RESSALVA Atendendo solicitação do(a) autor(a), o texto completo desta dissertação será disponibilizado somente a partir de 06/03/2027. UNIVERSIDADE ESTADUAL PAULISTA - UNESP CÂMPUS DE JABOTICABAL WEIGHT LOSS AND ȕ-HYDROXY-ȕ-METHYLBUTYRATE EFFECTS ON AEROBIC FITNESS OF OBESE DOGS Jhennifer de Castro Fenerick Médica veterinária 2025 UNIVERSIDADE ESTADUAL PAULISTA - UNESP CÂMPUS DE JABOTICABAL WEIGHT LOSS AND ȕ-HYDROXY-ȕ-METHYLBUTYRATE EFFECTS ON AEROBIC FITNESS OF OBESE DOGS Discente: Jhennifer de Castro Fenerick Orientador: Prof. Dr. Aulus Cavalieri Carciofi Coorientador: Prof. Dr. Guilherme de Camargo Ferraz Dissertação apresentada à Faculdade de Ciências Agrárias e Veterinárias – Unesp, Câmpus de Jaboticabal, como parte das exigências para a obtenção do título de Mestre em Ciências Veterinárias. 2025 F332w Fenerick, Jhennifer de Castro Weight loss and �-hydroxy-�-methylbutyrate effects on aerobic fitness of obese dogs / Jhennifer de Castro Fenerick. -- , 2025 64 p. : il., tabs. Dissertação (mestrado) - Universidade Estadual Paulista (UNESP), Faculdade de Ciências Agrárias e Veterinárias, Jaboticabal, Orientador: Aulus Cavalieri Carciofi Coorientador: Guilherme de Camargo Ferraz 1. Cães. 2. Metabolismo energético. 3. Aptidão física. 4. Variabilidade do batimento cardíaco. I. Título. Sistema de geração automática de fichas catalográficas da Unesp. Dados fornecidos pelo autor(a). IMPACTO POTENCIAL DESTA PESQUISA A pesquisa contribui para o bem-estar animal e a saúde pública, ao evidenciar os benefícios do emagrecimento em cães obesos, principalmente na melhora de parâmetros cardiovasculares e aptidão física, e ao orientar tutores sobre os riscos da obesidade, promovendo conscientização também sobre a obesidade humana e seus impactos. POTENTIAL IMPACT OF THIS RESEARCH This research contributes to animal welfare and public health by demonstrating the benefits of weight loss in obese dogs, especially in improving cardiovascular parameters and physical fitness, while also raising owner awareness about obesity risks in both pets and humans. DADOS CURRICULARES DO AUTOR Jhennifer de Castro Fenerick nasceu em Ribeirão Preto-SP no ano de 1998. Em 2017 ingressou no curso de Graduação em Medicina Veterinária na Faculdade de Ciências Agrárias e Veterinárias – UNESP, Campus de Jaboticabal. Durante a graduação participou do Grupo de Estudos em Medicina Felina (GEFel), realizou dois projetos de iniciação científica e foi monitora na disciplina de Nutrição de Cães e Gatos. Concluiu a graduação em outubro de 2022. Iniciou o programa de mestrado em Medicina Veterinária na área de Ciências Veterinárias com ênfase em Nutrição de Cães e Gatos em março de 2023. “E Deus viu que tudo era muito bom”. (Gênesis 1:31) AGRADECIMENTOS A Deus, por me conceder força, sabedoria e resiliência para seguir em frente, iluminando meu caminho e guiando cada escolha ao longo desta jornada. Aos meus pais, pelo amor incondicional, pelos ensinamentos que carrego comigo e pelo apoio inabalável em todas as fases da minha vida. Sem vocês, nada disso seria possível. Ao meu namorado, pelo carinho, paciência e compreensão nos momentos de desafios, e por ser minha companhia constante, incentivando-me a nunca desistir dos meus sonhos. À minha família, que sempre esteve ao meu lado, vibrando com cada conquista e me acolhendo nos momentos difíceis. Aos meus amigos, pelo suporte, pelas palavras de encorajamento e por me lembrarem da importância de celebrar cada etapa vencida. Ao meu orientador, professor Aulus, e ao meu coorientador, professor Guilherme, pela orientação, paciência e incentivo ao longo deste trabalho. Obrigada por compartilharem seus conhecimentos, por cada conselho e por acreditarem no meu potencial. Sem a dedicação e apoio de vocês, este projeto não teria sido possível. As minhas queridas amigas de profissão e de vida, Ticiane, Ariel, Maria Eduarda e Stephanie, pela parceria e por me incentivarem e celebrarem comigo em todos os momentos bons ou difíceis. Aos cães que participaram deste projeto, que tornaram cada momento do estudo ainda mais especial. Aos seus tutores, pelo compromisso, confiança e colaboração essencial para a realização desta pesquisa. Aos meus amigos do laboratório (pós-graduandos e funcionários) e as residentes da nutrição clínica, com quem tive o privilégio de conviver nesses anos, por toda a ajuda. À TSI Group pelo apoio financeiro, por acreditarem em mim para desenvolver esse grande projeto. Agradeço, ainda, a todos que, de alguma forma, contribuíram para a concretização