RESSALVA Atendendo solicitação do(a) autor(a), o texto completo será disponibilizado somente a partir de 28/08/2027 At the author's request, the full text will not be available online until August 28, 2027 UNIVERSIDADE ESTADUAL PAULISTA FACULDADE DE MEDICINA VETERINÁRIA E ZOOTECNIA CAMPUS DE BOTUCATU EFFECT OF ENVIRONMENTAL ENRICHMENT ON BEHAVIOR AND PERFORMANCE OF DAIRY CALVES JOÃO PEDRO DONADIO DA SILVA PEREIRA Dissertação apresentada ao Programa de Pós-graduação em Zootecnia como parte das exigências para obtenção do título de Mestre em Zootecnia BOTUCATU – SP Fevereiro, 2025 UNIVERSIDADE ESTADUAL PAULISTA FACULDADE DE MEDICINA VETERINÁRIA E ZOOTECNIA CAMPUS DE BOTUCATU EFFECT OF ENVIRONMENTAL ENRICHMENT ON BEHAVIOR AND PERFORMANCE OF DAIRY CALVES JOÃO PEDRO DONADIO DA SILVA PEREIRA Orientador: Prof. Dr. Matheus Deniz Co-orientadoras: Dra. Karolini Tenffen De-Sousa Dra. Teresa Cristina Alves Dissertação apresentada ao Programa de Pós-graduação em Zootecnia como parte das exigências para obtenção do título de Mestre em Zootecnia BOTUCATU – SP Fevereiro, 2025 Permitida a cópia total ou parcial deste documento, desde que seja citada a fonte. P436e Pereira, João Pedro Donadio da Silva Effect of Environmental Enrichment on Behavior and Performance of Dairy Calves / João Pedro Donadio da Silva Pereira. -- Botucatu, 2025 131 p. Dissertação (mestrado) - Universidade Estadual Paulista (UNESP), Faculdade de Medicina Veterinária e Zootecnia, Botucatu Orientador: Matheus Deniz 1. Zootecnia. 2. Produção Animal. 3. Etologia Aplicada. 4. Bovinocultura de Leite. 5. Bezerros Leiteiros. I. Título. Sistema de geração automática de fichas catalográficas da Unesp. Dados fornecidos pelo autor(a). VII BIOGRAFIA DO AUTOR João Pedro Donadio da Silva Pereira, nascido em 10 de abril de 1999, na cidade de Santos/SP, filho de Silmara Donadio da Silva e Roberto da Silva Pereira, ingressou no curso de Agronomia na Universidade Federal de Santa Catarina, em agosto de 2017, concluindo a graduação em dezembro de 2022. Durante a graduação, atuou como bolsista de iniciação científica no Laboratório de Etologia Aplicada e Bem-Estar Animal. Em março de 2023, iniciou o curso de Mestrado em Zootecnia na Faculdade de Medicina Veterinária e Zootecnia da Universidade Estadual Paulista “Julio de Mesquita Filho” campus de Botucatu, onde foi bolsista pela Fundação Coordenação de Aperfeiçoamento de Pessoal de Nível Superior. Durante o mestrado, recebeu o prêmio “Emerging Leaders in the Americas Program” que possibilitou o intercâmbio no Animal Welfare Program - University of British Columbia, no Canadá. Atua na área de Produção Animal, especificamente sobre comportamento e bem-estar na bovinocultura leiteira. Currículo Lattes: http://lattes.cnpq.br/6493860465511026 ORCID: https://orcid.org/0000-0002-6728-4373 E-mail: jpd.pereira@unesp.br http://lattes.cnpq.br/6493860465511026 https://orcid.org/0000-0002-6728-4373 mailto:jpd.pereira@unesp.br VIII PUBLICATIONS Articles DONADIO, J. P.; DE-SOUSA, K. T.; TORRES, R. N. S.; ALVES, T. C.; HÖTZEL, M. J.; DENIZ, M. A meta-analysis approach to evaluate the effects of early group housing on calf performance, health and behavior during the preweaning period. Journal of Dairy Science, 2024. Technological Innovations DENIZ, M.; OLIVEIRA, I. T. S.; DONADIO, J. P. VisionBrush. 2024, Brasil. Patente: Privilégio de Inovação. Número do registro: BR512024001540, título: "VisionBrush", Instituição de registro: INPI - Instituto Nacional da Propriedade Industrial. Depósito: 21/05/2024 Conference Proceedings ALCANTARA, B. D.; DONADIO, J. P.; TORRES, R. N. S.; DE-SOUSA, K. T.; HOTZEL, M. J.; DENIZ, M. Meta-analysis of the effect of milk feeding method on average daily gain, concentrate intake, and weight at weaning of dairy calves. In: American Dairy Science Association Annual Meeting, 2024, West Palm Beach. Abstracts of the 2024 American Dairy Science Association Annual Meeting, 2024. v. 107, p. 414. DONADIO, J. P.; DE-SOUSA, K. T.; TORRES, R. N. S.; ALVES, T. C.; HOTZEL, M. J.; DENIZ, M. Group housed dairy calves have greater body weight gain during preweaning than individually housed calves: A meta-analysis. In: American Dairy Science Association Annual Meeting, 2024, West Palm Beach. Abstracts of the 2024 American Dairy Science Association Annual Meeting, 2024. v. 107. p. 185. DONADIO, J. P.; DE-SOUSA, K. T.; HOTZEL, M. J.; ALVES, T. C.; DENIZ, M. Providing water in nipple bucket reduces the cross-sucking of dairy calves housed in groups in a pasture area. In: 57th Congress of the International Society for Applied Ethology, 2024, Curitiba. Book of abstracts of the 57th Congress of the International Society for Applied Ethology ISAE 2024, 2024. DENIZ, M.; ALCANTARA, B. D.; DONADIO, J. P.; DE-SOUSA, K. T.; TORRES, R. N. S.; HOTZEL, M. J. Effect of milk supply methods on the behavior of dairy calves: a meta-analysis approach. In: 57th Congress of the International Society for Applied Ethology, 2024, Curitiba. Book of abstracts of the 57th Congress of the International Society for Applied Ethology ISAE 2024, 2024. p. 210. ALVES, T.C.; DONADIO, J.P.; CHAMILLETE, S.A.M.; DE ALMEIDA, M.B., DE-SOUSA, K.T.; DENIZ, M. Criação de bezerros leiteiros em grupos e à pasto na Fazenda Canchim - área experimental da Embrapa Pecuária Sudeste em São Carlos-SP. Anais do IV Encontro Pan- americano sobre Manejo Agroecológico de Pastagens – PRV nas Américas, Florianópolis, Santa Catarina, Brasil. v. 19, n. 3, 2024. IX DONADIO, J.P.; DE-SOUSA, K. T.; ALVES, T.C.; DENIZ, M. O