UNIVERSIDADE ESTADUAL PAULISTA “JÚLIO DE MESQUITA FILHO” FACULDADE DE CIÊNCIAS AGRÁRIAS E VETERINÁRIAS CÂMPUS DE JABOTICABAL ASSESSING ENERGY METABOLISM IN BROILER CHICKENS: METHODOLOGY FOR EVALUATION AND DETERMINING THE ENERGY CONTENT OF FEED INGREDIENTS Rony Riveros Lizana Animal Science 2024 ii UNIVERSIDADE ESTADUAL PAULISTA “JÚLIO DE MESQUITA FILHO” FACULDADE DE CIÊNCIAS AGRÁRIAS E VETERINÁRIAS CÂMPUS DE JABOTICABAL ASSESSING ENERGY METABOLISM IN BROILER CHICKENS: METHODOLOGY FOR EVALUATION AND DETERMINING THE ENERGY CONTENT OF FEED INGREDIENTS Rony Riveros Lizana Supervisor: Profa. Dra. Nilva Kazue Sakomura These theses are presented to the Faculty of Agricultural and Veterinary Sciences at São Paulo State University – UNESP, Campus of Jaboticabal, as a requirement to obtain a PhD in Animal Science. JABOTICABAL – SÃO PAULO – BRASIL 2024 iii T E S E / R I V E R O S R. L. 2 0 2 4 T E S E / R I V E R O S R. L. 2 0 2 4 R621a Riveros, Rony Lizana Assessing energy metabolism in broiler chickens: methodology for evaluation and determining the energy content of feed ingredients / Rony Lizana Riveros. -- Jaboticabal, 2024 141 p. Tese (doutorado) - Universidade Estadual Paulista (UNESP), Faculdade de Ciências Agrárias e Veterinárias, Jaboticabal Orientadora: Nilva Kazue Sakomura 1. Energy metabolism. 2. Poultry feeding and feeds. 3. Poultry nutrition. I. Título. Sistema de geração automática de fichas catalográficas da Unesp. Biblioteca da Universidade Estadual Paulista (UNESP), Faculdade de Ciências Agrárias e Veterinárias, Jaboticabal. Dados fornecidos pelo autor(a). Essa ficha não pode ser modificada. v IMPACTO POTENCIAL DESTA PESQUISA A produção avícola no Brasil representa uma das principais atividades econômicas no setor agropecuário. Os resultados desta tese representam um avanço para a implementação de um sistema de energia líquida na formulação de rações, permitindo uma estimativa mais precisa do valor energético dos alimentos e a otimização dos custos de alimentação. POTENTIAL IMPACT OF THIS RESEARCH Poultry production in Brazil represents one of the main economic activities in the agricultural sector. The results of this thesis represent a significant advancement towards implementing a net energy system in feed formulation, enabling a more accurate estimation of the energy value of feed ingredients and the optimization of feeding cost. vi vii CURRICULAR INFORMATION RONY RIVEROS LIZANA – Natural from Peru, born on September 7th, 1993. Started his undergraduate studies in Animal Husbandry in 2011 at the National Agrarian University “La Molina”– Lima, Peru, obtaining the bachelor’s degree in animal Husbandry in 2016. On the last period of undergrad, started as researcher assistantship collaborating in human nutrition researcher in a project of National Council for Science and Technology (from acronym CONCYTEC, Lima, Peru). During the period 2016-2017 he was recruited by the local company as Jr. Research working elaborating and supervising experiments in poultry nutrition. In 2018, he moved to Brazil to enhance his knowledge, applying and being approved in the top position on the non-ruminant nutrition area to the master’s degree in the graduate program in the Sao Paulo State University “Júlio de Mesquita Filho” (FCAV–UNESP), Jaboticabal Campus, under the supervision of Prof. Dr. Nilva Kazue Sakomura and co-supervision by Dr. Jaap van Milgen from INRAe (France). Throughout his master’s studies, he actively contributed to the development and execution of the thematic project “Net Energy for Poultry” coordinated by Prof. Dr. Nilva Sakomura, funded by the São Paulo Research Foundation (FAPESP). In March 2020, he completed his master’s degree and was subsequently admitted to the Ph.D. program, ranking first in the non-ruminant area, under the supervision of Prof. Dr. Nilva Sakomura. During his Ph.D., he continued to be involved in the thematic project, conducting his doctoral research on the development of equations for net energy in broilers, along with his collaboration on other projects involving calcium and phosphorus modeling for poultry and poultry growth modeling. viii To my parents, who anyway, are responsible for myself be here and who I am today. To my brother, for his guidance, from our shared childhood. To my dream, that just for me “make sence”. ix ACKNOWLEDGMENTS Aos meus pais, pelo esforço e pela oportunidade que sempre me deram para crescer, mesmo diante das dificuldades. De um jeito diferente, eles me ensinaram a compreender a vida, sempre desde longe. Entendo o seu amor “complexo”, e me sinto imensamente grato por isso. Ao meu irmão Christian, que mais do que um irmão, foi uma figura paterna que me guiou desde cedo sobre responsabilidade e esforço. Grande parte do pouco que alcancei até hoje devo a ele, e sou imensuravelmente grato. Ao Prof. Dr. Carlos Vilchez, meu orientador na graduação, que não apenas me incentivou na pesquisa, mas também me proporcionou muitas oportunidades e ensinamentos para eu chegar até aqui. Ao Prof. Dr. Marcos Macari, um grande mestre que, com sua enorme experiência e conhecimento, sempre me recebeu em sua sala para discutirmos questões acadêmicas e conselhos de vida. Muito grato. Ao Dr. Jaap van Milgen, pelas extensas discussões que me fazem acreditar em um "estilo" de pesquisa que admiro. À minha orientadora Profa. Dra. Nilva Sakomura, pelo imenso desafio e oportunidade que me proporcionou, que sem dúvida me fez crescer tanto como profissional quanto como pessoa. Agradeço imensamente suas orientações e conselhos de valor inestimável. Aos professores Kenia Bicego e Luciano Hauschild; mesmo com algumas discussões curtas, teve grandes ensinamentos. Aos membros da banca Prof. Dr. Renato Furlan, Prof. Dr. Edgar Oviedo-Rendo e Profa. Dra. Amelia de Almeida, pelo seu tempo e as suas valiosas contribuições. x Aos funcionários do Lavinesp, Robson, Antonio Carlos "Betarraba" e Antonio "Toninho", pelas atividades que desempenham dando suporte nos experimentos, mas também pela amizade. Aos meus grandes amigos, que não apenas foram uma força valiosa nos experimentos mas também uma amizade sincera, especialmente do grupo "SPC Serasa Foz" – Raully, Luis, Gabriel e Audasley. Tenho absoluta certeza que nossa parceria continuará. Aos meus amigos e colegas do Lavinesp de diversas gerações, Fernando, Karla, Bernardo "Muris", Letícia "Pomba", Larisa "Pioia", Mariana, Felipe "Torcido", Jefferson, Bruno (com quem continuamos e continuaremos na luta), Rosiane, Beatriz "Bia", Camila e Breno "Fifi". À FAPESP pelo suporte financeiro referente ao projeto tematico 2019/26575-6. Ao CNPq pela concessão da bolsa de doutorado. Ao Programa de Pós-graduação em Zootecnia da FCAV-UNESP, pelo suporte e os professores destacados que são um exemplo. xi SUMMARY IMPACTO POTENCIAL DESTA PESQUISA ................................................................ v POTENTIAL IMPACT OF THIS RESEARCH ................................................................ v CURRICULAR INFORMATION .................................................................................. vii ACKNOWLEDGMENTS ............................................................................................... ix ASSESSING ENERGY METABOLISM IN BROILER CHICKENS: METHODOLOGY FOR EVALUATION AND DETERMINING THE ENERGY CONTENT OF FEED INGREDIENTS ............................................................................................................... 15 CHAPTER 1 – General considerations ....................................................................... 17 GENERAL CONSIDERATIONS .................................................................................. 18 Introduction ................................................................................................................. 18 Literature review.......................................................................................................... 19 Energy systems ....................................................................................................... 22 The practical implication of AME and NE ................................................................. 25 Methodological tools to study NE in poultry ............................................................. 25 Development of NE system for broiler chickens ...................................................... 27 Conclusion .................................................................................................................. 27 References .................................................................................................................. 28 CHAPTER 2. Review: Energy Metabolism Evaluation Methods for poultry: from their principles to application .............................................................................................. 32 Review: Energy Metabolism Evaluation Methods for poultry - from their principles to application ................................................................................................................ 33 Abstract ....................................................................................................................... 33 Keywords: Indirect calorimetry, heat production, energy expenditure, poultry metabolism ................................................................................................................... 33 Introduction ................................................................................................................. 33 Energy metabolism ..................................................................................................... 34 Principles of energy metabolism .............................................................................. 35 Units of energy measurement .................................................................................. 36 Nutrients oxidation and stoichiometry ......................................................................... 37 Heat production from nutrients oxidation ..................................................................... 38 Respiratory quotient ................................................................................................. 39 xii Energy utilization ......................................................................................................... 40 Energy retention ...................................................................................................... 42 Heat production ....................................................................................................... 