deste trabalho, seja com palavras de incentivo, apoio técnico ou simples gestos de motivação. Minha gratidão a cada um de vocês! i SUMÁRIO Página CERTIFICADO DE APROVAÇÃO DA CEUA................................................ ii RESUMO......................................................................................................... iii ABSTRACT..................................................................................................... iv CAPÍTULO 1 – Considerações Gerais 1. REVISÃO DE LITERATURA.......................................................................1 1.1. Obesidade................................................................................................1 1.2. Obesidade e aptidão física ....................................................................2 1.3. Variabilidade de frequência cardíaca e obesidade ............................ 4 1.4. Restrição calórica ................................................................................. 6 1.5. ȕ-hidroxi-ȕ-metilbutirato ...................................................................... 8 1.6. Teste de caminhada de 6 minutos ...................................................... 11 2. HIPOTESES E OBJETIVOS ..................................................................... 13 3. REFERÊNCIAS.......................................................................................... 14 CAPÍTULO 2 - Weight loss and ȕ-hydroxy- ȕ-methylbutyrate effects on aerobic fitness of obese dogs ABSTRACT................................................................................................... 22 INTRODUCTION........................................................................................... 23 MATERIALS AND METHODS...................................................................... 25 Animals …………......................................................................................... 25 Diets and experimental design.................................................................. 26 Body composition evaluation …………………………………..………...… 28 Treadmill habituation ……………………………………………………….… 29 Physical fitness assessment ……………………....................................... 30 Heart Rate and Heart Rate Variability ……………………………………… 30 Surface Infrared Termography ……………………………………………… 31 Lactate and glucose concentrations ………………………………………. 32 STATISTICAL ANALYSIS .......................................................................... 32 RESULTS..................................................................................................... 32 DISCUSSION................................................................................................ 34 CONCLUSION …………………………………………………………….…….. 38 REFERENCES.............................................................................................. 39 TABLES ……………………………………………………………………..….... 46 FIGURES ……………………………………………………………………..…... 52 ii Certificado da Comissão de Ética no Uso de Animais iii WEIGHT LOSS AND Ǻ-HYDROXY-Ǻ-METHYLBUTYRATE EFFECTS ON AEROBIC FITNESS OF OBESE DOGS RESUMO – A obesidade em animais de companhia tem se tornado problema crescente, afetando a longevidade e aumentando a incidência de doenças metabólicas. O excesso de gordura corporal está associado a inflamação crônica, resistência insulínica e disfunções musculoesqueléticas, impactando diretamente na aptidão física e no desempenho aeróbio. Redução da aptidão física e da capacidade cardiovascular em obesos é aspecto pouco estudado em cães, mas que apresenta correlação com qualidade de vida e saúde. A perda de peso é essencial para minimizar esses efeitos adversos, sendo normalmente alcançada por meio de restrição calórica e aumento da atividade física. No entanto, esse processo pode ocasionar perda de massa magra, comprometendo a saúde. Esta dissertação está apresentada em dois capítulos. No capítulo 1 é apresentada revisão de literatura sobre obesidade e seus fatores dietéticos, bem como suas implicações à aptidão física. No capítulo 2 é descrito estudo que avaliou o efeito do emagrecimento com ou sem suplementação de ȕ-hidroxi-ȕ- metilbutirato (HMB) sobre a composição corporal e a aptidão aeróbia de cães obesos alimentados com ração hipocalórica. Foram avaliados 18 cães incialmente obesos que foram submetidos a um programa de perda controlada de 20% do peso corporal. Estes foram divididos entre duas dietas, suplementada com HMB (n = 8) ou controle (n=10). A composição corporal dos cães foi determinada pelo método do óxido de deutério e a