uso de enriquecimento ambiental melhora os níveis de bem-estar de bezerros leiteiros. In: Jornada de Integração dos Alunos de Graduação e Pós-graduação (JOIA), Programa de pós-graduação em Biotecnologia animal, Faculdade de Medicina Veterinária e Zootecnia, Universidade Estadual Paulista “Júlio de Mesquita Filho”, campus de Botucatu, 2024. DONADIO, J.P.; DE-SOUSA, K.T.; TORRES, R.N.S.; HÖTZEL, M.J.; ALVES, T.C.; DENIZ, M. Efeito do alojamento em grupo no comportamento de bezerros leiteiros: uma meta- análise. In: XLI Encontro Anual de Etologia - Brasília, 2024. Disponível em: . DONADIO, J.P.; DE-SOUSA, K.T.; DE ALMEIDA, M.B.; ALVES, T.C.; HÖTZEL, M.J.; DENIZ, M. Rethinking environmental enrichment for dairy calves: improvements for basic biological needs? in Proceedings of the 3rd International Electronic Conference on Animals, 12–14 March 2025, MDPI: Basel, Switzerland. X AGRADECIMENTOS Agradeço aos meus pais, Silmara e Roberto, minha irmã, Anna, por estarem sempre presentes em minha vida, pelo apoio, carinho e pela força que me dão a cada dia. Ao meu orientador, Prof. Matheus Deniz, por ter me recebido como seu primeiro orientado de pós-graduação e por me permitir fazer parte do início de sua trajetória como professor. Juntamente às minhas coorientadoras, Dra. Karolini Tenffen De-Sousa e Dra. Teresa Cristina Alves, assim como os coautores dos trabalhos presentes na dissertação, Profa. Dra. Maria José Hötzel, Dr. Rodrigo Torres, Dra. Grazyne Tresoldi por todos os ensinamentos, parceria, apoio e dedicação, que foram fundamentais para a minha formação acadêmica, profissional e pessoal. Aos amigos que continuaram comigo e aos que fiz durante os dois anos de mestrado, por todo o apoio e torcida. Em especial à minha companheira, Isabela, pelo carinho e paciência durante essa fase. Aos colegas do Grupo de Estudos em Bovinocultura Leiteira (GEBOL) por todas as atividades realizadas durante meu período de mestrado, que me ajudaram e contribuíram muito para a minha formação. Tenho muito orgulho de ter feito parte do início deste brilhante grupo. Ao Programa de Pós-Graduação em Zootecnia da Faculdade de Medicina Veterinária e Zootecnia da UNESP, Campus de Botucatu, pelo suporte e pela oportunidade de estudar em um programa de excelência. O presente trabalho foi realizado com apoio da Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Código de Financiamento 001. À Embrapa Pecuária Sudeste, seus pesquisadores, trabalhadores e estudantes, por me receberem de peito aberto e por todo o apoio durante o meu experimento. I am grateful to EduCanada, University of British Columbia (UBC), Animal Welfare Program (AWP), Professors Marina von Keyserlingk and Daniel Weary and the Dairy Education and Research Centre (DERC) team for the amazing opportunity to enhance my research skills, immerse myself in a new culture, and build meaningful connections with international researchers. To all the friends I made during this experience, for the shared experiences, memories and all support. XI RESUMO GERAL As práticas convencionais de criação de bezerros leiteiros, como o alojamento individual em ambientes com poucos estímulos, são amplamente adotadas, mas seus impactos negativos no bem-estar dos animais têm sido alvo de críticas da sociedade. Essas práticas restringem comportamentos naturais essenciais para o desenvolvimento dos bezerros, como o contato social, exploração e a sucção, podendo resultar em estresse e frustração. O enriquecimento ambiental envolve modificações que buscam aumentar a relevância biológica do ambiente de criação. Este estudo teve como objetivo avaliar os efeitos do enriquecimento ambiental – por meio do alojamento em grupo durante o pré-desmame, do enriquecimento físico e da oferta de água por bicos – no comportamento, saúde e desempenho de bezerros leiteiros. Primeiramente, realizamos uma revisão sistemática com abordagem meta-analítica de estudos que compararam a criação de bezerros leiteiros em pares ou grupos com a criação individual. Os resultados indicaram que o alojamento precoce (< 14 dias de idade) em grupo traz benefícios significativos, como maior ganho de peso e consumo de concentrado, além de favorecer comportamentos ativos, como alimentação e brincadeira, e reduzir comportamentos de autolimpeza. Na segunda parte, uma revisão sistemática foi conduzida para compilar estudos sobre enriquecimento físico para bezerros leiteiros, como o aumento do espaço disponível, acesso a áreas externas e adição de objetos no ambiente de criação. As práticas de enriquecimento físico mostraram-se eficazes em elevar os níveis de atividade, promover comportamentos exploratórios e reduzir comportamentos anormais, como a mamada cruzada. Por fim, conduzimos um experimento para avaliar o impacto do enriquecimento nutricional, fornecendo água por bicos, no comportamento de bezerros alojados em grupo à pasto. Nossos resultados sugerem que a mudança na oferta de água influencia a atividade dos bezerros ao longo do tempo, alterando o padrão de ingestão de água, com maior consumo durante a noite. Observamos também que a ausência de estímulo para a sucção, ao utilizar baldes abertos para a oferta de água, contribuiu para o aumento da mamada cruzada. Em síntese, os estudos destacam a importância de modificar o ambiente de criação para atender às necessidades comportamentais dos bezerros, evidenciando os benefícios do alojamento em grupo, do enriquecimento físico e da oferta de estímulos para a sucção na melhoria do bem-estar desses animais. É