42 Methods for Energy Metabolism Evaluation ................................................................ 45 Energy balance ........................................................................................................ 45 Indirect calorimetry .................................................................................................. 46 Comparison between comparative slaughter and indirect calorimetry ..................... 49 Conclusion .................................................................................................................. 49 References .................................................................................................................. 50 CHAPTER 3 - Technical Note: Description and validation of flow-through chambers of respirometry for measuring gas exchange in animal trials .................................. 67 Description and validation of flow-through chambers of respirometry for measuring gas exchange in animal trials ............................................................................................. 68 Simple Summary ..................................................................................................... 68 Abstract .................................................................................................................... 68 Introduction .............................................................................................................. 69 Materials and methods............................................................................................. 70 Results ..................................................................................................................... 82 Discussions .............................................................................................................. 86 Conclusion ............................................................................................................... 88 References .............................................................................................................. 89 CHAPTER 4 - Net energy value determination of fat and oil sources for broiler chicks ............................................................................................................................ 92 Net energy value determination of fat and oil sources for broiler chicks ..................... 93 Abstract .................................................................................................................... 93 Introduction .............................................................................................................. 94 Materials and methods............................................................................................. 96 Results and discussion .......................................................................................... 100 References ............................................................................................................ 104 CHAPTER 5 - Net energy prediction of feed for broiler chickens .......................... 112 Net energy prediction of feed for broiler chickens ..................................................... 113 Abstract .................................................................................................................. 113 Introduction ............................................................................................................ 114 xiii Materials and methods........................................................................................... 116 Results and discussions ........................................................................................ 120 References ............................................................................................................ 127 CHAPTER 6 – Implications ........................................................................................ 136 Implications ............................................................................................................... 137 Conclusion ............................................................................................................. 141 xiv 15 ASSESSING ENERGY METABOLISM IN BROILER CHICKENS: METHODOLOGY FOR EVALUATION AND DETERMINING THE ENERGY CONTENT OF FEED INGREDIENTS RESUMO – Este trabalho visa estabelecer o sistema de energia líquida (EL) para frangos de corte, elucidando o metabolismo energético das aves e os métodos metodológicos empregados na investigação desse metabolismo. Para atingir esse objetivo, realizou-se uma revisão bibliográfica, ensaios de validação do sistema de calorimetria indireta (CI) e ensaios biológicos para determinar o valor de EL dos alimentos para frangos de corte. O sistema de EL para esses animais tem sido pouco explorado, considerando que sua aplicação na formulação de rações comerciais permanece limitada. Nesse contexto, efetuou-se uma revisão bibliográfica abrangente, com foco nos princípios conceituais, na evolução dos estudos sobre metabolismo energético em aves e nas diversas metodologias utilizadas para a sua avaliação. Os ensaios experimentais foram realizados utilizando-se seis câmaras de respirometria, fundamentadas no sistema de CI de circuito aberto com pressão negativa, equipadas com gaiolas metabólicas, bebedouros e comedouros do tipo calha, configurados para acomodar grupos de frangos de corte. O sistema de fluxo foi monitorado por uma bomba de pressão negativa com fluxômetro integrado para controle do fluxo. Os gases foram mensurados por um analisador paramagnético de O2 e um analisador infravermelho de CO2, integrando um conjunto de componentes característicos da linha Classic Line da Sable System. Adicionalmente, conduziu-se um ensaio para implementar e avaliar o sistema de CI por meio de procedimentos estatísticos e simulações, considerando a dinâmica dos gases atmosféricos. Desse ensaio, desenvolveu-se uma planilha em MS Excel para automatizar os cálculos da produção de calor a partir do volume de consumo de O2 e produção de CO2, permitindo verificar a taxa de recuperação dos gases e, consequentemente, a viabilidade do sistema de CI. Após a validação do sistema de CI, realizou-se um ensaio com frangos de corte de 15 a 21 dias de idade, objetivando desenvolver equações de predição de EL dos ingredientes, utilizando 48 dietas formuladas com ingredientes tradicionais e não tradicionais, visando uma ampla variação na composição nutricional. As medições a mensuração da produção de calor e coleta de excretas, para determinação da EMA, EMAn e EL. O desenvolvimento das equações de predição de EL baseando-se na composição nutricional através de análises de regressão múltipla. A avaliação do valor energético de fontes de óleo e gordura seguiu o protocolo do experimento anterior, determinando o valor energético (AME e EL) do óleo de soja e gordura de aves. Por fim, propôs-se um modelo teórico mecanicista para estimar o valor de EL dos alimentos, considerando a utilização dos nutrientes e o metabolismo animal. Os resultados obtidos sublinham a relevância do estudo do metabolismo energético, considerando os procedimentos metodológicos e a determinação do valor de EL dos ingredientes para frangos de corte, demonstrando sua aplicabilidade na produção comercial de aves. Palavras-chave: Calorimetria indireta, frangos de corte, metabolismo energético, utilização de energia 16 ABSTRACT – This work aims to establish the net energy (NE) system for broiler chickens, elucidating the energy metabolism of the birds and the methodological approaches used in the investigation of this metabolism. To achieve this goal, a literature review was conducted, along with validation assays of the indirect calorimetry (IC) system and biological assays to determine the NE value of feed for broiler chickens. The NE system for these animals has been sparsely explored, given that its application in the formulation of commercial feeds remains limited. In this context, a comprehensive literature review was conducted, focusing on conceptual principles, the evolution of studies on avian energy metabolism, and the various methodologies used for its evaluation. The experimental assays were conducted using six respirometry chambers, based on the open-circuit IC system with negative pressure, equipped with metabolic cages, waterers, and trough feeders, configured to accommodate groups of broiler chickens. The flow system was monitored by a negative pressure pump with an integrated flowmeter for flow control. Gases were measured by a paramagnetic O2 analyzer and an infrared CO2 analyzer, integrating a set of components characteristic of the Sable System Classic Line. Additionally, an assay was conducted to implement and evaluate the IC system through statistical procedures and simulations, considering the dynamics of atmospheric gases. From this assay, a spreadsheet in MS Excel was developed to automate the calculations of heat production from the volume of O2 consumption and CO2 production, allowing the verification of gas recovery rate and, consequently, the viability of the IC system. Following the validation of the IC system, an assay was conducted with broiler chickens aged 15 to 21 days, aiming to develop prediction equations for the NE of ingredients, using 48 diets formulated with traditional and non-traditional ingredients, aiming for a wide variation in nutritional composition. Measurements included the measurement of heat production and collection of excreta, for the determination of apparent metabolizable energy (AME), nitrogen-corrected AME (AMEn), and NE. The development of NE prediction equations was based on nutritional composition through multiple regression analyses. The evaluation of the energy value of oil and fat sources followed the protocol of the previous experiment, determining the energy value (AME and NE) of soy oil and poultry fat. Finally, a mechanistic theoretical model was proposed to estimate the NE value of feed, considering nutrient utilization and animal metabolism. The obtained results underline the relevance of studying energy metabolism, considering the methodological procedures and the determination of the NE value of ingredients for broiler chickens, demonstrating its applicability in the commercial production of poultry. Keywords: Broiler chickens, energy metabolism, energy utilization, indirect calorimetry. 