avaliação da aptidão aeróbia pela concentração de lactato, glicose, temperatura superficial da carúncula lacrimal, mensuração de frequência cardíaca e variabilidade de frequência cardíaca antes e após os cães serem submetidos à caminhada de seis minutos com carga de peso. Com o emagrecimento cães reduziram a massa gorda em 18.8±1.97%, elevando sua massa magra em 15.9±1.23%. Não houve diferença significativa entre as dietas (P>0,05) para nenhuma das variáveis de aptidão física analisadas. As concentrações médias de lactato e glicose em repouso diminuíram com o emagrecimento (P<0,05). Foi observado menor aumento da temperatura da carúncula lacrimal após o exercício com a perda de peso (P<0,05). O intervalo RR, RMSSD e potência HF aumentaram com o emagrecimento (P<0,05), enquanto a FC em repouso diminuiu (P<0,05). Conclui-se dentro do presente modelo de estudo que a inclusão de HMB em dietas formuladas para perda de peso não demonstrou benefícios à composição corporal ou parâmetros aeróbios. Entretanto, a perda de peso acarretou benefícios relevantes à saúde, modulando a função cardiovascular e melhorando a aptidão física dos cães. Palavras-chave: Cães, metabolismo energético, aeróbio, variabilidade de frequência cardíaca iv WEIGHT LOSS AND Ǻ-HYDROXY-Ǻ-METHYLBUTYRATE EFFECTS ON AEROBIC FITNESS OF OBESE DOGS ABSTRACT - Companion animal obesity has become a growing problem, affecting longevity and increasing the incidence of metabolic diseases. Excess body fat is associated with chronic inflammation, insulin resistance, and musculoskeletal dysfunctions, directly impacting physical fitness and aerobic performance. The reduction in physical fitness and cardiovascular capacity in obese dogs is a poorly studied aspect but has a strong correlation with quality of life and overall health. Weight loss is essential to minimize these adverse effects and is typically achieved through caloric restriction and increased physical activity. However, this process can lead to lean mass loss, compromising overall health. This dissertation is presented in two chapters. Chapter 1 provides a literature review on obesity, its dietary factors, and its implications for physical fitness. Chapter 2 describes a study that evaluated the effect of weight loss, with or without ȕ-hydroxy-ȕ-methylbutyrate (HMB) supplementation, on body composition and aerobic fitness in obese dogs fed a hypocaloric diet. A total of 18 initially obese dogs underwent a controlled 20% body weight loss program. They were divided into two diet groups: one supplemented with HMB (n = 8) and a control group (n = 10). The dogs' body composition was determined using the deuterium oxide dilution method, and aerobic fitness was assessed by measuring lactate concentration, glucose levels, surface temperature of the lacrimal caruncle, heart rate (HR), and heart rate variability (HRV) before and after a six-minute weighted walk test. With weight loss, the dogs reduced their fat mass by 18.8±1.97% and increased their lean mass by 15.9±1.23%. No significant differences were observed between diets (P>0.05) for any of the physical fitness variables analyzed. Mean resting lactate and glucose concentrations decreased with weight loss (P<0.05). A lower increase in lacrimal caruncle temperature after exercise was observed following weight loss (P<0.05). The RR interval, RMSSD, and HF power increased with weight loss (P<0.05), while resting HR decreased (P<0.05). Within the present study model, it is concluded that the inclusion of HMB in weight loss diets did not demonstrate benefits for body composition or aerobic parameters. However, weight loss resulted in significant health benefits, modulating cardiovascular function and improving the physical fitness of the dogs. Keywords: Dogs, energy metabolism, aerobic, heart rate variability 1 CAPÍTULO 1 - CONSIDERAÇÕES GERAIS 1. Revisão de Literatura 1.1 Obesidade A necessidade de se tratar e conhecer adequadamente o tema obesidade dentro da sociedade atual é relevante. O aumento do número de obesos em todo o mundo é alarmante, constituindo importante problema de saúde pública. Na medicina veterinária, a obesidade é uma das enfermidades nutricionais mais frequentes que acometem os animais de companhia. Nos últimos anos, aumentou o número de animais de companhia obesos. Estimativas sugerem que a incidência de obesidade em cães e gatos oscilam entre 35 a 60%(Chandler et al., 2017; Porsani et al., 2020; Montoya et al., 2025). A obesidade é definida como acúmulo de excesso de tecido