fundamental que as práticas de manejo levem em conta esses aspectos para promover a saúde e o desenvolvimento adequado dos bezerros, contribuindo, assim, para a sustentabilidade das fazendas leiteiras. Palavras-chave: bem-estar animal; bovinocultura leiteira; etologia aplicada; experiências positivas; produção animal; resiliência. XII ABSTRACT Conventional dairy calf-rearing practices, such as individual housing in low-stimulation environments, are widely used but have increasingly come under societal scrutiny due to their negative impact on animal welfare. These practices restrict natural behaviors essential for calf development, including social contact, exploration, and sucking, which can lead to stress and frustration. Environmental enrichment includes modifications aimed at enhancing the biological relevance of the rearing environment. This study sought to evaluate the effects of environmental enrichment—through group housing during the pre-weaning phase, physical enrichment, and water provision through teats—on the behavior, health, and performance of dairy calves. First, we conducted a systematic review and meta-analysis of studies comparing pair or group housing for dairy calves with individual housing. Results showed that early group housing provides significant benefits, such as increased weight gain and concentrate intake, as well as promoting active behaviors, like feeding and play, and reducing self-cleaning behaviors. In the second part, a systematic review compiled studies on physical enrichment for dairy calves, such as increased available space, access to outdoor areas, and the addition of objects to the environment. Physical enrichment practices were found to effectively increase activity levels, encourage exploratory behaviors, and reduce abnormal behaviors, such as cross-sucking. Finally, we conducted an experiment to assess the impact of nutritional enrichment, by providing water through teats, on the behavior of group-housed and pasture-raised calves. Our results suggest that changing the method of water provision influences calf activity patterns over time, altering water intake with increased consumption at night. We also observed that the absence of a sucking stimulus, when open buckets were used for water provision, contributed to an increase in cross-sucking behavior. In summary, these studies emphasize the importance of modifying the rearing environment to meet calves' behavioral needs, highlighting the benefits of group housing, physical enrichment, and providing sucking stimuli to improve calf welfare. It is essential for management practices to consider these factors to support calf health and development, thereby contributing to the sustainability of dairy farms. Keywords: animal production; animal welfare; applied ethology; dairy cattle; positive experiences; resilience. XIII LIST OF ILLUSTRATIONS Figure 1. Flowchart following PRISMA guidelines (Page et al., 2021) showing the process to identify and screen articles for eligibility and inclusion in this systematic review and meta-analysis. .............. 37 Figure 2. Subgroup (weaning age covariate) analysis effects of group housing on calves’ average daily gain. WMD = weighted mean differences between group-housed and individually housed calves. .... 40 Figure 3. Forest plot with the subgroup analysis of the effects of (A) sex class (male, female, and male and female) and (B) group size (pair, 3 to 6 calves, and 8 to 12 calves) on the average daily gain parameter (Effect = Weighted mean difference (WMD). The x-axis shows the effect size of the WMD. Diamonds to the left of the solid line represent a reduction in the measure, whereas diamonds to the right of the line indicate an increase. Each diamond represents the mean size effect for that study, and the diamond’s size reflects the study’s relative weighting to the overall size effect estimate, with larger diamonds representing greater weight. The lines connected to the diamond represent the upper and lower 95% confidence interval for the group housing effect. The dotted vertical line represents the overall estimated effect size. The diamond at the bottom represents the mean response across the studies, and the solid vertical line represents a mean difference of zero or no effect. Note: weights and between- subgroup heterogeneity tests are from the random-effects model. ....................................................... 41 Figure 4. Forest plot with subgroup analysis of the effects of age, sex, housing type, behavioral test, and group size on calves’ behavioral test parameters (Effect = standard mean difference (SMD). The x- axis shows the effects of SMD. Diamonds to the left of the solid line represent a reduction in the measure, whereas diamonds to the right of the line indicate an increase. Each diamond represents the mean size effect for that study, and the diamond's size reflects the study's relative weighting to the overall size effect estimate with larger diamonds representing greater weight. The lines connected to the diamond represent the upper and lower 95% confidence interval for the group housing effect. The dotted vertical line represents the overall size effect estimate. The diamond at the bottom represents the mean response across the studies, and the solid vertical line represents a mean difference of zero or no effect. Note: Weights and between-subgroup heterogeneity tests are from the random-effects model. .................... 