M^ C a r a b a ñ o Lúe Dr. Ingeniero Agrónomo. C a r l o s de B l a s B e o r l e g u i Dr. Ingeniero Agrónomo.

(3) ^. mis cuatto abu£.lob.

(4) AGRADECIMIENTOS (fi (JisÔL ct^~ '€fl'íO-l>0-ñ.o u '^t.loi La liealLzación. de eita teiii peío 'ceaala'c/ne lot môtet. ^l íuenoi. S-avieí. tamhién., pot iu amiitadu. añoi de mi todavía. ^t c^- êiúi. îllamide.pot.. âxcía. que compatiimoi. ^l (/Ina. ftpinoia. tet. mi masitíapeío. coniôi.. ^taciai. datante. a ¿oi doi pot. ¡tohíe todo... pot ¿ e l una t>aen.a amia,a.. pot tu auuda. dentzo u fueía. po'c ¡tu valioia. iaiioi. qua me kan. dedicado. "cotta " vida.. u ^íu.'z.ia ^fícodemui. momentoi. Lat conât. de. ^Lat pot todo &l iiampo. de nueifco. en. el laho'cato'cio u po'c todoí> loi deipacAo.. anuda en el Íaíoíatotí-O. u tu dedicación. en el manô. de. canuladat.. ^t. î.at.ia. îlvit.. u âtlot. ^l. S.avie'c ônzález. ^¡{gdîauez. l"âttu'cab")po'c. tu apoup incondicional. u tinc&'ca. amittad.. cont/cctación. en lat. po'c iui pacientei. fptapo'c. ^fl êpe,. B.avie'c ^{gd'd.gu.ez,. ^. toda la auudap'cettada ôlanda. en lat navetupox u ^{gta ^llatet. dificilei.. ^ntonio. ^lotet. u a Lot compañe'coi. m.it mát intünat âta. animada. "aquellot. almue'czot",. po'c tu colabo'cación. en. diitintot. tetit. / at del "ôuífw. po'c tu CU30U0 u aactencia en ¿a tecta final de eita ^. u tu. comidat.. (/i 3.uan. atp&ctot de eita. ta'cdei •Pcente al e^pecfcofotómetfco. votottat. arntaai ^naela. mi atatitud. detde hace ya m-uchah añat vueitta. and ^{ahbit. îsiewcck. ^ntte.". tetit.. u fya, tin ellai lat co¿a¿ kahtian. iido mucho mái. ei doble: ataciat poí bel co/no toi^, pot. h.abe'une b'zindado amittad. u pot kaJie'zjne auudado tanto en lot momentot. mát. dutoi. ^ ketmanot, i/otottot. toda mi familia, ^ngel. y. ^wcia. cuatto:. ^'caciatpo'c. ^pWca. Întonio. pe'co etpecialmente.. a mit pad'cet,. (flngel. poi^que te lo meicecen y kan tábido ayuantatme. u ^íatibel. u a. pacientemente.. mit ^. todo.. '^kacobo po'c tu eno'cme entiba,. poí tu catino. u po'c que'ce'c como. quie'ce..

(5) INDEX.

(6) General Index. INDEX INDEX. 1. RESUMEN. 5. SUMMARY. 11. CHAPTER I: Literature Recent. advances. review. in nitrogen. and objectives. nutrition. 16. in rabbits.. 16. 1. Introduction. 17. 2 . Crude protein and total a m i n o a c i d s. 17. 3 . Digestible crude protein and a m i n o acids. 21. 3 . 1 . Faecal balance. 21. 3.2. Ileal balance. 25. 3.3. Apparent v e r s u s true digestibility. 28. 4. Objectives. 30. 5. R e f e r e n c e s. 30. CHAPTER II: Experiment Effect. of type. and amino. of diet. acidflotu. 1 and. 36 caecotrophy. in rabbits. on ileal. endogenous. nitrogen 36. 1. Abstract. 38. 2 . Introduction. 39. 3 . Materials and M e t h o d s. 40. 3 . 1 . Diets. 40. 3.2. Animáis a n d h o u s i n g. 42. 3.3. Experimental procedure. 43. 3.4. Analytical m e t h o d s. 45. 3.5. Statistical analysis. 46. 4. R e s u l t s. 47. 5. D i s c u s s i o n. 54.