adiposo corporal (German et al., 2010; Mao et al., 2013; Alonso et al., 2017), sendo associada a diversas doenças que comprometem a qualidade de vida e reduzem a longevidade dos animais. Diversos fatores contribuem para desenvolvimento da obesidade, como idade, sexo, estado reprodutivo e predisposição genética (Carciofi et al., 2005; McGreevy et al., 2005; Colliard, 2006; Coucier et al. 2010; Mao, 2013; Hoenig et al., 2002). No entanto, o manejo alimentar inadequado, associado ao sedentarismo, são os principais fatores que contribuem para a obesidade em cães. O consumo excessivo de energia, aliado ao baixo gasto energético, decorrente da falta de atividade física e da castração, resultam em um balanço energético positivo e, consequentemente, acúmulo de gordura corporal (Bland et al., 2010; Munoz-Prieto et al., 2018). Estudos sugerem que o estilo de vida dos tutores influencia diretamente a alimentação e o nível de atividade física dos cães, uma vez que tutores com estilos de vida sedentária tendem a ter cães menos ativos e com maior risco de obesidade, reforçando a hipótese de que a relação humano-animal impacta diretamente a saúde do pet e que a atividade física é fundamental à saúde dos mesmos (Kienzle, 2002; Mcgreevy et al., 2005; Freeman et al., 2006; Bland et al., 2010; Munoz-prieto et al., 2018). O indivíduo obeso apresenta estado constante de inflamação corporal que frequentemente resulta em várias desordens sistêmicas (Odegaard e Chawla, 2011). A hipertrofia dos adipócitos, condição essencial para desenvolvimento da 2 obesidade, é caracterizada por uma série de alterações metabólicas, como a infiltração de macrófagos, ativação das células endoteliais, aumento do estresse oxidativo e acúmulo de ácidos graxos livres. Estas condições promovem ativação do estado pró-inflamatório, especialmente pela ativação da via Fator nuclear kappa B (NF-kB) (Skurk et al., 2007; Maury e Brichard, 2010). Além do estado inflamatório, os aumentos dos depósitos corporais de gordura estão relacionados com profundas alterações das funções fisiológicas (Gayet et al., 2004). Estas, em sua grande maioria, estão associadas direta ou indiretamente a problemas cardiorrespiratórios, osteoarticulares e desordens metabólicas como dislipidemias, redução da tolerância à glicose, resistência insulínica, e em felinos um dos pontos negativos mais relevantes é a associação entre obesidade e o desenvolvimento de diabetes mellitus (Gayet et al., 2004; German, 2006; German et al., 2009; Cave et al., 2012; Verbrughee et al., 2012; Pereira-Neto et al., 2018). Foge ao objetivo deste texto, no entanto, uma revisão mais extensa das alterações associadas à obesidade. 38 6 CONCLUSION The dose of 87±8.7 mg/kg metabolic weight of HMB in diets formulated for weight loss in dogs showed no benefits in body composition or aerobic parameters of obese dogs in weight loss. Obesity in dogs can induce HRV impairment and weight loss is beneficial in cardiac modulation attributable to possible increase in parasympathetic activity. Short-term HRV testing might have significant potential as a screening tool for cardiac autonomic dysfunction in obese dogs. Further studies with larger sample sizes need to be conducted to confirm the hypotheses generated in this study. Furthermore, the adapted 6-MWT protocol that was applied in the present study proved efficient in generating physical effort in obese dogs and being safe and practical since it induced transient changes in the physiological variables analysed. 39 7 REFERENCES 1. World Health Organization. Taking action on childhood obesity (No. WHO/NMH/PND/ECHO/18.1). World Health Organization (2018). 2. Montoya-Alonso JA, Bautista-Castaño I, Peña C, Suárez L, Juste MC, Tvarijonaviciute A. Prevalence of canine obesity, obesity-related metabolic dysfunction, and relationship with owner obesity in an obesogenic region of Spain. Front Vet Sci. (2017);4:248075. 3. Farcas AK, Michel KE. Small animal obesity. Vet Clin North Am Small Anim Pract. (2016);46(5):Elsevier Health Sciences, USA. 4. Porsani MYH, Teixeira FA, Oliveira VV, Pedrinelli V, Dias RA, German AJ, et al. Prevalence of canine obesity in the city of São Paulo, Brazil. Scientific Reports. (2020);10(1). 5. Montoya M, Péron F, Hookey T, et al. Overweight and obese body condition in :million cats seen at primary practices across the USA 1.3׽ million dogs and 4.9׽ Prevalences by life stage from early growth to senior. Prev Vet Med. (2025);235:106398. 