43 Figure 5. Diagram of knowledge gaps in literature regarding environmental enrichment for dairy calves. References: Mandel et al. (2016); Veissier et al. (2024); Costa et al. (2016); Donadio et al. (2024); Meagher et al. (2019); Beaver et al. (2019); Roland et al. (2016); Mota-Rojas et al. (2024); Welk et al. (2023); Khan et al. (2016); Miller-Cushon and Devries (2015); Miller-Cushon and Jensen (2025). ... 68 Figure 6. Flowchart following Preferred Reporting Items for Systematic Review and Meta-Analyses guidelines (Page et al., 2021) showing the process to identify and screen articles for eligibility and inclusion in this systematic review. ...................................................................................................... 71 Figure 7. Graphical representation of different space allowances (m² per calf) and social housing evaluated in 18 studies. Each group size evaluated is represented by a symbol. References: Dellmeier et al. (1985); Fisher et al. (1985); Friend et al. (1985); Jensen et al. (1997, 1998); Ferrante et al. (1998); Wilson et al. (1999); Jensen and Kyhn (2000); Tapkı et al. (2006); Færevik et al. (2008); Tripon et al. (2012); Rushen and De Passillé (2014); Sutherland et al., (2014a, b); Calvo-Lorenzo et al. (2016, 2017); Hulbert et al. (2019); Neave et al. (2021). ............................................................................................ 73 XIV Figure 8. Graphical illustration of different flooring types used to enrich dairy calves rearing environment in 20 studies included in this review. Note: All studies evaluated more than one flooring type. ...................................................................................................................................................... 77 Figure 9. Graphical illustration of differents object types used to enrich dairy calves rearing environment in 19 studies included in this review. ............................................................................... 80 Figure 10. Schematic representation (not to scale) of the paddock allocation for experimental groups. a) Nipple water trough (Nipple-WT) treatment paddock. b) Open water trough (Open-WT) treatment paddock. ................................................................................................................................................ 97 Figure 11. Frequency (%) of cross-sucking events performed on different body parts of pre-weaned dairy calves (n=21) under different water delivery methods [Open water trough (Open-WT) vs. Nipple water trough (Nipple-WT)]. Circle size represents the relative frequency of cross-sucking events for each body part. .................................................................................................................................... 103 Figure 12. Heatmap of frequency (descriptive analysis) of cross-sucking events for each hour under different water delivery methods [Open water trough (Open-WT) and Nipple water trough (Nipple- WT)]. .................................................................................................................................................. 103 Figure 13. Heatmap of frequency of visits to water trough per hour for each hour per treatment [Open water trough (Open-WT) and Nipple water trough (Nipple-WT)]. .................................................... 104 Figure 14. Body measures parameters (Chest girth; Withers height; Rump height; Rump Width; Rump length; Body length) throughout the experimental period for each treatment. Points and boxes in red (circles) refer to calves in Nipple water trough (Nipple-WT), and in blue (triangles) to calves in Open water trough (Open-WT). ................................................................................................................... 107 XV LIST OF TABLES Table 1. Population, intervention, comparison, and outcome of search term strings used for the final search. ................................................................................................................................................... 32 Table 2. Effects of group housing on the performance, feed intake, and health parameters found in the studies included in the meta-analysis. ................................................................................................... 38 Table 3. Meta-regression of the effect of group housing for dependent variables with weighted mean differences (WMD; average daily gain, concentrate intake) and variables with standardized mean differences (SMD; lying behavior) evaluated in the studies included in this review. ........................... 39 Table 4. Effects of group housing on behavior parameters found in the studies included in the meta- analysis. ................................................................................................................................................ 42 Table 5. Population and outcome search term strings were used for the final search in the systematic review. .................................................................................................................................................. 69 Table 7. Average ± SD of the water intake (L) per calf raised on pasture during preweaning under different water delivery method [Open water trough (Open-WT; n = 11) vs. Nipple water trough (Nipple-WT; n = 10)] and period of the day (Daytime vs. Nighttime). .............................................. 