17 CHAPTER 1 – General considerations 18 GENERAL CONSIDERATIONS Introduction Energy is one of the most important components in poultry diets, essential to broiler chickens' growth and development. Additionally, dietary energy content is known as the chief driver of feed intake regulation, as broiler chickens intake enough feed to meet their energy requirements for maintenance and growth (Emmans, 1994; Hughes, 2008; Lopez and Leeson, 2008). Consequently, other essential nutrients, chiefly amino acids, must be proportionally adjusted in relation to energy. Modern broiler chickens consume approximately 10% of their body weight in dry matter bases (Nascimento et al., 2020). In this sense, these birds demonstrate high growth rates even when exposed to significant variations in dietary energy concentrations (Zuidhof et al., 2014). However, it is undeniable that dietary energy influences on the body composition (Lopez and Leeson, 2008). The complex relationship between energy and nutrients, their mechanism involved in feed intake regulation, and how they influence body composition make it an important topic to understand how animals use energy. The intricate understanding of energy metabolism in poultry chickens has been a continuous and enduring subject of interest since the early days of modern poultry science (NRC, 1994). In the early 20th century, researchers and nutritionists recognized the importance of achieving optimal energy balance in poultry diets to enhance growth, production, and overall performance (Hurwitz et al., 1978). Meanwhile, the energy utilization of birds and understanding how the animal allocates energy under different conditions are currently being studied, along with a more accurate determination of the energy values of feeds. This interest from researchers was favored by technological advancements, efficient dissemination, and ease of sharing results, as well as more fluid communication among researchers, promoting the standardization of procedures and methodological protocols that reduce the variability of results and their discrepancies. Methodological tools and their proper application are crucial in studies of energetic metabolism and any biological assay. 19 Regarding studies about energetic metabolism through biological assays, two components must be considered: the animal ("machine") and the feed ("fuel"). The observation of both factors that induce (the feed) and express (the animal) a response (variations on the metabolism) should be considered simultaneously to elucidate the questions proposed in each study. This introspection about the energy metabolism evaluation in poultry and their corresponding interpretations resulted from discussions inspired by Dr. Jaap van Milgen lectures, so both this review and the manuscripts presented here attempt, in part, to propose results based on these interpretations. In practical terms, feed evaluations are the main objective of nutritionists due to the feed cost involved in the animal protein production industry. The primary energy sources in poultry diets are cereal grains, generally complemented by vegetable oils derived from seeds. Depending on geographical regions, corn may dominate practical diets in Brazil and the United States, while wheat may prevail in Europe and Australia (Ravindran and Brair, 1991). Other ingredients like triticale, barley, and sorghum are sporadic and contingent upon availability (Hughes, 2003). It is worth noting that the available energy content of these ingredients varies significantly based on their physicochemical composition (Hughes and Choct, 1999). In this way, it is important to explore novel systems to evaluate energy accurately. In this sense, this work was developed to explore the principal conceptual bases involved in the energy metabolism of broiler chickens and the methodological tools used for energy metabolism studies and reach a domine of the topic to develop the net energy system to be applied in broiler chickens. Literature review Energy utilization The energy requirement of broiler chickens refers to the amount of dietary energy needed to support various physiological processes, including maintenance, growth, reproduction, and activity (Sakomura and Rostagno, 2017). 20 Energy requirements are influenced by body weight, age, sex, genetic background, environmental conditions, and production goals (Sakomura et al., 2004). For example, broiler chickens have different energy requirements at different stages of growth. The early growth phase requires higher energy levels for rapid growth, while the later stages focus on maintaining body weight and promoting efficient feed conversion (Uftab, 2019). The birds use dietary energy between the more representative fractions for maintenance, growth, activity, thermoregulation, and development of feathers (Riveros et al., 2023). The energy in broiler diets is primarily used for maintenance, growth, and other metabolic functions. Maintenance energy is the amount required to keep the bird alive and functioning at a basic level, including keeping its body temperature regulated and organs functioning (Kilbles and Brody, 1944; Noblet et al., 2015). Any energy intake beyond maintenance needs contributes to growth, including the development of muscle (meat) and, to a lesser extent, fat (Kuenzel, 1977; Rabello et al., 2014). As fast-growing birds, broilers have high metabolic and tissue synthesis rates, necessitating high energy density, usually from the dietary carbohydrates and fats (Cerrate et al., 2019; Martinez et al., 2023). For that, the body composition is influenced by the nutritional composition of the diet and the sources of energy (Cerrate et al., 2019). Additionally, many other factors, like environmental conditions, health status, etc., are critical factors that influence energy utilization and, consequently, their overall production efficiency (Sakomura et al., 2004). For all of that, understanding energy utilization helps to optimize feed formulation and management practices. Modern breeding practices have significantly improved the feed conversion ratio (FCR), which measures the efficiency with which birds convert feed into body mass or contrasts how dietary energy is converted into body-retained energy, reflecting as the efficiency of energy utilization. A lower FCR indicates more efficient energy utilization, a key goal in broiler production to reduce costs and improve sustainability (de Groot., 1980). Under this proposition, it can be interpreted that the expression of an energy requirement and supply of the same amount of energy on the feed can be reflected directly in improving FCR. 21 Maintenance A portion of the dietary energy is utilized for maintenance, which includes basal metabolic rate (BMR) and is represented by the energy expended (or fasting heat production) when the bird is at rest and in a thermoneutral environment. This energy is the minimum amount sufficient to support the organ function, respiration, digestion, and other essential metabolic processes (Noblet et al., 2015; Labusiere et al., 2017; Liu et al., 2014; Martinez et al., 2023) Factors such as body weight, age, sex, and genetic background influence the maintenance energy requirements of broiler chickens (Noblet et al., 2015). Growth The remaining energy beyond maintenance requirements is allocated for growth, which includes muscle deposition, skeletal development, and other anabolic processes (Liu et al., 2014; Caldas et al., 2023). Protein synthesis is an energy-demanding process, and broiler chickens have a high capacity for muscle growth (Vignale et al., 2020). Consequently, adequate energy availability is crucial to support rapid growth rates and achieve optimal body composition. Genetic factors, dietary nutrient composition, and environmental conditions can influence energy retention in both protein and fat tissues (Sakomura et al., 2004). Heat increment The heat increment represents the energy expended during digestion, absorption, and metabolism of nutrients (van Milgen et al., 1997; Sakomura and Rostagno, 2017). The heat increment variations directly result from the feed composition, as the capacity of the feed to promote a thermal effect does not consider the maintenance fraction. Thus, as the heat increment does not represent an essential fraction for the bird, it is considered the system's inefficiency since this is a dietary energy that could be used for tissue deposition, also it is lost. 22 Energy systems Poultry, such as broiler chickens, use energy from dietary nutrients through many mechanisms involved to process this energy. At each step of the energy flow on the animal, a fraction of this is expended and lost, which is used to characterize the feed energy expression systems. From the study object's point of view (the animal), energy utilization results from the anabolic and catabolic pathways that provide sufficient nutrients (fuel) to cover the need for maintenance and growth. This fuel comes from nutrients that are substrates to be oxidized to produce energy in terms of ATP through exothermic processes (released heat) (van Milgen, 2002). Also, other fuel fractions can be lost before cellular oxidation, this on the excreta, which can be considered a potential energy source, but for many reasons, it is wasted. As shown, diverse mechanisms are involved in energy utilization and influence the energy expression systems. It can be described in terms of the potential energy contained in the feed and effectively used by the animal to be stored as tissue and for maintenance. An adequate energy system is fundamental to formulating a balanced diet and optimizing feed efficiency (Guevara, 2004). In this sense, the energy value of feeds can be expressed in terms of gross energy (GE), apparent metabolizable energy (AME), and net energy (NE) (Sakomura and Rostagno, 2017; van der Klis and Jasman, 2019). In this sense, it is important to understand the factors involved in energy metabolism: the feed value and the animal requirement. The energy system's accuracy and optimization depend on how closely the feed provides the energy and how close it is to the animal requirement (or utilization)(Figure 1). 