6. German AJ. The growing problem of obesity in dogs and cats. J Nutr. (2006);136:1940S-1946S. 7. Laflamme DP. Understanding and managing obesity in dogs and cats. Vet Clin North Am Small Anim Pract. (2006);36:1283-1295. 8. Arch JR, Hislop D, Wang SJ, Speakman JR. Some mathematical and technical issues in the measurement and interpretation of open-circuit indirect calorimetry in small animals. Int J Obes. (2006);30(9):1322-31 9. Grzemski A, Stachowiak M, Flisikowski K, Mankowska M, Krzeminska P, Gogulski M, et al. FTO and IRX3 genes are not promising markers for obesity in Labrador retriever dogs. Ann Anim Sci. (2019);19:343-357. 10. Jeusette I, Greco D, Aquino F, Detilleux J, Peterson M, Romano V, et al. Effect of breed on body composition and comparison between various methods to estimate body composition in dogs. Res Vet Sci. (2010);88:227-232. 11. Pogány Á, Torda O, Marinelli L, Lenkei R, Junó V, Pongrácz P. The behaviour of overweight dogs shows similarity with personality traits of overweight humans. Royal Society Open Science. (2018) Jun;5(6):172398. 12. Laflamme DP. Nutritional care for aging cats and dogs. Vet Clin North Am Small Anim Pract. (2012);42:769-791. 13. Robertson ID. The association of exercise, diet and other factors with owner- perceived obesity in privately owned dogs from metropolitan Perth, WA. Preventive Veterinary Medicine. (2003);58(1-2):75–83. 40 14. Corbee RJ. Obesity in show dogs. J Anim Physiol Anim Nutr.(Berl) (2013); 97(5):904-910. 15. Jeusette I, Detilleux J, Cuvelier C, Istasse L, Diez M. Ad libitum feeding following ovariectomy in female Beagle dogs: Effect on maintenance energy requirement and on blood metabolites. J Anim Physiol Anim Nutr (Berl). (2004);88:117-121. 16. Spain CV, Scarlett JM, Houpt KA. Long-term risks and benefits of early-age gonadectomy in cats. Journal of the American Veterinary Medical Association. (2004);224(3):372–9. 17. Larson BT, Lawler DF, Spitznagel EL, Kealy RD. Improved glucose tolerance with lifetime diet restriction favorably affects disease and survival in dogs. J Nutr. (2003);133:2887-2892. 18. Nijland ML, Stam F, Seidell JC. Overweight in dogs, but not in cats, is related to overweight in their owners. Public Health Nutrition. (2010);13:102–106. 19. Muñoz-Prieto A, Nielsen LR, Dąbrowski R, Bjørnvad CR, Söder J, Lamy E, et al. European dog owner perceptions of obesity and factors associated with human and canine obesity. Sci Rep. 2018;8(1):13353. 20. German AJ, Hervera M, Hunter L, Holden SL, Morris PJ, Biourge V, Trayhurn P. Improvement in insulin resistance and reduction in plasma inflammatory adipokines after weight loss in obese dogs. Domest Anim Endocrinol. (2009);37(4):214-226. 21. German AJ, Ryan VH, German AC, Wood IS, Trayhurn P. Obesity, its associated disorders and the role of inflammatory adipokines in companion animals. Vet J. (2010);185:4-9. 22. Lund EM, Armstrong PJ, Kirk CA, Klausner JS. Prevalence and risk factors for obesity in adult dogs from private US veterinary practices. Int J Appl Res Vet Med. (2006);4:177-186. 23. Tropf M, Nelson OL, Lee PM, Weng HY. Cardiac and Metabolic Variables in Obese Dogs. Journal of Veterinary Internal Medicine. (2017);31(4):1000–7. 24. Tvarijonaviciute A, Ceron JJ, Holden SL, Cuthbertson DJ, Biourge V, Morris PJ, et al. Obesity-related metabolic dysfunction in dogs: a comparison with human metabolic syndrome. BMC Veterinary Research. (2012);8(1):147. 25. Trayhurn P, Wood IS. Adipokines: inflammation and the pleiotropic role of white adipose tissue. British Journal of Nutrition. (2004);92(3):347–55. 26. Lawler DF, Evans RH, Larson BT, Spitznagel EL, Ellersieck MR, Kealy RD. Influence of lifetime food restriction on causes, time, and predictors of death in dogs. J Am Vet Med Assoc. (2005);226:225-231. 41 27. Manens J, Ricci R, Damoiseaux C, Gault S, Contiero B, Diez M, Clercx C. Effect of body weight loss on cardiopulmonary function assessed by 6-minute walk test and arterial blood gas analysis in obese dogs. J Vet Intern Med. (2014);28:371- 378. 28. Rhyu H, Cho SY. The effect of weight loss by ketogenic diet on the body composition, performance-related physical fitness factors and cytokines of Taekwondo athletes. Journal of Exercise Rehabilitation. (2014) Oct 31;10(5):326– 31. 29. Weiss EP, Jordan RC, Frese EM, Albert SG, Villareal DT. Effects of Weight Loss on Lean Mass, Strength, Bone, and Aerobic Capacity. Medicine & Science in Sports & Exercise. (2017);49(1):206–17. 30. Carciofi AC, Gonçalves KNV, Vasconcellos RS, Bazolli RS, Brunetto MA, Prada F. A weight loss protocol and owners participation in the treatment of canine obesity. Ciênc Rural. (2005);35(6):1331-1338. 