104 Table 8. Effect of water delivery method, week, and its interaction on the frequency (% ± standard error, SE) of the behaviour of pre-weaned group-housed dairy calves (n=21) raised on pasture ....... 105 XVI LIST OF ABBREVIATIONS, ACRONYMS, AND SYMBOLS ADG Average daily gain AGP Alpha-1-Acid Glycoprotein BHB Beta-Hydroxybutyrate BW Body weight CAPES Coordenação de Aperfeiçoamento de Pessoal de Nível Superior CI Confidence interval CNPq Conselho Nacional de Desenvolvimento Científico e Tecnológico FMVZ Faculdade de Medicina Veterinária e Zootecnia GLMM General linear mixed model ID Identification IFN-γ Interferon Gamma IgG Immunoglobulin G NA Not applicable NEFA Non-Esterified Fatty Acids REML Restricted Maximum Likelihood Estimate SMD Standardized mean difference THI Temperature-Humidity Index UNESP Universidade Estadual Paulista WT Water trough WMD Weighted mean difference XVII SUMMARY INTRODUCTION .................................................................................................................. 19 HYPOTHESES ....................................................................................................................... 22 OBJECTIVES ......................................................................................................................... 22 GENERAL OBJECTIVE ................................................................................................................................ 22 SPECIFIC OBJECTIVES ............................................................................................................................... 22 REFERENCES ....................................................................................................................... 23 CHAPTER 1 ............................................................................................................................ 28 ABSTRACT ................................................................................................................................................ 30 INTRODUCTION ........................................................................................................................................ 31 METHODS ................................................................................................................................................. 32 Search strategy ............................................................................................................................................. 32 Study inclusion criteria and screening .......................................................................................................... 32 Data extraction strategy and synthesis procedures ...................................................................................... 33 Statistical analysis ........................................................................................................................................ 34 Weighted mean difference and standardized mean difference ...................................................................... 34 Heterogeneity and publication bias .............................................................................................................. 35 Meta-regression and subgroup analysis ....................................................................................................... 35 RESULTS .................................................................................................................................................. 36 Systematic analysis of the included studies ................................................................................................... 36 Calves’ performance, feed intake, and health parameters ............................................................................ 37 Calf behavioral parameters .......................................................................................................................... 42 Behavior and Cognitive test parameters ....................................................................................................... 42 DISCUSSION .............................................................................................................................................. 44 Performance ................................................................................................................................................. 44 Behavior ........................................................................................................................................................ 46 Behavioral tests ............................................................................................................................................ 46 Health parameters ........................................................................................................................................ 47 CONCLUSION ............................................................................................................................................ 48 NOTES ...................................................................................................................................................... 48 REFERENCES ............................................................................................................................................ 50 SUPPLEMENTARY MATERIAL ................................................................................................................... 60 CHAPTER 2 ............................................................................................................................ 