Utilization and Metabolism of Ammonium Citrate in Growing Pigs Fed Diets Deficient in Non-Essential Amino Acid-Nitrogen
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Utilization and metabolism of ammonium citrate in growing pigs fed diets deficient in non-essential amino acid-nitrogen by Wilfredo D. Mansilla A Thesis presented to The University of Guelph for the degree of Doctor of Philosophy in Animal Biosciences Guelph, Ontario, Canada ©Wilfredo D. Mansilla, August, 2017 ABSTRACT Utilization and metabolism of ammonium citrate in growing pigs fed diets deficient in non-essential amino acid-nitrogen Wilfredo D. Mansilla Advisors: University of Guelph, 2017 Dr. C. F. M. de Lange Dr. J. P. Cant With reductions in crude protein (CP) levels and higher supplementation of crystalline essential amino acids (EAA) in swine diets, the supply of non-essential amino acids (NEAA), or nitrogen (N) required for the synthesis of NEAA, is also reduced increasing the ratio between EAA-N and total N in the diet. When diets are deficient in NEAA-N, non-protein N (NPN) could supply additional N required for the endogenous synthesis of NEAA. The main objective of the present thesis was to assess the efficiency of ammonia for providing extra N when diets are deficient in NEAA-N. Secondary objectives were to determine the effects of ammonia supplementation on the amino acid (AA) profile of retained protein as an indicator of AA requirements and quantification of ammonia absorption and metabolism in the portal-drained viscera (PDV) and liver. Added ammonia to a NEAA-N deficient diet increased body weight (BW) gain and N retention and rendered similar efficiencies as supplemented Glu and a mix of NEAA indicating that the capacity for NEAA synthesis was not limiting. Moreover, ammonia did not modify (P > 0.05) the AA profile of retained protein compared to the NEAA mix implying that feeding ammonia, and consequently increasing the endogenous synthesis of NEAA, does not alter utilization of EAA for protein deposition, maintaining estimated requirements for EAA in growing pigs. When added in the diets, ammonia was rapidly absorbed by the PDV with the peak of absorption within 15 min after feed intake. Recovery of ammonia in the portal vein only accounted for 53% of added ammonia without increasing urea net flux, implying ammonia utilization in the PDV. Across splanchnic organs, appearance of Ala, Glu and citrulline (Cit) increased with ammonia supplementation. The results demonstrate that dietary ammonia is an efficient N source when diets are deficient in NEAA-N, and that the capacity for de novo synthesis of NEAA is not limiting under these circumtances. The latter should be considered when estimating minimal inclusion levels of NEAA in the diet. RESUMEN Uso y metabolismo del citrato de ammonio en cerdos en crecimiento alimentados con una dieta deficiente de nitrógeno proveniente de amino ácidos no esenciales Wilfredo D. Mansilla Asesores: Universidad de Guelph, 2017 Dr. C. F. M. de Lange Dr. J. P. Cant Con la reducción de los niveles de proteína y la mayor inclusión de amino ácidos esenciales (AAE) sintéticos en las dietas de cerdos, el contenido de amino ácidos no esenciales (AANE), o del nitrógeno (N) necesario para la síntesis de AANE, se reduce. Cuando las dietas son deficientes en N de AANE, el N no proteico podría proveer el N adicional requerido para la síntesis endógena de AANE. El objetivo principal de la presente tesis es el de evaluar la eficiencia de ammonia para proveer N adicional cuando las dietas son deficientes en N de AANE. Los objetivos secundarios son determinar el efecto de suplementar ammonia en la proporción de amino ácidos (AA) de la proteína retenida como indicador de los requerimientos de AAE y cuantificar la absorpción y el metabolismo de ammonia en los órganos drenados por la vena porta (ODVP) y el hígado. La amonia añadida en la dieta incrementó ganancia de peso diaria y la retención de N, y tuvo una eficiencia similar a la suplementacion de glutamato y la mezcla de AANE indicando que la capacidad para la síntesis endógena de AANE no limitó el cremimiento. La supplementación de amonia no modificó la proporcición de AAE en la proteína retenida comparado a mezcla de AANE suplementada indicando que añadir amonia en la dieta, y por consiguiente el incremento en la síntesis endógena de AANE, no altera la utilización de los AAE para la síntesis de proteína, manteniendo los requerimientos de AAE. La amonia añadida en la dieta fue rápidamente absorbida por los ODPV, con un pico de absorbción dentro de 15 min después de consumir el alimento. La aparicion de ammonia en la sangre de la vena porta representó solo 53% del total de amonia consumida sin aumentar la aparicion de úrea indicando que la amonia fue utilizada por aquellos órganos. Post-metabolismo de los ODPV y del hígado, la aparicion de Ala, Glu