31. Diez M, Nguyen P. Obesity: epidemiology, pathophysiology and management of the obese dog. In: Encyclopedia of Canine Clinical Nutrition. Aimargues: Royal Canin.(2006);p. 2-53. 32. German AJ, Holden SL, Bissot T, Hackett RM, Biourge V. Dietary energy restriction and successful weight loss in obese client-owned dogs. J Vet Intern Med. (2007);21(6):1174-1180. 33. Luis LW, Goloni C, Theodoro SS, Tozato MEG., Pacheco LG, Monti M, Carciofi AC. Intake of energy, protein, amino acids and minerals by dogs under energy restriction for body weight loss when fed with commercial weight loss diets. Journal of animal physiology and animal nutrition. (2023);107 Suppl 1, 1–10. 34. Humbert B, Martin L, Dumon H, Darmaun D, Nguyen P. Dietary protein level affects protein metabolism during the postabsorptive state in dogs. J Nutr. (2002);132(6 Suppl 2):1676S-1678S. 35. Jackson JR, Laflamme DP, Owens SF. Effects of dietary fiber content on satiety in dogs. Vet Clin Nutr. (1997);4:130-134. 36. Jewell DE, Edwards GL, Azain MJ, et al. Fiber but not conjugated linoleic acid influences adiposity in dogs. Vet Ther. (2006);7:78-85. 37. Arciero PJ, Edmonds R, He F, Ward E, Gumpricht E, Mohr A, et al. Protein- pacing caloric-restriction enhances body composition similarly in obese men and women during weight loss and sustains efficacy during long-term weight maintenance. Nutrients. (2016);8(8):476. 38. Panton LB, Rathmacher JA, Baier S, Nissen S. Nutritional supplementation of the leucine metabolite ȕ-hydroxy-ȕ-methylbutyrate (HMB) during resistance training. Nutrition. (2000);16:734–739. 42 39. Jowko E, Ostaszewski P, Jank M, Sacharuk J, Zieniewicz A, Wilczak J, Nissen S. Creatine and ȕ-Hydroxy-ȕ-Methylbutyrate (HMB) Additively Increase Lean Body Mass and Muscle Strength During a Weight-Training Program. Nutrition. (2001);17:558-566. 40. Wilson JM, Fitschen PJ, Campbell B, Wilson GJ, Zanchi N, Taylor L, et al. International Society of Sports Nutrition Position Stand: beta-hydroxy-beta- methylbutyrate (HMB). Journal of the International Society of Sports Nutrition. (2013); 2:10(1). 41. Knitter AE, Panton L, Rathmacher JA, Petersen A, Sharp R. Effects of ȕ- hydroxy ȕ-methylbutyrate on muscle damage following a prolonged run. J Appl Physiol. (2000);89:1340-1344. 42. Lamboley CR, Royer D, Dionne IJ. Effects of beta-hydroxy-beta- methylbutyrate on aerobic performance components and body composition in college students. Int J Sport Nutr Exerc Metab. (2007);17:56-69. 43. Vukovich MD, Dreifort GD. Effect of beta-hydroxy beta-methylbutyrate on the onset of blood lactate accumulation and VO2 peak in endurance-trained cyclists. Journal of Strength and Conditioning Research. (2001);15:491–497. 44. Fédération Européenne De L'industrie Des Aliments Pour Animaux Familiers. FEDIAF. Nutritional Guidelines for Complete and Complementary Pet Food for Cats and Dogs. Brussels (2021). 45. Ferrioli E, Pfrimer K, Cruz BM. Uso de isótopos estáveis leves em ciências nutricionais. In: Dutra JEO, Marchini JS, editors. Ciências Nutricionais. 2nd ed. São Paulo: Sarvier; (2008); p. 443-465. 46. Goloni C, Peres FM, Senhorello IL, Di Santo LG, Mendonça FS, Loureiro BA, et al. Validation of saliva and urine use and sampling time on the doubly labelled water method to measure energy expenditure, body composition and water turnover in male and female cats. Br J Nutr. (2020);124(4):457-469. 47. Schoeller DA, Hnilicka JM. Reliability of the doubly labeled water method for the measurement of total daily energy expenditure in free-living subjects. The Journal of nutrition. (1946);126(1), 348S–354S. 48. Ellis KJ, Wong WW. Human hydrometry: comparison of multifrequency bioelectrical impedance with 2H2O and bromine dilution. Journal of applied physiology. (1998); 85(3), 1056–1062. 49. Pace N, Rathbun EN. Studies on body composition. 3. The body water and chemically combined nitrogen content in relation to fat content.(1945) 50. Cerqueira JA, Restan WAZ, Fonseca MG, Catananti LA, de Almeida MLM, Junior WHF, et al. Intense exercise and endurance-training program influence serum kinetics of muscle and cardiac biomarkers in dogs. Res Vet Sci. (2018);121:31-39. 43 51. Pelosi A, Rosenstein D, Abood SK, Olivier BN. Cardiac effect of short-term experimental weight gain and loss in dogs. Veterinary Record. (2012);172. 52. Swimmer RA, Rozanski EA. Evaluation of the 6-minute walk test in pet dogs. Journal of veterinary internal medicine. (2011); 25(2), 405–406. 