64 ABSTRACT ................................................................................................................................................ 66 INTRODUCTION ........................................................................................................................................ 67 METHODS ................................................................................................................................................. 68 Search strategy ............................................................................................................................................. 68 Study inclusion criteria and screening .......................................................................................................... 69 Data extraction ............................................................................................................................................. 70 RESULTS .................................................................................................................................................. 70 Overview ....................................................................................................................................................... 70 Space allowance ........................................................................................................................................... 73 Enclosure types ............................................................................................................................................. 75 Flooring ........................................................................................................................................................ 76 Objects .......................................................................................................................................................... 78 DISCUSSION .............................................................................................................................................. 80 CONCLUSIONS .......................................................................................................................................... 83 NOTES ...................................................................................................................................................... 83 REFERENCES ............................................................................................................................................ 85 CHAPTER 3 ............................................................................................................................ 92 ABSTRACT ................................................................................................................................................ 94 INTRODUCTION ........................................................................................................................................ 95 XVIII MATERIALS AND METHODS ....................................................................................................................... 96 Location and thermal environment .............................................................................................................. 96 Experimental area and management ............................................................................................................ 96 Animals and treatments ................................................................................................................................ 98 Measurements .............................................................................................................................................. 99 Behaviour ..................................................................................................................................................... 99 Growth ......................................................................................................................................................... 99 Statistical analysis ...................................................................................................................................... 100 RESULTS ................................................................................................................................................. 102 Cross-sucking ............................................................................................................................................. 102 Behaviour ................................................................................................................................................... 104 Growth ....................................................................................................................................................... 106 DISCUSSION ............................................................................................................................................ 107 Limitations and Future Directions ............................................................................................................. 110 CONCLUSION .......................................................................................................................................... 110 CONFLICT OF INTEREST STATEMENT ...................................................................................................... 