(7) General Index. 6. Acknowledgements. 57. 7. References. 57. CHAPTER IIJ: Experiment Comparíson. of different. 2 units for nitrogen. 63 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 2.2. Proteína bruta digestible y aminoácidos digestibles. IV VIII.

(8) 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. 4.1 Digestibilidad fecal versus ileal 4.2. Digestibilidad aparente versus verdadera 5. Referencias bibliográfícas. XIV XV XVII XVIII.

(9) RESUMEN.

(10) Resumen. RESUMEN El objetivo general de esta tesis doctoral ha sido el tratar de establecer las bases para u n 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 u n 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 u n a cánula en T de vidrio.. Los animales alimientados con el pienso PF mostraron u n 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 u n a relación lineal entre el consumo total de MS (pienso + cecotrofos) y el ñujo ileal de nitrógeno.

(11) 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 u n 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 u n a 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 u n a 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)..

(12) 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 u n a 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 u n balance positivo de proteína de 0.558 g/día (~ u n 18% de lo que llegaba al íleon). Este balance positivo se podría explicar por u n a entrada de urea al ciego procedente del catabolismo de los aminoácidos en el hígado a través del torrente sanguíneo. Todos los alimentos mostraron u n a digestibilidad fecal aparente de la proteína mayor que la ileal, al.

(13) Resumen. reciclarse u n 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 u n 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.

(14) 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 u n a 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 u n a 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 u n a 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.

(15) SUMMARY. 11.

(16) 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.

(17) 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.

(18) Summary. alfalfa hay (AH), barley grain (BG), wheat bran (WB) and sunñower meal (SM). For this experiment animáis were surgicaiiy fitted witti a giass Tcannula 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.

(19) Summary observed for threonine digestibility, which tended to be lower a t ileal t h a n at faecal level. This could be a n effect of the particularly 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.

(20) CHAPTER I: Literature review and objectives. Recent advances in nitrogen nutrition in rabbits.. 16.

(21) 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.

(22) 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 methionine,. of the most limiting amino acids in rabbit diets:. 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.

(23) 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. MJ. 11.1. 10.5. 10.5. Crude protein. g. 184 (153-198)a. 153 (145-162). 159 (154-162). Digestible protein. g. 129 (114-139). 107 (102-113). 111 (108-113). Total. g. 8.4 (100)^^. 7.5 (100). 8.0. Digestible. g. 6.5 (100). 5.9 (100). 6.3. Total. g. 6.5 (77). 5.4 (72). 6.0. Digestible. g. 5.0 (75). 4.1 (70). 4.6. Total. g. 7.0 (83). 6.4 (85). 6.8. Digestible. g. 4.8 (73). 4.4 (75). 4.7. Digestible energy. Lysine ^. Sulphurb.d. Threonine t'.e. ^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.

(24) Chapter I: Literature review and objectives The p r e s e n t r e c o m m e n d a t i o n s , relative to lysine, for total s u l p h u r a m i n o acids a n d , especially, for total threonine (Table 1) are higher t h a n t h e amino acid composition of whole body a n d milk (Table 2). These differences show t h e limitations of the ideal protein, a s it a s s u m e s t h a t metabolic efficiency is similar for all the amino acids, a n d does not take into a c c o u n t t h e m a i n t e n a n c e r e q u i r e m e n t s . There is not Information a b o u t a m i n o acid r e q u i r e m e n t s for m a i n t e n a n c e in rabbits. However, in pigs, the m a i n t e n a n c e r e q u i r e m e n t s , relative to lysine, are especially high in t h r e o n i n e a n d s u l p h u r amino acids (Williams, 1995).. Table 2. Amino acid composition (mg/gN) of the whole body (53 day oíd growing rabbits) and of the doe milk (Moughan et al, 1988; Nicodemus et al, unpublished).. Amino acids. Whole body Absoluta Relativa to valué lysine. Milk Absoluta valué. Ralative to lysine. Lysine. 383. 100. 451. 100. Alanine. 365. 74. 228. 50. Arginine. 415. 108. 328. 73. Aspartic acid. 467. 121. 451. 100. Histidine. 193. 50. 159. 35. Isoleucine. 194. 51. 304. 67. Leucine. 429. 112. 567. 125. Methionine. 77. 20. 150. 33. Cystine. 158. 41. 175. 39. Glutamic acid. 788. 205. 1220. 270. Glycine. 466. 121. 106. 23. Phenylalanine. 249. 65. 281. 62. Serine. 283. 74. 228. 50. Threonine. 245. 64. 305. 67. Tyrosine. 192. 50. 332. 73. Valine. 239. 62. 382. 85. 20.