y Citrulina incrementó con la suplementación de amonia. Éstos resultados demuestran que la amonia es una fuente de N eficiente cuando las dietas son deficientes en N de AANE, y que la capacidad para la síntesis endógena de AANE no es limitante en las condiciones estudiadas. Lo último debe ser considerado cuando se recomienden niveles mínimos de AANE en la dieta. In memory of Kees Thanks for giving me this opportunity vi ACKNOWLEDGEMENTS My most sincere gratitude to Dr. Kees de Lange for giving me the opportunity of pursuing and completing this dream. He was not only an excellent advisor, but a great person; I will always consider a privilege being one of his students. I would like to thank my second advisor, Dr. John Cant, for his guidance during the last year of my doctorate; this thesis would have not been completed without his advice and our long hours of discussion in his office. Thanks to the members of my advisory committee, Drs. Brian McBride, Anna-Kate Shoveller, and Hélène Lapierre, for their patience and support during the process of writing this thesis; they all have made important contributions during analysis of the data and interpretation of the results. To Dr. John K. Htoo, from Evonik Nutrition & Care, for his suggestions during the design of the studies and for providing all the crystalline amino acids needed in all the experiments. To Dr. Martin Nyachoti, from University of Manitoba, for opening the doors of his lab and for collaborating in our research. Many thanks to all the technicians and former and present students from our lab, The de Lange lab; conducting an animal experiment goes far beyond from what the reader can see in this thesis. I could have not done it without their invaluable help. I have to individually thank Julia Zhu, our technician and surgeon master, for her input during the development of the multicatheter technique presented in this thesis. Thanks to all other students, summer students, and volunteers for their help, it is always well appreciated. To my family for their unconditional support through all this time and distance. Special thanks to my mother for her idea on improving the pockets that hold the catheters in place; her small ideas always made big difference. To my former advisor in Perú, Dr. Carlos Vílchez, for initiating this amazing experience. The studies presented in this thesis were funded by Evonik Nutrition & Care, Ontario Pork, OMAFRA and Swine Innovation Porc, thanks to them all. vii LIST OF CONTENTS CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW ....................................... 1 1.1. Introduction ......................................................................................................................... 1 1.2. Amino acid requirements in growing pigs .......................................................................... 3 1.2.1. Requirements for essential amino acids .................................................................... 4 1.2.2. Requirements for non-essential amino acids ............................................................. 4 1.3. Estimation of amino acid bioavailability ............................................................................ 6 1.3.1. Amino acid digestibility ............................................................................................. 6 1.4. Essential amino acid nitrogen to total nitrogen ratio .......................................................... 8 1.4.1. Optimum essential amino acid nitrogen to total nitrogen ratio ................................ 9 1.5. Utilization of non-protein nitrogen in non-ruminant diets ................................................ 11 1.5.1. Incorporation of non-protein nitrogen into amino acids and body tissues ............. 11 1.5.2. Non-protein nitrogen as a nitrogen supplement ...................................................... 12 1.6. Ammonia metabolism in the gastrointestinal tract............................................................ 13 1.6.1. Ammonia production in the gastrointestinal tract ................................................... 13 1.6.2. Ammonia absorption in the gastrointestinal tract ................................................... 14 1.7. Ammonia metabolism in the liver ..................................................................................... 15 1.7.1. Ammonia capturing pathways ................................................................................. 15 1.7.1.1. Glutamate dehydrogenase ................................................................................ 16 1.7.1.2. Glutamine synthetase ........................................................................................ 17 1.7.1.3. Urea cycle ......................................................................................................... 18 1.8. Non-essential amino acid production