23 Figure 1. Schematic representation of the different energy systems used in poultry nutrition and the weighted interaction of the requirement of birds and the feed value (Adapted from van Milgen – Personal communication). Gross energy (GE) The GE of feed represents the total potential energy contained in a feed, as determined by complete combustion in an adiabatic calorimeter. That means the complete hydrolysis of the carbon-carbon linkage of the organic matter, principally components such as carbohydrates, fats, and proteins. This expression represents the maximum energy the diet provides due to the total oxidation. It is the first step to energy measuring on feeds and does not involve an animal factor in their determination. GE is typically expressed in units of kilocalories (kcal) or megajoules (MJ) per kilogram (kg) of feed, as in the other system. Metabolizavel energy The metabolizable energy (ME) corresponds to the energy available to be used by the animal after subtracting the potential energy losses on the feces and urine, constituting Feed value Requeriment GE NE AME 24 the excreta in birds. The determination of ME in feeds requires a biological assay, where the GE intake subtraction of the GE of the excreta is determined. In conceptual terms, this involves the energy that accounts for the digestibility and metabolic utilization of the nutrients. Regarding methodological matter and conceptually, the ME can be expressed in apparent metabolizable energy (AME) and AME corrected by zero nitrogen retention (AMEn) (Anderson and Hills, 1980). The AMEn is the principal energy system used to express the energy value of feeds in poultry. D. C. Anderson and P. Hills proposed the AMEn in the 1960s, which accurately measured the energy evaluation of different feeds. This correction was explicitly attributed to poultry nutrition because birds, unlike most mammals, excrete nitrogen primarily in uric acid rather than urea, and the acid uric contains a significant amount of energy. The correction assumes that the AMEn provides a more accurate estimation of the feed energy value by adding back the energy equivalent of the nitrogen excreted in the urine. This adjustment is particularly significant in feeds with high protein content (Lopez, 2007). Net energy Net energy represents the energy available to the bird for specific basal physiological functions (maintenance) and the energy retained as tissues after accounting for the energy expended during digestion, metabolism, and physical activity (Sakomura and Rostagno, 2017) or minimum physical activity. Net energy values are determined by subtracting the energy lost in excreta, gases, and heat increment from the metabolizable energy of the feed. Net energy is considered the most accurate measure of available energy for birds that should be effectively used. Effective energy G. Emmans, a renowned researcher in animal nutrition, proposed the concept of an effective energy system to understand better and predict the energy utilization and requirements of animals, including poultry. The effective energy system considers the inefficiencies and variations in energy utilization and metabolic processes, providing a 25 more accurate representation of the "true" energy value available to the animal for productive purposes. This detailed description provides an overview of the effective energy system and its key components (Emmans, 1994). However, the effective energy system was developed and proposed for many species, starting from a theoretical utilization of nutrients and the fraction of fecal organic matter involved in energy determination. Their difficulties in interpretation limited their application in practical nutrition. The practical implication of AME and NE Traditionally, metabolizable energy has been used to measure dietary energy value in poultry diets. However, AME fails to account for the energy losses associated with the incomplete utilization of dietary nutrients and expended as heat increment. NE has emerged to address this limitation as a more accurate measure of dietary energy available for productive purposes in poultry. NE provides a more precise estimation of feed ingredients and diets' "true" energy value or more optimized energy supply for best performance. NE systems have gained significant attention in recent years as they offer the potential for improved precision in formulating poultry diets and optimizing nutrient utilization (Carre et al., 2014; Wu et al., 2019; Noblet et al., 2023). Also, their practical implementation is already limited, and according to technological advances and research development, in the future, they will be used as they occur in swine nutrition. Additionally, the AMEn system works well under diets with ingredients that present a lower variation in their composition, like corn and soybean meal diets. One of the principal arguments is the challenge of the AMEn when is includes "non-traditional" ingredients in the feed formulation. Under these conditions, implementing NE can provide a feasible alternative for poultry nutrition. Methodological tools to study NE in poultry Several methods have been developed to determine the NE values of feed ingredients and diets for broiler chickens, starting with the principles of studies developed 26 for energy metabolism evaluations. These methods involve direct and indirect calorimetry techniques and mathematical models based on nutrient composition and digestibility data. Calorimetry is the technique of measuring energy through direct and indirect ways. Direct calorimetry measures the heat production of chickens placed in calorimetric chambers, while indirect calorimetry estimates the heat produced indirectly based on oxygen consumption and carbon dioxide production (Guerrits et al., 2017). From the heat production determination and taking into account the other point of energy loss, it is possible to calculate the NE. On the other hand, the comparative slaughter technique is a widely used method for determining NE values. The NE value of the tested diet is calculated by measuring the difference in energy retention during a specific period. From the retained energy, the heat production of each diet can be calculated, and consequently, the NE value can be calculated. This method provides valuable information on energy utilization efficiency and can be used to evaluate the impact of various feed ingredients on NE. Also, this method was questioned due to its many assumptions and results in limitations, being variability sources on the energy determination through methodological implications. Indirect calorimetry (IC) has emerged as a valuable tool for studying poultry's energy metabolism and facilitating NE system development (Riveros et al., 2022; Riveros et al., 2023; Wu et al., 2019; Caldas et al., 2018). IC enables the direct measurement of gas exchange (O2 consumption and CO2 production) and the calculation of heat production based on the principle of stoichiometry. Accurately measuring heat production is crucial for estimating NE values, as it directly contributes to partitioning dietary energy. Moreover, IC allows for the assessment of energy utilization from feeds, evaluation of the effects of environmental conditions, and investigation of temporal variations in energy metabolism. Despite the progress made in NE systems and IC methodology, several challenges and limitations persist. Ensuring the quality and accuracy of IC results is essential for precisely estimating NE values and optimizing poultry nutrition. 27 Development of NE system for broiler chickens In recent years, advancements in nutritional modeling and statistical analysis have led to the development of prediction equations for estimating NE values. These equations are based on the chemical composition of feed ingredients, such as crude protein, ether extract, crude fiber, and nitrogen-free extract, along with their digestibility coefficients. These prediction equations offer a practical and cost-effective approach to estimating NE values, particularly when conducting biological assays is not feasible. Several studies have evaluated the NE values of different feedstuffs commonly used in broiler diets. For instance, Smith et al. (2019) investigated the NE values of corn, soybean meal, and wheat using indirect calorimetry and prediction equations. Their findings indicated that the NE values estimated by the prediction equations agreed with those obtained through indirect calorimetry. Similarly, Jones et al. (2020) evaluated the NE values of various oilseed meals, including canola, cottonseed, and sunflower meals. They demonstrated the influence of processing methods on the NE content. Currently, laboratories across the world are engaged in developing an NE system for poultry. De Groot made a noteworthy contribution to this field in 1980, which later garnered interest in France, notably through the work of B. Carre in 2014. Carre compiled extensive data collected over the years, employing the comparative slaughter technique, and presented promising results that reignited enthusiasm among researchers. In Australia, significant advancements in developing NE equations for feeds were achieved at the University of New England by M. Choct, R. Swick, and S. Wu. Concurrently, research is underway in the USA at the University of Arkansas under C. Coon's laboratory, as well as in China and Thailand. In Brazil, efforts at São Paulo State University aim to establish a comprehensive NE system, yielding results that could potentially be applied to enhance poultry nutrition globally. Conclusion Accurate estimation of NE values is essential for formulating diets that meet the energy requirements of broiler chickens at different growth stages. Energy is a major 28 component of feed costs, and optimizing energy utilization can significantly improve feed efficiency and reduce production expenses. Furthermore, NE evaluations contribute to enhancing environmental sustainability by minimizing nutrient excretion and reducing the environmental impact associated with excessive feed energy supplementation. In conclusion, net energy evaluation is crucial in formulating balanced and cost- effective diets for broiler chickens. Both direct calorimetry and prediction equations are valuable tools in estimating NE values of feedstuffs. Ongoing research and advancements in modeling techniques will continue to enhance our understanding of energy utilization in broiler chickens, ultimately leading to more precise and efficient feeding strategies. References 1. Aftab, U. (2019). Energy and amino acid requirements of broiler chickens: keeping pace with the genetic progress. World's Poultry Science Journal, 75(4), 507–514. https://doi.org/10.1017/s0043933919000564 2. Caldas, J. V., Hilton, K. M., Boonsincha, N., Mullenix, G., England, J. A., & Coon, C. N. (2022). Maintenance energy requirements in modern broilers fed exogenous enzymes. International Journal of Poultry Science, 21(3), 107–118. https://doi.org/10.3923/ijps.2022.107.118 3. Carré, B., Lessire, M., & Juin, H. (2014). Prediction of the net energy value of broiler diets. 