(25) 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.

(26) Chapter I: Literature review and objectives. observed for storage proteins located in grains or s e e d s (cereals, legume a n d oil seeds, a n d oil seed meáis) a n d the lowest for proteins protected by a cell wall with a high degree of lignification (forages a n d fibrous by products). In fact, insoluble nitrogen in NDF or ADF solution s e e m s to be one of t h e best single predictor of protein digestibility (Martínez a n d Fernández,. 1980;. Villamide a n d Fraga, 1998).. Table 3 s h o w s c r u d e a n d digestible protein c o n t e n í of t h e m a i n protein s o u r c e s u s e d in rabbit diets. Crude protein overestimates the valué of absorbable protein of all ingredients, b u t mainly in those with digestibility, a s w h e a t b r a n or alfalfa hay. Although digestible. lower protein. i n c r e a s e s t h e a c c u r a c y of diet formulation, t h e u s e of t h i s u n i t is restricted d u e to the scarce Information of digestible content of r a w materials. Alfalfa is t h e best-evaluated ingredient, a s a total of 2 3 s a m p l e s (14 d e h y d r a t e d a n d 9 a s hay) have been studied.. Table 3. Crude and digestible protein content (g/kg) of some protein sources rabbit's diets (Villamide et al, 1998).. Sunflower meal Wheat bran Bar ley grain Alfalfa hay. Crude protein Absolute Relative 1 1 valué valué. 320. 100. 160. in. Digestible protein Absolute r-. i î 1 Relative valué valué. 270. 100. 50. 107. 39. 120. 33. 90. 33. 180. 56. 117. 43. However, for other dietary ingredients (sunflower meal, barley, a n d oats), no more t h a n 4-6 b a t c h e s have been evaluated, being the m o s t frequent situation t h a t the referenced valué of c r u d e protein digestibility comes j u s t from one batch.. Recently,. Villamide. and. Fraga. (1998). have. proposed. e q u a t i o n s to predict the digestible protein c o n t e n t of feed. different ingredients. 22.

(27) 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 number^. Intercept. B. N. R^. RSD. 1. 0.83 ± 0 . 0 6 0.75 ± 0 . 0 5. 26. 2. -39.4 ± 11.1 -2.3 ± 7 . 3. 27. 0.892 0.911. 3.44 3.90. 3. -55.3 ± 2 4 . 6. 0.94 + 0.06. 18. 0.936. 7.14. 4. -19.2 ± 18.4. 0.72 ± 0 . 1 5. 17. 0.617. 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 byproducts. 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.