53. Tarvainen MP, Niskanen JP, Lipponen JA, et al. Kubios HRV—heart rate variability analysis software. Comput Methods Programs Biomed. (2014);113:210– 220. 54. Restan AZ, Camacho AA, Cerqueira JA, Zacché E, Kirnew MD, Loureiro BA, et al. Effect of a lactateǦguided conditioning program on heart rate variability obtained using 24-Holter electrocardiography in Beagle dogs. Bryner RW, editor. PLOS ONE. (2020) Jun 1;15(6):e0233264. 55. Rizzo M, Arfuso F, Alberghina D, Giudice E, Gianesella M, Piccione G. Monitoring changes in body surface temperature associated with treadmill exercise in dogs by use of infrared methodology. Journal of Thermal Biology. (2017);69:64– 8. 56. Albert FJ., Morente-Sánchez J, Ortega FB., Castillo MJ., Gutiérrez Á. Usefulness of ȕ-hydroxy-ȕ-methylbutyrate (HMB) supplementation in different sports: an update and practical implications. Nutr. Hosp. (2015);32( 1 ): 20-33. 57. Arazi H, Asadi A, Hoseini K. The effect of HMB supplementation on cardiovascular risk factors after four weeks of resistance training in amateur athletes. J Sport Med Phys Fitness. (2015);55(1-2):89-93. 58. Oktaviana J, Zanker J, Vogrin S, Duque G. The Effect of ȕ-Hydroxy-ȕ- Methylbutyrate (HMB) on Sarcopenia and Functional Frailty in Older Persons: A Systematic Review. The journal of nutrition, health & aging. (2018);23(2):145–50. 59 Yang C, et al. Effects of beta-hydroxy-beta-methylbutyrate supplementation on older adults with sarcopenia: a randomized, double-blind, placebo-controlled study. The Journal of nutrition, health and aging. (2023); 27(5):329-339 60. Ransone J, Neighbors K, Lefavi R, Chromiak J. The effect of beta-hydroxy beta-methylbutyrate on muscular strength and body composition in collegiate football players. Journal of strength and conditioning research. (2003); 17(1):34– 39. 61. Slater G, Jenkins D, Logan P, Lee H, Vukovich M, Rathmacher JA, Hahn A. G. Beta-hydroxy-beta-methylbutyrate (HMB) supplementation does not affect changes in strength or body composition during resistance training in trained men. International journal of sport nutrition and exercise metabolism. (2001);11(3):384– 396. 44 62. Thomson JS, Watson PE, Rowlands DS. Effects of nine weeks of beta- hydroxy-beta- methylbutyrate supplementation on strength and body composition in resistance trained men. Journal of strength and conditioning research. (2009); 23(3):827–835. 63. Durkalec-Michalski K, Jeszka J, Podgórski T. The effect of a 12-week beta- hydroxy-beta-methylbutyrate (HMB) supplementation on highly-trained combat sports athletes: A randomised, double-blind, placebo-controlled crossover study. Nutrients. (2017);9(7):753. 64. Durkalec-Michalski K, Jeszka J. The effect of ȕ-hydroxy-ȕ-methylbutyrate on aerobic capacity and body composition in trained athletes. J Strength Cond Res. (2016);30(9):2617-2626. 65. Robinson EH, Stout JR, Miramonti AA, Fukuda DH, Wang R, Townsend JR, et al. High-intensity interval training and ȕ-hydroxy-ȕ-methylbutyric free acid improves aerobic power and metabolic thresholds. Journal of the International Society of Sports Nutrition. (2014);11:16. 66. Blüher M. Adipose tissue dysfunction contributes to obesity-related metabolic diseases. Best Pract Res Clin Endocrinol Metab. (2013);27(2):163-177. 67. Trayhurn P. Hypoxia and Adipose Tissue Function and Dysfunction in Obesity. Physiological Reviews. (2013);93(1):1–21. 68. Wood IS, de Heredia FP, Wang B, Trayhurn P. Cellular hypoxia and adipose tissue dysfunction in obesity. Proceedings of the Nutrition Society. (2009); 68(4):370–7. 69. Chondronikola M, Volpi E, Børsheim E, Porter C, Annamalai P, Enerbäck S, et al. Effect of progressive weight loss on lactate metabolism: a randomized controlled trial. Obesity.(Silver Spring) (2018);26(4):683-688. 70. Gayet C, Bailhache E, Dumon H, Martin L, Siliart B, Nguyen P. Insulin resistance and changes in plasma concentration of TNFalpha, IGF1, and NEFA in dogs during weight gain and obesity. Journal of animal physiology and animal nutrition. (2004);88(3-4): 157–165. 71. Jones TE, et al. Plasma lactate as a marker of metabolic health: Implications of elevated lactate for impairment of aerobic metabolism in the metabolic syndrome. Surgery. (2019);166(5):861-866. 72. Santoro A, McGraw TE, Kahn BB. Insulin action in adipocytes, adipose remodeling, and systemic effects. Cell Metabolism. (2021);33(4):748–57. 73. Yarnell K, Fleming J, Stratton TD, Brassington R. Monitoring changes in skin temperature associated with exercise in horses on a water treadmill by use of infrared thermography. Journal of Thermal Biology. (2014); 45: 110-116. 