111 ACKNOWLEDGMENTS ............................................................................................................................. 111 REFERENCES .......................................................................................................................................... 112 SUPPLEMENTARY MATERIAL .................................................................................................................. 118 FINAL CONSIDERATIONS AND IMPLICATIONS ..................................................... 120 SUPPLEMENT A – MANUSCRIPT PUBLISHED IN JOURNAL OF DAIRY SCIENCE .............................................................................................................................. 121 SUPPLEMENT B – PATENT: COMPUTER PROGRAM: VISIONBRUSH .............. 122 SUPPLEMENT C – ABSTRACT PUBLISHED IN THE “AMERICAN DAIRY SCIENCE ASSOCIATION ANNUAL MEETING” ........................................................ 123 SUPPLEMENT D – ABSTRACT PUBLISHED IN THE “AMERICAN DAIRY SCIENCE ASSOCIATION ANNUAL MEETING” ........................................................ 124 SUPPLEMENT E – ABSTRACT PUBLISHED IN THE “57TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR APPLIED ETHOLOGY” .................................... 125 SUPPLEMENT F – ABSTRACT PUBLISHED IN THE “57TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR APPLIED ETHOLOGY” .................................... 126 SUPPLEMENT G – ABSTRACT PUBLISHED IN THE “ANAIS DO IV ENCONTRO PAN-AMERICANO SOBRE MANEJO AGROECOLÓGICO DE PASTAGENS – PRV NAS AMÉRICAS” ............................................................................................................... 127 SUPPLEMENT H – ABSTRACT PUBLISHED IN THE “XLI ENCONTRO ANUAL DE ETOLOGIA” ................................................................................................................. 128 SUPPLEMENT I – ABSTRACT PUBLISHED IN THE “PROCEEDINGS OF THE 3RD INTERNATIONAL ELECTRONIC CONFERENCE ON ANIMALS” ............... 129 SUPPLEMENT J – APPROVAL PROTOCOL OF THE ANIMAL ETHICS OF SÃO PAULO STATE UNIVERSITY - BOTUCATU ............................................................... 130 SUPPLEMENT K – APPROVAL PROTOCOL OF THE ANIMAL ETHICS EMBRAPA ........................................................................................................................... 131 19 INTRODUCTION In commercial dairy calf rearing, conventional practices such as individual housing, social isolation, early weaning, and separation from the mother often compromise calf welfare (COSTA et al., 2019; MEE, 2013; VON KEYSERLINGK et al., 2009). Despite being widely adopted, these practices are increasingly criticized for their adverse effects on calf welfare (PERTTU; VENTURA; ENDRES, 2020; PLACZEK; CHRISTOPH-SCHULZ; BARTH, 2021; SIROVICA et al., 2022). Calves in conventional systems may experience stress and frustration due to a lack of stimuli, such as social isolation, restricted movement, and limited opportunities to express natural behaviors (HULBERT; MOISÁ, 2016; WHALIN; WEARY; VON KEYSERLINGK, 2021). The ability to express species-specific natural behaviors is crucial for animal welfare, as it reflects their capacity to adapt and cope with environmental challenges (BROOM, 1986). When these behaviors are disrupted, it may indicate that the animal is struggling to meet its biological and psychological needs (WEINER, 1992). Stress plays a critical role in influencing behavior (MOBERG, 2000), particularly through activation of the hypothalamic-pituitary-adrenal axis, which triggers the release of glucocorticoids such as cortisol (TSIGOS; CHROUSOS, 2002). This stress response is vital for short-term survival, enabling the animal to deal with immediate threats or challenges. However, when stress becomes chronic or recurrent (distress), it can have detrimental effects on the animal's health, growth, and productivity (GRANDIN; DEESING, 2002; UNDERWOOD, 2002). In dairy calves, stressors such as early maternal separation, poor housing conditions, restricted social interactions, and inadequate nutrition can disrupt biological functions, making calves more susceptible to disease, impairing growth, and altering metabolic processes (STAFFORD; MELLOR, 2011; WEARY; JASPER; HÖTZEL, 2008). While cortisol levels, measured in serum, plasma, saliva, feces, urine, or hair, are commonly used to assess stress (HEIMBÜRGE; KANITZ; OTTEN, 2019; MÖSTL; PALME, 2002), they offer only part of the picture. Changes in behavior, such as increased vocalizations, restlessness, and reduced play, are important factors that should be considered to provides a more comprehensive view of the animal's ability to cope with stress (ALADOS; ESCOS; EMLEN, 1996; RUTHERFORD et al., 2004). Natural behaviors are essential indicators of an animal's needs and its ability to cope with its environment (FRASER et al., 1997). These behaviors have evolved to fulfill biological functions related to survival, social bonding, and overall well-being. Recognizing the origins and motivations behind such behaviors helps inform management practices that minimize the 20 negative impacts of confinement and improve animal welfare (NEWBERRY, 1995). In dairy calves, behaviors such as social contact, scratching, and suckling are not only instinctive but also highly motivated, meaning that calves actively seek opportunities to express them (DE PASSILLÉ, 2001; EDE; WEARY; VON KEYSERLINGK, 2022; MCCONNACHIE et al., 2018). When these behaviors are restricted, such as through