(28) Chapter I: Literature review and objectives. D a t a obtained in r a b b i t s for alfalfa hay agree with observations m a d e in pigs. In this way, Table 5 shows the a p p a r e n t c r u d e protein a n d amino acid faecal digestibility of three b a t c h e s of alfalfa differing in NDF a n d protein c o n t e n t (from 38.7 to 55.0% on DM, a n d from 17 to 2 2 % on DM, respectively). Although amino acids followed t h e s a m e trend t h a n crude protein digestibility, the differences between extreme b a t c h e s of alfalfa were higher in lysine a n d threonine digestibility (17 a n d 13.5%, respectively) t h a n for c r u d e protein digestibility (10%). F u r t h e r m o r e , lysine, methionine a n d threonine digestibilities tended to be higher t h a n c r u d e protein digestibility, mainly in the alfalfa of better quality.. Table 5. Effect oftype of alfalfa hay on apparent faecal and amino acids (96) (García et al, 1995b) Alfalfa hayí. digestibility. of protein. SEM. P. 57.4 c. 1.0. 0.001. 76.0 b. 57.2 c. 2.0. 0.001. 79.8 a. 72.5 b. 72.5 b. 2.0. 0.001. 77.4 a. 72.2 b. 57.0 c. 2.0. 0.001. A. B. C. Crude protein. 74.4 a. 71.5 b. Lysine. 81.4 a. Methionine Threonine. 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 t h e s a m e way, T a b o a d a et al. (1994 a n d 1996) a n d de Blas et al. (1998) observad t h a t digestibility of synthetic a m i n o acids is higher (from 0.93 to 1.0) t h a n t h e corresponding a m i n o acids contained in raw materials (from 0.54 to 0.80 a s average). These r e s u l t s show t h a t t h e u s e of faecal digestible a m i n o acids could improve t h e characterisation of nitrogen valué of feeds, b u t also t h e definition of r e q u i r e m e n t s . Some discrepancies in total amino acid r e q u i r e m e n t s. reported. in the literature could be. partially. explained by differences of amino acid digestibility in t h e diets u s e d to determine t h e m . Considering a diet with the s a m e supply of digestible threonine (4.8 g / k g , Table 1), total dietary t h r e o n i n e c o n t e n t should vary 24.

(29) 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 t o t a l s u p p l y , r e s p e c t i v e l y . O n t h e c o n t r a r y , a d i e t f o r m u l a t e d a c c o r d i n g to t o 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 o r 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 i g e s t i b i l i t y is h i g h e r o r l o w e r t h a n 0 . 6 8 6 (Table 1). A c c o r d i n g to D e B l a s e t a l . ( 1 9 9 8 ) , b o t h s i t u a t i o n s will l e a d to a r e d u c t i o n i n 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 i g e s t i b l e l y s i n e , 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 r o w i n g 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 i d e a 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 t o u s e t h i s u n i t i n d i e t f o r m u l a t i o n is t h e l a c k of d a t a of d i g e s t i b l e a m i n o a c i d c o n t e n t of f e e d s . At t h i s p o i n t , it c o u l d b e c l e a r t h a t a m a i n objective 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.

(30) 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; MottaFerreira 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.

(31) 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. Proto, 1976 Nicodemus, 1999 SF. HF. SF. -. -. 1.30 3.87 5.69 4.74 1.58 3.59 5.26 3.33 5.05. 1.71 4.10 6.20 4.56 1.13 4.19 4.94 2.80 4.38. 1.41 1.35 3.94 5.61 5.05 1.70 3.41 5.35. 5.21 3.15 9.15 10.25 4.47 3.84 3.89. 5.19 3.57 8.66 10.25 5.19 5.21 4.40. 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. -. 5.35 5.87 2.72 9.35 11.53 4.74 -. 4.31. 27.

(32) 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<¿>" 10. 12. ''. THRE0NINE=21 14. 16. 18. 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.

(33) 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.

(34) 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.

(35) 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 Llysine 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.

(36) 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 Nutrition. 54: 285-292. 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 nutrient intake of fattening rabbits. Journal of Animal Science 69: 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 Bureau, Wallingford, UK. Fraga, M.J. 1998b. Protein requirements. In The Nutrition of the Rabbit (ed. J.C.. de. Blas. and. J.. Wiseman),. pp.. 133-143.. Commonwealth. Agricultural Bureau, Wallingford, UK. Fu:adación Española para el Desarrollo de la Nutrición Animal (FEDNA). 2003. Tablas FEDNA de composición y valor nutritivo de alimentos para la fabricación de piensos compuestos. Universidad Politécnica de Madrid, Spain. Ganuza, J. M., Balcells, J., Pérez, J.F., Fondevila, M. and Parker, D.S. 1999.Nutritive valué of caecum microorganism and caecotrophes in rabbits. Proceedings of the British Society of Animal Science, 223. 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: 267275. 32.

(37) 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.