45 74. Elias B, Starling M, Wilson B, McGreevy P. Influences on Infrared Thermography of the Canine Eye in Relation to the Stress and Arousal of Racing Greyhounds. Animals. (2021);11(1):103. 75. Dai F, Fusi J, Vantini S, Gatto F, Cavallari P, Faustini M, et al. Validation of a fear test in sport horses using infrared thermography. J Vet Behav. (2015);10(2):128-136. 76. Stewart M, Stafford KJ, Dowling SK, Schaefer AL, Webster JR. Eye temperature and heart rate variability of calves disbudded with or without local anaesthetic. Physiology & Behavior. (2008);93(4-5):789–97. 77. Riemer S, Assis L, Pike TW, Mills DS. Dynamic changes in ear temperature in relation to separation distress in dogs. Physiology & Behavior. (2016) Dec;167:86– 91. 78. Travain T, Colombo ES, Heinzl E, Bellucci D, Prato Previde E, Valsecchi P. Hot dogs: Thermography in the assessment of stress in dogs (Canis familiaris)— A pilot study. Journal of Veterinary Behavior. (2015);10(1):17–23. 79. Travain T, et al. How good is this food? A study on dogs' emotional responses to a potentially pleasant event using infrared thermography. Physiology & Behavior. (2016); 159: 80-87. 80. Valera M, Bartolomé E, Sánchez MJ, Molina A, Cook N, Schaefer A. Changes in Eye Temperature and Stress Assessment in Horses During Show Jumping Competitions. Journal of Equine Veterinary Science. (2012);32(12):827–30. 81. McNicholl J, Howarth GS, Hazel SJ. Influence of the Environment on Body Temperature of Racing Greyhounds. Frontiers in veterinary science. (2016);3:53. 82. Novelli ELB, Diniz YS, Galhardi CM, Ebaid GMX, Rodrigues HG, et al. Anthropometrical parameters and markers of obesity in rats. Laboratory animals. (2007);41(1):111-119. 83. Allerton TD, Primeaux S.D. High-fat diet differentially regulates metabolic parameters in obesity-resistant S5B/Pl rats and obesity-prone Osborne-Mendel rats. Canadian journal of physiology and pharmacology. (2016);94(2):206-215. 84. Strüven A, Holzapfel C, Stremmel C, Brunner S. Obesity, nutrition and heart rate variability. International journal of molecular sciences. (2021);22(8):4215. 85. Oliveira MS, Muzzi RA, Araújo RB., Muzzi LA, et al. Heart rate variability parameters of myxomatous mitral valve disease in dogs with and without heart failure obtained using 24-hour Holter electrocardiography. The Veterinary Record. (2012);170(24):622-627. 86. Pongkan W, Jitnapakarn W, Phetnoi W, Punyapornwithaya V, Boonyapakorn C. Obesity-induced heart rate variability impairment and decreased systolic function in obese male dogs. Animals (Basel). (2020);10:1383. 46 87. Vieira AB, Restrepo MA, Auzenne D, Molina K, O’Sullivan M, Machado MV, et al. Mild to moderate overweight in dogs: is there an impact on routine hematological and biochemical profiles, echocardiographic parameters and cardiac autonomic modulation? Vet Res Commun. (2022);46:527–35. 88. Bogucki S, Noszczyk-Nowak A. Short-term heart rate variability (HRV) in healthy dogs. Pol J Vet Sci. (2015);18(2):307-312. 89. Manzo A, Ootaki Y, Ootaki C, Kamohara K, Fukamachi K. Comparative study of heart rate variability between healthy human subjects and healthy dogs, rabbits and calves. Lab Anim. (2009);43(1):41-45. 90. Filho, M. S, et al. Influence of weight loss on heart rate variability in dogs. Brazilian Journal of Veterinary Medicine.(2019);41(1). 91. Farah BQ, Prado WL, Tenório TR, Ritti-Dias RM. Heart rate variability and its relationship with central and general obesity in obese normotensive adolescents. Einstein (Sao Paulo). (2013);11(3):285-90. 92. Karason K, Mølgaard H, Wikstrand J, Sjöström L. Heart rate variability in obesity and the effect of weight loss. Am J Cardiol. (1999);83(8):1242-7. 93. Poirier P, Bogaty P, Philippon F, Garneau C, Fortin C, Dumesnil JG. Preclinical diabetic cardiomyopathy: relation of left ventricular diastolic dysfunction to cardiac autonomic neuropathy in men with uncomplicated well-controlled type 2 diabetes. Metabolism. (2003);52(8):1056-61. 94. Cooper TM, McKinley PS, Seeman TE, Choo TH, Lee S, Sloan RP. Heart rate variability predicts levels of inflammatory markers: Evidence for the vagal anti- inflammatory pathway. Brain Behav Immun. (2015);49:94-100. 95. Pinto AM, Sanders TA, Kendall AC, Nicolaou A, Gray R, Al-Khatib H, Hall WL. A comparison of heart rate variability, n-3 PUFA status and lipid mediator profile in age- and BMI-matched middle-aged vegans and omnivores. Br J Nutr. (2017);117(5):669-685.