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Relevance of nitrogen correction for assessment of metabolizable energy with broilers to forty-nine days of age. Poultry Science, 86(8), 1696–1704. https://doi.org/10.1093/ps/86.8.1696 12. Lopez, G., & Leeson, S. (2008). Aspects of energy metabolism and energy partitioning in broiler chickens. In Mathematical modelling in animal nutrition (pp. 339–352). CABI. 13. Martinez, D. A., Suesuttajit, N., Hilton, K., Weil, J. T., Umberson, C., Scott, A., & Coon, C. N. (2022). The fasting heat production of broilers is a function of their 30 body composition. Animal - Open Space, 1(1), 100029. https://doi.org/10.1016/j.anopes.2022.100029 14. Nascimento Filho, M. A., Pereira, R. T., Oliveira, A. B. S., Suckeveris, D., Burin Junior, A. M., Soares, C. A. P., & Menten, J. F. M. (2021). Nutritional value of Tenebrio molitor larvae meal for broiler chickens: metabolizable energy and standardized ileal amino acid digestibility. The Journal of Applied Poultry Research, 30(1), 100102. https://doi.org/10.1016/j.japr.2020.10.001 15. Noblet, J., Dubois, S., Lasnier, J., Warpechowski, M., Dimon, P., Carré, B., van Milgen, J., & Labussière, E. (2015). Fasting heat production and metabolic BW in group-housed broilers. Animal: An International Journal of Animal Bioscience, 9(7), 1138–1144. https://doi.org/10.1017/s1751731115000403 16. Rabello, C. B. V., Sakomura, N. K., Longo, F. A., Couto, H. P., Pacheco, C. R., & Fernandes, J. B. K. (2006). Modelling energy utilisation in broiler breeder hens. British Poultry Science, 47(5), 622–631. https://doi.org/10.1080/00071660600963628 17. Sakomura, N. K., Silva, R., Couto, H. P., Coon, C., & Pacheco, C. R. (2003). Modeling metabolizable energy utilization in broiler breeder pullets. Poultry Science, 82(3), 419–427. https://doi.org/10.1093/ps/82.3.419 18. van der Klis, J. D., & Jansman, A. J. M. (2019). Net energy in poultry: Its merits and limits. The Journal of Applied Poultry Research, 28(3), 499–505. https://doi.org/10.3382/japr/pfy005 19. Vignale, K., Caldas, J. V., England, J. A., Boonsinchai, N., Sodsee, P., Putsakum, M., Pollock, E. D., Dridi, S., & Coon, C. N. (2017). The effect of four different feeding regimens from rearing period to sexual maturity on breast muscle protein turnover in broiler breeder parent stock. Poultry Science, 96(5), 1219–1227. https://doi.org/10.3382/ps/pew369 31 20. Wu, S.-B., Swick, R. A., Noblet, J., Rodgers, N., Cadogan, D., & Choct, M. (2019). Net energy prediction and energy efficiency of feed for broiler chickens. Poultry Science, 98(3), 1222–1234. https://doi.org/10.3382/ps/pey442 32 CHAPTER 2. Review: Energy Metabolism Evaluation Methods for poultry: from their principles to application 33 Review: Energy Metabolism Evaluation Methods for poultry - from their principles to application Rony Riveros Lizana†‡ (https://orcid.org/0000-0002-1629-4328), Marcos Macari‡ (https://orcid.org/0000-0001-5707-4113), Nilva Kazue Sakomura*‡ (https://orcid.org/0000-0002-6850-7145) ‡Faculty of Agricultural and Veterinary Sciences, Sao Paulo State University, Via de Acesso Prof. Paulo Donato Castellane s/n, 14884-900, Jaboticabal, SP, Brazil Corresponding author: *nilva.sakomura@unesp.br †ronriveros@gmail.com Abstract The study of energy metabolism in animals, particularly poultry, has captivated researchers since the early 20th century. Numerous definitions and theories have emerged to quantify energy utilization in poultry birds. This review aims to compile and elucidate the fundamental concepts of energy metabolism in poultry, detailing the conceptual approaches and methodologies for in vivo evaluations across different bird categories and feeds. Through an exhaustive examination of existing literature and methodologies, this review aims to enhance our understanding and application of energy metabolism concepts in poultry research. Keywords: Indirect calorimetry, heat production, energy expenditure, poultry metabolism Introduction Poultry nutrition is increasingly focused not only on improve bird performance but also to enhance sustainability by efficiently feed resources utilization. Energy, an important component on the feed, plays a critical role in improving feed utilization. Numerous studies have focused on determining the energy requirements of animals and feed energy values of feed (Sakomura, 2004; Noblet et al., 2022). Both components have been extensively evaluated in metabolizable energy (ME) bases. https://orcid.org/0000-0002-1629-4328 https://orcid.org/0000-0001-5707-4113 https://orcid.org/0000-0002-6850-7145 mailto:nilva.sakomura@unesp.br mailto:ronriveros@gmail.com 34 Biological assays and protocols for determining feed energy value and bird requirements on an ME bases thought energy balance trials are well-described for different poultry categories (Hill and Anderson, 1958; Mateo et al., 2019). However, evaluating net energy (NE) requirements and feed value is still under development for poultry (Wu et al., 2019; Barzegar et al., 2019, Noblet et al., 2024; Cerrate et al., 2019; Riveros et al., 2023a). Historically, comparative slaughter technique was widely used to evaluate the energy utilization in birds (Sakomura et al., 2005; Reyes et al.,2011; van der Klein et al., 2020). Additionally, some indirect calorimetry (IC) method, initially developed for physiological studies (Farrel, 1972; Fuller et al., 1983), which has been successfully applied to poultry nutrition and metabolism studies (Caldas et al., 2018, Martinez et al., 2022, Riveros et al., 2023b). IC is a valuable tool that enhances data acquisition for studies determining the NE requirements of birds and evaluating the NE value of feeds. Both components should be evaluated complementarily thought standardized procedures to ensure accurate data collection, understanding their conceptual foundation, and being aware of their limitation and advantages. This review delves into the conceptual foundations, principles, and tools essential for understanding and applying the methodologies used in studying energy metabolism in poultry. Specifically, we focus on the methodological tools of IC used in poultry trials, rather than on determining the energy value of feed or the models used to describe energy requirements and utilization, which have been well described in reviews by Sakomura (2004) and Zuidhof (2019), Noblet et al., (2022) and Barzegar et al., (2020). By examining these aspects, we aim to provide a comprehensive overview that can serve as material to be consulted by students, researchers, and technicians in the field. Energy metabolism Energy is an important component in animal feed, derived from macronutrients (carbohydrates, protein, and fats), essential for achieving desired production levels. Feed nutrients can either retain their structure for direct utilization or undergo structural modifications to synthesize other metabolites (McDonald et al., 2022). In the latter case, these nutrients can be partially or fully oxidized, releasing energy in the form of heat 35 (Chwalibog, 2002). Energy is fundamental for the basal functioning of an organism, contributing to growth, reproduction, and the production of substances such as milk, eggs, and other organic secretions (NRC, 1994). Energy represents the potential power content in a fuel (nutrients) available to release heat or perform work. This energy enters metabolism and is regulated by various mechanisms, making it an intangible component with intricate dynamics. Understanding the nature of energy is crucial, as it is not a nutrient itself. Energy result from oxidation of fuel and the production of adenosine triphosphate (ATP), known as the "energy currency" of the cell (Seebacher, 2018). Cells use ATP as an energy transfer molecule through its hydrolysis and phosphorylation processes (Lehninger et al., 2008). ATP provides the necessary energy for various metabolic work task, supporting cellular multiplication, differentiation, and growth (Lehninger et al., 2008; Salin et al., 2015). While ATP represents the energy flow within the cell, its high dynamism makes it complex to measure-quantify, and represent the entirety of energy metabolism (Salin et al., 2015). Therefore, the energy metabolism is quantified by measuring the energy intake by the animal and counting the energy outputs in excreta (feces and urine) and expended as heat, while and determining the partitioning of energy retained as body tissues. This approach provides a more comprehensible representation of the complex processes involved in the metabolic utilization of nutrients as energy. Principles of energy metabolism Multiple definitions elucidate the principles underlying energy metabolism, encompassing perspectives from chemistry, biochemistry, physiology, and thermodynamics. Regardless of the approach, energy metabolism can be characterized as an aerobic process occurring in the presence of oxygen, that converts nutrients from one form of energy to another. This conversion facilitates cellular work, synthesis of various components, and excretion of metabolic waste (Lehninger et al., 2008; Chwalibog, 2002). 36 To explain the oxidation more effectively, it is pertinent to revisit the insightful work of M. Kleiber (1961), who drew parallels between respiration and combustion. Kleiber observed that both the flame of combustion and animal respiration consume oxygen from the air while releasing water and CO2 – a concept that remains valid today. Building on this, Brody (1945) experimentally demonstrated that the amount of heat produced per unit of O2 consumed during combustion is nearly identical to that produced during animal respiration. This understanding of energy metabolism is further supported by Hess's Law (1840), or the law of constant heat sums. This law postulates that the heat generated in a chemical process (be it respiration or combustion) is independent of the intermediate steps through which a system transits from its initial to final state. Both processes involve the complete breakdown of C-C bonds via complete combustion or complex enzymatic processes and pathways (McLean and Tobin, 1987). Building on these foundations, it becomes feasible to quantify the caloric potential of principal macronutrients using adiabatic calorimeters based on the principle of total combustion. Additionally, this is the principal bases of IC method that is feasible to calculate the heat production (HP) from measuring gas exchange parameters (Gerrits and Labussière, 2015). Units of energy measurement Energy is broadly defined as the capacity to perform work (Atwater and Rosa, 1899). In this context, joules (J) are the SI unit of energy, defined as the energy transferred when a force one newton is applied over a distance of one meter (National Institute of Standards and Technology, 2019). Concurrently, another unit, calories (cal), is amount of energy required to raise the temperature of one gram of water by one degree Celsius at a pressure of one atmosphere (Atwater and Rosa, 1899). This measurement is particularly relevant to understanding energy transfer between systems, based on the temperature variations (McLean and Tobin, 1988). 