(38) 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. National Academy Press. Washington D.C. 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 growing pigs: a review. Canadian Journal of Animal Science 77: 149163. 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. In Digestivo Physiology in Pigs (ed. M.W.A. Verstegen, J. Huisman and L. A. den Hartog), pp. 147-166. EAAP Publication No.54, PUDOC, Wageningen, The Netherland. Spreadbury, D. 1978. A study of the protein and amino acid requirements of growing New Zealand White rabbit with emphasis on lysine and sulphur-containing amino acids. British Journal of Nutrition 39: 601613. 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.

(39) Chapter I: Literature review and objectives. Tabeada, E., Méndez, J. and De Blas, J.C. 1995. The response of highly productive. rabbits. to dietary. sulphur. amino. acid. contení. for. reproduction and growth. Reproduction Nutrition Developement 35: 191-203. Trocino, A., Xiccato, G., Queaque, P.I. and Sartori, A. 2000. Feeding plans at different protein levéis: effects on growth performance, meat quality and nitrogen excretion in rabbits. Proceedings of the 7th World Rabbit Congress vol C: 467-474. Valencia, Spain. Villamide, M.J. and Fraga, M.J. 1998. Prediction of digestible crude protein and protein digestibility of feed ingredients for rabbits from chemical analysis. Animal Feed Science and Technology 70: 211-224. Villamide, M.J., Maertens, L., De Blas, J.C. and Pérez, J.M. 1998. Feed evaluation. In The Nutrition of the Rabbit (ed. J.C. de Blas and J. Wiseman),. pp.. 89-101.. Commonwealth. Agricultural. Bureau,. Wallingford, UK. Williams, P.E.V. 1995. Digestible amino acids for non-ruminant animáis: theory and recent challenges. Animal Feed. Science and Technology. 53: 173-187. Xiccato, G. 1996. Nutrition of lactating does. In Proceedings of the sixth world rabbit congress (ed. F. Lebas), 1: 29-47. Toulouse, France. Xiccato, G. 1999. Feeding and meat quality in rabbits: A review. World Rabbit Science 7: 75-86. Yoshida, T., Pleasants, J.R., Reddy, B. and Wostman, B.S. 1968. Efficiency of digestión in germ-free and conventional rabbits. British Journal of Nutrition 22: 723-737. Yoshida, T., Pleasants, J.R., Reddy, B. and Wostman, B.S. 1971. Amino acid composition. of caecal. contents. and. faeces. in. germ. free. and. conventional rabbits. Journal of Nutrition 101: 1423-1429. Zebrowska, T. 1973. Digestión and absorption of nitrogenous compounds in the large intestine of pigs. Roczniki Nauk Rolnizych B95: 85-90.. 35.

(40) 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.

(41) Chapter II: Experiment 1. Effect of type of diet (casein-based or protein-free diet) a n d caecotrophy on ileal e n d o g e n o u s nitrogen a n d a m i n o acid flow in rabbits^. A.I. García*, J . C . de Blas, a n d R. C a r a b a n o. Departamento. de Producción Animal, E. T. S. Ingenieros. Universidad. Politécnica 28040, Madrid,. Agrónomos,. Spain. t Financial s u p p o r t w a s provided by t h e S p a n i s h Comisión Interminesterial de Ciencia y Tecnología (Project AGF199-1109) * Present a d d r e s s : Nutreco Poultry and Rabbit Research del Monte, 45950,. Toledo,. Centre,. Casarrubios. Spain.. 37.

(42) 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.

(43) 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 h a s 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 h a s 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 J a n s m a n 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.

(44) 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.