individual housing or bucket feeding, calves may develop signs of frustration and engage in abnormal behaviors, including excessive licking, cross-sucking, or stereotypies (JENSEN; BUDDE, 2006; VEISSIER; CARÉ; POMIÈS, 2013). Understanding these biological drives is crucial for designing housing and management systems that allow for the expression of natural behaviors, ultimately supporting healthier physical and psychological development in dairy calves. Natural behaviors can be encouraged by adopting environmental enrichment, which involves modifications to enhance the biological relevance of captive animals’ environment (NEWBERRY, 1995) and contributes to positive animal welfare (RAULT et al., 2025). Environmental enrichment is categorized into sensory, occupational (cognitive), nutritional, social, and physical dimensions (BLOOMSMITH; BRENT; SCHAPIRO, 1991). This concept has been widely applied in captive animal management, from zoo animals to farm livestock (CARLSTEAD; SHEPHERDSON, 2000; HERRON; BUFFINGTON, 2012; MAPLE; PERDUE, 2013; VAN DE WEERD; DAY, 2009). There is increasing interest in environmental enrichment for dairy calves. However, research on this topic remains limited and dispersed (MANDEL et al., 2016). For example, social contact plays a crucial role in the emotional and cognitive development of calves (BERTELSEN; JENSEN, 2019). The stress caused by social isolation makes calves more fearful than those with social contact (CREEL; ALBRIGHT, 1988; GAILLARD et al., 2014). On the other hand, social contact allows calves to engage in natural behaviors like play, running, and grooming, which are essential for their social and physical development (LI et al., 2020; MINTLINE et al., 2013). Group housing has been widely studied to understand the role of social interactions in calf development, as these interactions promote emotional and cognitive growth, reduce stress, and enhance resilience (BREEN et al., 2023; MALÁ et al., 2023; ZHANG; JUNIPER; MEAGHER, 2021). Also, enriching the physical environment—by increasing space, providing access to outdoor areas, or adding items like grooming brushes or objects for exploration—can significantly improve calf welfare (CALVO-LORENZO et al., 2017; SINNOTT et al., 2022; SUTHERLAND; WORTH; STEWART, 2014; ZHANG; JUNIPER; MEAGHER, 2022). Outdoor calf-rearing systems have been shown to offer benefits, including improved health outcomes (LORENZ et al., 2011; WÓJCIK; NAŁĘCZ-TARWACKA; GOŁĘBIEWSKI, 2013) 21 and reduced fearfulness (FIELD et al., 2024), compared to conventional indoor settings. Physical changes to calves’ environment can boost activity levels (LI et al., 2020), encouraging play and exploratory behaviors, and reduce abnormal behaviors such as non-nutritive oral behaviors (ZHANG; JUNIPER; MEAGHER, 2021). For environmental enrichment to be effective, however, it must provide stimuli from which animals can acquire information, pleasure, and enhance behavioral adaptability, in other words, improve animal’s welfare (VEISSIER et al., 2024). This growing interest in environmental enrichment for dairy calves highlights the need for a systematic and comprehensive understanding of how such strategies can enhance calf welfare. Non-nutritive behaviors in calves, such as cross-sucking and other oral stereotypies, are usually related to negative emotional states (DE PASSILLÉ; RUSHEN; JANZEN, 1997; LIDFORS, 1993) and animals can hurt themselves and others performing this abnormal behavior (LALANDE; BEAUCHEMIN; H. FAHMY, 1979). Using nipple-based feeders instead of open buckets for milk feeding has been shown to reduce the frequency of cross- sucking, rewarding them with milk when they perform this behavior (JENSEN; BUDDE, 2006; REIPURTH et al., 2020). For instance, many other modifications to rearing environments and management strategies have been explored to reduce cross-sucking behavior (WELK; OTTEN; JENSEN, 2023). 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APPLIED ANIMAL BEHAVIOUR SCIENCE 237. https://doi.org/10.1016/j.applanim.2021.105295 120 Final considerations and implications Our findings underscore the benefits of early group housing, physical enrichment, and nipple provision in supporting dairy calves’ growth, health, and natural active behaviors. Together, these studies show that well-designed enrichment positively impacts calf behavior and welfare, advocating for a comprehensive approach to calf-rearing those addresses both physical and social needs. Effective management practices that meet calves' natural motivations contribute to healthier development and support sustainable dairy farming. In this thesis, environmental enrichment is defined as modifications that improve biological functioning, yet for calves, some of these practices serve to fulfill basic needs. Calves are strongly motivated to engage in behaviors like social interaction, suckling, and grazing; however, barren environments (usually adopted in commercial dairy farms) often lack the necessary stimuli for these behaviors, leading to the development of abnormal behaviors. The dairy industry should shift toward establishing baseline environments that inherently fulfill calves' basic needs, such as providing social contact, supporting natural suckling behavior, and ensuring essential welfare standards. With these foundational needs met, environmental enrichment can then go beyond simply addressing innate behaviors, focusing instead on additional stimulation that fosters exploration and enhances welfare.