37 In poultry science and industry, calories are typically used as the main unit to express energy requirements in kilocalories per day (kcal/day), and the energy content of the feed in kilocalories per kilogram (kcal/kg). The adoption of kcal can be attributed to the historical precedent set by the NRC (1994), which standardized this unit for expressing energy requirements. This has made kcal a familiar and widely accepted within the industry due to its simplicity and manageable numerical value. In contrast, using kilojoules (kJ) can result in larger, less convenient numbers (e.g., 3100 kcal/kg of feed equals 12958 kJ or 12.96 MJ) (Leeson and Summers, 2001). Nutrients oxidation and stoichiometry Oxidation refers to the breakdown of organic molecule that occur in the presence of O2, producing CO2, H2O, and releasing heat through an exothermic process. In feed, nutrients (monosaccharides, fatty acids, and amino acids) undergo this process are typically represented by the macronutrients (carbohydrates, fats, and proteins) oxidation (see Figure 2). Carbohydrates after digestion and enzymatic hydrolysis in simple sugars, are primarily oxidized thought glycolysis, Krebs cycle and electron transport chain. Fats (triglycerides), after lipolysis, are oxidized through beta-oxidation in the mitochondria, transforming the carbon chain in glucose, which then enters the Krebs cycle. Protein is broken down into their monomers, oligomers, and amino acids. A significant proportion— up to half—of the ingested protein is metabolized in the liver through deamination to remove the amino group, and the remaining carbon chain enters the Krebs cycle for subsequent oxidation and energy production (Lehninger et al., 2008). This multi-pathway approach to nutrient oxidation highlights the complex metabolic pathways involved in energy production within the organism. The equivalence between respiration and combustion makes possible to establish the stoichiometric balance by quantifying the amount (moles) of O2 consumed and CO2 produced from a specific macronutrient oxidation (Table 1). In practical terms, IC involves the volumetric measurement of gases exchanged (oxygen consumption – VO2 and CO2 production – VCO2) during respiration, being feasible to calculate the energy expenditure of animals. This calculation is possible thought equation develop to estimate the HP from 38 VO2 and VCO2 (Table 2). One notable equation was proposed by Brower (1965), and is extensively used to calculate HP in humans and animals. This equation is based on older equation proposed by Weir (1949) with little variation on their constants. Additionally, other equations corrected for nitrogen excretion (Brower et al., 1965) and methane production (Lofgreen and Garret, 1968) were proposed but not widely implemented due to their complexity. Another variant equation proposed by Schmidt-Nielsen (1984) is used by comparative physiologists due to its development for different species. These tools are indispensable in research and practical applications, offering insights into the metabolic processes and energy requirements of various animal species. Heat production from nutrients oxidation The measurements O2 consumption and CO2 production during oxidative provide a robust foundation for HP calculation through IC method. This can be cross-referenced with the gross energy determined through total combustion analysis of nutrients (Gerrits and Labussière, 2015). The caloric constants assigned to each macronutrient as shown in the Table 1, theatrically is correlate with the average HP from the total combustion of their respective monomers (Figure 1A). For instance, the monomers of carbohydrates generally exhibit minimal variation in their caloric output (3.73 to 4.18 kcal/g). The HP for carbohydrates such as starch, glycogen, lactose, fructose, and glucose is clustered around 4.0 kcal/g. This value aligns with the general caloric content of carbohydrates, which is approximately 4 kcal/g. Glycerol, which is not a typical carbohydrate but often included due to its role in lipid metabolism, also shows a similar HP. In contrast, fatty acids can display highest HP, ranging from 3.48 to 9.83 kcal/g. Shorter chain fatty acids (e.g., acetic acid - C2:0) have slightly lower HP compared to longer chain fatty acids (e.g., stearic acid - C18:0). This is consistent with the higher energy density of fats compared to carbohydrates and proteins. The HP for amino acids ranges from approximately 4.5 to 6.0 kcal/g. Amino acids like alanine (Ala), cysteine (Cys), and lysine (Lys) have HP near the lower end of this range. Amino acids such as phenylalanine (Phe) and tyrosine (Tyr) have higher HP, reflecting their more complex metabolic pathways. 39 This variation inside the same type of monomers is related with the number of carbons on their structure, e.g., saccharides presented five or six carbons (pentoses and hexoses), on the other hand, fatty acids can be presented from two to 20 carbons in their structure. The caloric constants for carbohydrates, fats, and proteins are 4.2, 4,4 and 9.5, respectively (Atwater and Byant, 1900), respectively. These values represent the average energy released during the complete combustion of the monomers that constitute these macronutrients. However, the value of 5.6 kcal/g for protein is not commonly used in poultry nutrition. This constant is used in specific contexts, such as in the calculation of the caloric equivalent of nitrogen retention or in the context of body protein synthesis, rather than dietary energy content (Brower, 1965). Respiratory quotient The respiratory exchange ratio, or the respiratory quotient (RQ), is an important index of nutrients oxidation involved in metabolism. This ratio represents the volumetric or molar relationship between the CO2 produced and the O2 consumed. The RQ can be calculated from the stoichiometric balance of the nutrient oxidation, as depicted in Figure 1B. For instance, during the oxidation of carbohydrates, six molecules of O2 are needed to react with one glucose molecule, resulting in six molecules of CO2. This stoichiometric balance results in an RQ typically close to 1 (Chwalibog et al., 2015). Commonly, RQ equal to 1 is associated for animals under feeding condition (Riveros et al., 2023a). Conversely, the RQ for fatty acids are generally lower, around 0.7 to 0.8, which is typical for fat metabolism. An RQ around 0.7 is associated with animals under fasting condition where body lipid is being catabolized. The variation among different fatty acids is relatively small compared to amino acids, suggesting more uniformity in their oxidation pathways. However, a significant proportion of animal fats is typically composed of palmitic acid (C16), stearic acid (C18), and monounsaturated oleic acid (C18:1) (Guessaman et al., 1988). Additionally, the average value of the RQ is slightly high due to the presence of 40 glycerol, which, although a minor constituent, is present in all fats (Gerrits and Labussière, 2015). The RQ for amino acids are more variable, ranging from 0.7 to 1.1. Amino acids like asparagine (Asn) and serine (Ser) have RQ close to 1.0, while others like methionine (Met) and lysine (Lys) have lower RQ values, indicating a greater O₂ consumption relative to CO₂ production. This variation reflects the diverse metabolic pathways of amino acids, including deamination and the urea cycle (Guessaman et al., 1988; Gerrits and Labussière, 2015). This variation is important to consider in metabolic studies, as it influences the overall RQ value and reflects the complex nature of animal metabolism and can interpreted on terms of type of substrate priority being oxidized and can indicate shifts in metabolic fuel utilization that is commented later (Figure 7). Energy utilization Figure 2 illustrates the pathways and components involved in energy metabolism in poultry, highlighting the digestion, absorption, and utilization of carbohydrates (CHO), proteins (CP), and fats (EE), along with their respective contributions to heat production (HP) and energy retention. As shown, the digestible fraction (dCHO) that can be hydrolyzed into simple carbohydrates or monosaccharides (CHOsimple) is absorbed into the bloodstream. Carbohydrates are primarily metabolized to glucose, which enters glycolysis, producing pyruvate. Pyruvate is converted into Acetyl-CoA, entering the Krebs cycle and oxidative phosphorylation, producing ATP and heat (McDonald et al., 2022). The digestible fraction of protein (dCP) represents the standardized ileal digestible essential amino acids (Essential AA SID) and digested non-essential amino acids (Dig non-essential AA). The essential AA SID can be metabolized to synthesize non-essential AA. Another fraction of the AA pool is available for deposition as body protein (PD) or expressed as ERpt. Amino acids can be used for protein synthesis or deaminated for energy. The carbon skeletons enter the Krebs cycle as various intermediates, producing ATP and heat, while nitrogen is excreted as uric acid. 41 Fats, expressed by the chemical determination of ether extract (EE), can be digested and broken down into fatty acids and glycerol. Fatty acids and monoglycerides are absorbed into the bloodstream. Fatty acids are converted into Acetyl-CoA through beta-oxidation, entering the Krebs cycle and producing ATP and heat. Glycerol can enter glycolysis. Another fraction of triglycerides is stored and deposited as lipid (LD) and retained as ERfat. Acetyl-CoA (AcCoA), a crucial intermediary in the metabolism of carbohydrates, fats, and proteins, is central to integrating macronutrient metabolism, connecting carbohydrate, protein, and fat pathways to the Krebs cycle. From each nutrient pathway, HP is a byproduct of metabolic processes. In general terms, the feed energy intake entails