(45) Chapter II: Experiment 1. in t h e diet formulation. Sunflower hull a n d w h e a t s t r a w were included in the diets to r e a c h a correct t r a n s i t time a n d r a t e of p a s s a g e of t h e digesta t h r o u g h t h e intestinal tract w h e r e a s pectin a n d cellulose were a d d e d to e n s u r e a m i n i m u m fermentable s u b s t r a t e e n t r a n c e into t h e c a e c u m for t h e m a i n t e n a n c e of microbial metabolism. Both diets also i n c l u d e d 2 g / k g of fibre m o r d a n t e d with y t t e r b i u m according to t h e p r o c e d u r e of Uden et al. (1980). Ytterbium w a s u s e d a s indigestible m a r k e r . The m a r k e d fibre w a s p r o d u c e d from a mixture of sunflower hulls a n d w h e a t straw (1:1) w a s h e d with detergent in a n a u t o m a t i c w a s h i n g m a c h i n e . Diets were pelleted a n d a n i m á i s were given ad libitum access to feed a n d w a t e r d u r i n g all t h e experiment.. Table 1. Ingredient. composition. ofthe experimental. diets. ítem C diet PF diet Ingredient, (g/kg) Purified casein 150 Maize s t a r c h 300 460 Lard 15 15 Sunflower h u l l s 170 170 Wheat straw 170 170 Purified cellulose 70 70 Pectinst 60 60 Sodium chloride 5.0 5.0 Calcium c a r b o n a t e 10 10 Dicalcium p h o s p h a t e 10 10 Sepiolite* 23 23 V i t a m i n / m i n e r a l premix§ 5 5 Sunflower h u l l / w h e a t straw + 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; vitamin E, 20 l U / k g diet; thiamine, 1; vitamin A, 8,375 l U / k g , a n d vitamin D3, 7 5 0 l U / k g . * Sunflower hull a n d w^heat stravi? (1:1) m a r k e d with Ytterbium.. 41.

(46) Chapter II: Experiraent 1 Table 2. Chemical composition. ofthe experimental. ítem Nitrogen Starch NDF ADF ADL NDF-Nt GE ( M J / k g DM) DE ( M J / k g DM) Arginine Cystine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Valine Alanine Aspartic acid Glutamic acid Glycine Proline Serine Tyrosine t NDF-N = nitrogen bound to the NDF. 3.2. Anim.als and. diets. C diet 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. fe/kg DM basis) PF diet 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. housing. Twenty eight New Zealand White x Californian doe r a b b i t s weighing 3 7 9 1 ± 141 g were surgically fitted with a glass T - c a n n u l a a t ileum, 10 to 15 cm before t h e ileo-caeco-colic junction, according to t h e procedure described by Gidenne et al. (1988), a n d following the principies for care of a n i m á i s in experimentation (Spanish Royal Decree 2 2 3 / 8 8 , 1988). After a 5 weeks. 42.

(47) 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.

(48) Chapter II: Experiment 1. diet followed the s a m e procedure described above for ileal sampling b u t wearing a wooden collar from two days before starting the ileal collection period (Figure 1).. Figure 1 .•Experimentalprocedure. ofthe casein-based. free diet (PF-diet) allotuing or not. caecotrophy. C-Dict. diet (C-diet). Ileal Collection Adaptation Period. Practicing caecotrophy. ll:O0h. lOd. 0(1. lid. andprotein-. Soft Facces Collection. 20:00h. 14d. 17d. 20d. Ileal Collection. Adaptation Period. lliOOh. 2Q:00h. Not practicing caecotrophy Od. PF-Diet Ileal Collection ' ii;o();. Adaptation Period. Soft Faeces Collection. Practicing caecotrophy 4d. Sd. 6d. 12d. Ileal Collection Adaptation Period Not practicing caecotrophy Od. 2d. 4d. 5d. 6d. Ileal flows of nitrogen a n d amino acids were d e t e r m i n e d by the dilution t e c h n i q u e of a m a r k e r (mordant ytterbium). The indigestible fraction of nitrogen contained in t h e fibre s o u r c e s (NDF-N) of t h e experimental diets constitute a source of exogenous losses at the e n d of t h e ileum which w a s t a k e n into a c c o u n t according to Schulze et al. (1994). The a m o u n t of e n d o g e n o u s ileal nitrogen w a s calculated a s foliows:. 44.

(49) 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. NDF-NF. ENF (g/d). = nitrogen bound to NDF flow. = TNF - N D F - N F. 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. E N F (g/d). =TNF-NDF-NF. 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.

(50) 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). Neutraldetergent. 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.