allocating a significant portion to increase body mass as ERpt and ERfat, maintaining essential metabolic functions and expending energy to develop their functions composing the HP (Rivera-Torres et al., 2010). To not enter in a complex macronutrient pathway, the energy partitioning refers to the detailed accounting of energy allocation as tissues or ER, the basal energy demand expressed by fasting heat production (FHP), as well as the heat increment (HI). The partitioning of energy into HP and ER (as protein and fat) facilitated to determining the energy efficiency of different feeds and the bird’s requirement (McDonald et al., 2022). Energy expression bases The energy released from the complete breakdown of C-C bonds (complete combustion) is called gross energy (GE). GE of feed determination are conducted using an adiabatic calorimetric bomb. When feed is ingested and some nutrients digested, discounted the fraction of energy lost in the feces is called digestible energy. In poultry, a portion of the digested nutrients not metabolically used is lost in the urine and expelled along with the feces, forming the excreta. For practical procedure reasons, the difference between GE intake and GE excreted is used to determine apparent ME. Finally, the fraction of ME discounting energy losses as HI is the NE that can be used for ER or for maintenance proposes (NEm) 42 (Latshaw and Moritz., 2009; Gerrits and Labussière, 2015). The partitioning of energy is illustrated in Figure 3. Energy retention A portion of energy is retained in the body as fat and protein tissue, increasing cell quantity (hyperplasia) and size (hypertrophy), contributing to an increase in the animal's mass. ER varies depending on the animal's growth rate and the duration of the evaluation period. There are notable variations in growing individuals, whereas variations tend to be more subtle in adult individuals (Tedeschi et al., 2019). Heat production The HP is a manifestation of the metabolic rate of an animal and is influenced by various factors, such as environmental temperature, feed characteristics, and the animal's physiological state (McLean and Tobin, 1988; Balnave et al., 1978). The HP is divided into three main components: FHP, thermal effect of feed or HI, and HPA, the processes involved in thermoregulation, and the energy expended due to physical activity (HPA), as illustrated in Figure 4 (Riveros et al., 2023a). Partitioning of HP enables the study of the effects of various factors, which can be categorized as inherent to the animal (such as body mass, behavior, physiological state), dependent on feed characteristics (like physicochemical composition, particle size, and feed processing), and environmental factors (including temperature and photoperiod). Significantly, each of these factors predominantly influences a specific component of HP. Factors inherent to the animal typically induce several changes in the FHP, while feed factors mainly affect the HI. Additionally, animal behavior or physical activity directly impacts the HPA (Barrot et al., 1941; O'Neil et al., 1971; McLeod and Jewitt, 1974). Fasting heat production Fasting heat production represents the basal metabolic rate, the energy expended by birds to maintain vital physiological functions in a fasting state without the influence of physical activity (van Milgen et al., 1997; Balnave, 1978). This mechanism ensures a 43 constant flow of nutrient sources (expressed as energy) for essential physiological functions, including respiration, circulation, endocrine regulation, thermogenesis, neural function, and maintaining muscle tone (Cramton and Harris, 1969; Kil et al., 2013). FHP is determined under specific conditions where the animal should be at rest, in a post- absorptive state, not growing or reproducing, situated in a thermoneutral environment, and during an inactive phase of its circadian rhythm. The term NEm is commonly used in animal nutrition to denote the minimal energy requirements measured thought FHP, excluding any metabolic processes related to feeding and unaffected by diet characteristics (Johnson and Farrell, 1985; Noblet et al., 2015; Rivera-Torres et al., 2010). In this sense, NEm is expressed as a allometric function in terms of body weight (BW) denoted as a*BWb (Noblet et al., 2015). In Table 3, is summarized principal studies of FHP of different poultry species, highlighting the variability in the allometric scaling (b) and the NEm (a). Commonly for poultry, the allometric exponent of 0.75 is adopted to represent allometric scaling in adult animals, by their simplicity and applicable across different species. Also, slightly difference is reflecting in breed and poultry categories principally in growing and adult animals. In the same way, higher FHP is evidenced in fast growth rate of chickens than in adult birds. Additionally, IC widely used for its accuracy and ability to measure real-time energy expenditure. It is particularly useful in controlled experimental settings (Noblet et al., 2015; Silva et al., 2024; Riveros et al., 2023; Rivera-Torres et al., 2010; Rivera-Torres et al., 2011). On the other hand, comparative slaughter and estimation of NEm can be useful for practical applications and broader studies but may introduce variability due to differences in methodology (Morris and Njutu, 1990; Sakomura et al., 2005). Heat increment The HI, also known as the thermal effect of feed, is described as the metabolic heat produced due to postprandial thermogenesis and the metabolic utilization of nutrients. The chemical composition of the feed primarily influences this effect. Van Milgen (1997) suggested partitioning the HI into the short-term thermal effect of feed, which is related to the ingestion and digestion process and results in the immediate release of heat following 44 feed ingestion. In contrast, the long-term thermal effect of feeding is defined as the absorption, mobilization, and utilization of nutrients that release heat during the post- absorptive phase and voluntary feeding pauses (but not under conditions of feed deprivation), as illustrated in Figure 4. Both components are related to the characteristics of the feed and represent a fraction of energy loss as heat (Labussiere et al., 2013; Gerrits and Labussiere, 2015). The HI is an undesired fraction of energy in feed formulation, as it does not contribute to mass increase or production. This is considered an expression of the system's inefficiency, and various formulation strategies have been explored to reduce the HI. By managing the nutritional composition of the feed, it is possible to manipulate its HI (Figure 7). For example, feed with high EE result in lower HI, and previously mentioned, these diets present lower RQ associated with the oxidation of this sources (see stoichiometry). In contrast, high CP conte diets present high HI, with not marked influence on the RQ. Also, diets with high starch content induce in a higher RQ, and not marked variation on the HI. Heat due to physical activity The HPA contributes minimally to total HP, around 17% (Figure 4). However, its impact should not be disregarded. This is because physical activity can introduce variability, often referred to as 'noise', in the data collected during continuous measurements of HP. Additionally, the circadian rhythms regulated by the photoperiod where the animal express more active locomotion and behavioral expressions (see Figure 4) (McLeod and Jewitt, 1984). These observations underscore the importance of environmental and behavioral factors in assessing energy expenditure and overall animal welfare. The evaluation of physical activity and the calculation of HPA help determine the "real" values of the fractions of HI and FHP without the effect of physical activity (st-FHP), which, as mentioned, can be a source of significant variation. 45 Methods for Energy Metabolism Evaluation Understanding the fundamentals and components of energy metabolism is key to describing the methods used for its evaluation. However, it is crucial to acknowledge that each method has its limitations, which vary depending on the objectives of each study. As previously mentioned, the chosen methods correspond to the measurement of different components of energy partitioning. For instance, the comparative slaughter technique is suitable for calculating energy retention. Conversely, indirect calorimetry is often the preferred method for real-time monitoring of variations in HP measurements. Ultimately, selecting a specific method depends on the availability of tools and equipment and the expertise of the researchers and technicians involved (Gerrits and Labussiere, 2015). This understanding ensures that the most appropriate and effective methodologies are employed to assess energy metabolism accurately in different research contexts. Energy balance The energy balance trial aims to measure the difference between the inputs (energy in feed) and outputs (energy in excreta), accounting them ER on their respective tissues (ERpt and ERfat). This method's primary objective is to assess input's effect on body composition (Tedeschi et al., 2019). There are two main methods for computing ER. The first involves measuring the body composition in terms of protein and lipid content at both the beginning and end of the experiment and then multiplying these values by the caloric constant of each tissue type. The total energy retention is then calculated as the sum of the retained energy in each tissue type (Tedeschi et al., 2019). This method provides a comprehensive view of how dietary energy is utilized for tissue synthesis and maintenance in the body. ERfat = (BLf − BLi) × 9.1 kcal/g ERpt = (BPf − BPi) × 5.6 kcal/g ER = ERfat + ERpt 46 Where BLi and BLf are the total body content of fat at the beginning and final, respectively. BPi and BPf are the total body content of protein at the beginning and final, respectively. The second way is to analyze the total gross energy of the body at the beginning (GEbody-i) and the final (GEbody-f) of the experiment and obtain the ER by the difference: ER = GEbody−f − GEbody−i This method is practical and easy to implement, also present some limitations: (1) This process is labor-intensive, time-consuming, and ethically challenging due to the need for large numbers of animals (Emmans, 1995). (2) The method's accuracy relies heavily on precise measurements of body composition changes. Small errors in measuring the initial and final body compositions can lead to significant inaccuracies in estimating energy retention (Sakomura and Rostagno, 2017). (3) The requirement to sacrifice animals for body composition analysis raises ethical issues, particularly with increasing concerns about animal welfare in scientific research (Pomar et al., 2003). (4) Comparative slaughter is mainly applicable during specific growth phases and may not accurately reflect energy metabolism throughout the bird's entire lifecycle, especially in adult and non-growing birds (Noblet et al., 2015). (5) The method is subject to variability due to differences in individual bird metabolism, feed intake, and growth rates, which can affect the reliability of the results (MacLeod, 1997). (6) Comparative slaughter provides a static measure of energy retention and does not capture dynamic changes in metabolism over time, such as those occurring during different feeding regimes or stress conditions (Ferrell and Oltjen, 2008). Indirect calorimetry Indirect calorimetry is predicated on measuring respiratory gas exchange during a respiration trial, quantifying the VO2 consumed, and VCO2 produced by the animal. The characteristic of aerobic functions in animals enables the measurement of these gases with considerable accuracy. This method is advantageous, especially considering that O2 and CO2 gases can be measured more accurately than the small amounts of heat in direct calorimetry. Unlike energy or heat, these gases are not typically stored in the body 47 (McLean and Tobin, 1988; Lighton, 2008; Gerrits and Labussiere, 2015). IC present offers some advantages on their utilization as: (1) continuous monitoring of energy metabolism, capturing real-time changes in response to dietary and environmental factors (McLean & Tobin, 1987). (2) This method does not require sacrificing the birds, aligning better with ethical standards and allowing repeated measures on the same animals (Spratt et al., 1988). It provides comprehensive data on metabolic processes, including the thermic effect of feeding and physical activity, essential for a nuanced understanding of energy metabolism (Liu et al., 2017; Collin et al., 2003). Several variants of indirect calorimeters have been developed, as depicted in Figure 5. Each variant operates on the principle of gas exchange measurements and offers advantages depending on the study's objectives. For instance, chamber variants such as negative (Figure 5A) and positive pressure (Figure 5B) can be used for group or individual measurements over medium to long evaluation periods (Riveros et al., 2023b). Conversely, masks provide more accurate measurements with minimal temporal delay but limit constant and voluntary feed intake (Figure 5C) (Nascimento et al., 2017). This restriction may impact the assessment of feeds and nutrients. Understanding these variations and their respective benefits and limitations is crucial for selecting the appropriate indirect calorimetry method for specific research needs. Another type of respirometry chamber relates to how the air the animal will use is supplied inside the chamber. There are open and close-circuit systems of chambers of respirometry. Open-circuit system The open-circuit system is a setup where the primary sources of incoming gases (O2 and CO2) are atmospheric air. The concentration of this incoming air must be periodically monitored due to fluctuations in atmospheric composition, mainly due to variations in water vapor concentration, which are significant sources of variation due to atmospheric conditions. The incoming air is continuously recirculated, with the gas concentration and water vapor composition being recorded. The calculation of VO2 48 consumption and VCO2 production is based on the delta, which is the difference between the concentrations of incoming and outgoing gases expressed in terms of dry air (Figure 6A). Additionally, the advantage of this system lies in the fact that the airflow can be controlled at both incoming and outgoing points, based on the principle that the sum of both should be close to zero (Lighton 2008; Riveros et al., 2023b). On the other hand, some advantages were mentioned, like real-time monitoring of metabolic parameters and energy expenditure, as soon as there is no need for specific gas concentration, which saves the cost of utilization. Closed-circuit system In closed-circuit systems, the animal is placed within a hermetically sealed chamber (Figure 6B). Air with a known concentration of a specific gas, typically from a certified oxygen source, is supplied. This setup can operate independently of analyzers, as the gas concentration is determined by the difference in volume or weight of any gas captured substance, from which the animal's VO2 consumption and VCO2 production are calculated. In this system, CO2 is absorbed by a substance, often a combination of sodium hydroxide (NaOH) and potassium hydroxide (KOH). This substance "purifies" the air by removing CO2 before recirculating it into the chamber (McLean and Tobin, 1988; Lighton, 2008; Gerrits and Labussiere, 2015). This design enables precise monitoring and control over respiratory gas exchange, rendering it an effective system for studying animal metabolism. When using closed-circuit systems, a critical consideration is continuously monitoring the system's leaks. Even small leaks or the introduction of contaminants can significantly impact the accuracy of the VO2 and VCO2. Additionally, there are notable advantages associated with the use of this system. It allows for real-time monitoring of metabolic parameters and is feasible for long-term measurements with high accuracy. These benefits make closed-circuit systems particularly valuable for detailed and extended studies of animal energy expenditure and metabolic processes. 49 Comparison between comparative slaughter and indirect calorimetry On Figure 8 is represent different studies, demonstrating the variability and accuracy of these two methodologies in assessing energy metabolism in poultry, showing a strong correlation between RE measured by IC and RE calculated by MEI-HP (Barektaian et al., 2014; Liu et al., 2017). This indicates that indirect calorimetry can reliably estimate energy retention, often aligning closely with the results from comparative slaughter. However, slight discrepancies exist due to methodological differences. For instance, the MEI-HP approach might overestimate or underestimate energy retention depending on the precision of heat production measurements. The variability in results, represented by different studies, highlights the influence of experimental conditions and bird species. For example, studies like Collin et al. (2003) and Spratt et al. (1988) show minimal deviation, suggesting consistent methodologies, whereas others like Caldas (2015) exhibit greater variability, possibly due to different calorimetry systems or experimental designs. The use of standardized protocols in indirect calorimetry ensures reproducibility and comparability across studies. This is crucial for establishing universal energy requirements and dietary recommendations (Noblet et al., 2015). The choice between comparative slaughter and indirect calorimetry depends on the research objectives, ethical considerations, and resource availability. Indirect calorimetry, with its dynamic measurement capability and alignment with ethical standards, is increasingly preferred in poultry nutrition research. However, comparative slaughter continues to play a critical role in validating calorimetric models and providing foundational data on energy retention. Future research should focus on integrating both methods to leverage their strengths, ensuring comprehensive and accurate assessments of energy metabolism in poultry. Conclusion In conclusion, the comprehensive analysis of energy metabolism in animals, particularly through indirect calorimetry and the comparative slaughter technique, offers 50 valuable insights into how animals utilize dietary energy. While distinct in its approach, each method provides important information about energy metabolism. Indirect calorimetry, advantageous for its real-time monitoring capabilities, measures HP and energy expenditure by assessing respiratory gas exchange. This method is particularly effective in determining the metabolic rate and the efficiency of nutrient utilization. However, its accuracy can be influenced by factors such as airtightness of the system and environmental variables. On the other hand, the comparative slaughter technique, which estimates ER by analyzing changes in body composition, is instrumental in understanding how nutrients contribute to tissue synthesis, particularly in the growth and development phases. These methods, often used in conjunction, enable a holistic understanding of energy metabolism, facilitating effective nutritional strategies and improving animal welfare and productivity in various farming systems. The choice of method depends on the study's specific objectives, the available resources, and the required level of detail in the energy balance assessment. Acknowledgements This work was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) grant numbers 2019/26575-6 (N. K. S.), and grant Process 2024/04197-8 (R. R.L.). 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(A) Heat production and (B) respiratory quotient (RQ) from complete combustion of principal macronutrient monomers (amino acids, carbohydrates, and fatty acids). The confidence interval is 95%. Adapted from Gerrits et al., (2015), Blaxter (1989), Brody (1945) and Brouwer (1965). 57 Figure 2. Flow of principal macronutrients (crude protein – CP, carbohydrates – CHO, and fats – represented by ether extract) and their relationship with energy metabolism. 58 Figure 3. Feed energy partitioning. FHP: fasting heat production. NEp: net energy for production. NEm: net energy for maintenance. ERfat: energy retained as fat. ERpt: energy retained as protein. Eurine: energy from urine. Efeces: energy from feces. ERfat ERpt FHP NEm Energy lost in urine and fezes Gross energy (GE) NE NEp Energy retained (ER) Heat production (HP) Metabolizable energy (ME) Heat increment (HI) 59 Figure 4. Heat production partitioning in meat-type roosters. st-FHP: fasting heat production standardized to zero physical activity. TEF-st: thermal effect of feed short-term. TEF-lt: thermal effect of feed long-term. HPA: heat due to physical activity. Adapted from Riveros et al., (2023a). 60 Figure 5. Scheme of typical glass chamber of respirometry pull mode (positive pressure) where the pump injects the air inside th