UNIVERSIDADE ESTADUAL PAULISTA FACULDADE DE MEDICINA VETERINÁRIA E ZOOTECNIA CAMPUS DE BOTUCATU BEHAVIOR AND PRODUCTIVE INDICATORS FOR BROILER CHICKENS: IS ENVIRONMENTAL ENRICHMENT ALWAYS POSITIVE? MARCONI ITALO LOURENÇO DA SILVA BOTUCATU, SP August, 2023 Thesis submitted to the Graduate Program in Animal Sciences in fulfillment of the requirements for the degree of Doctor of Philosophy in Animal Sciences UNIVERSIDADE ESTADUAL PAULISTA FACULDADE DE MEDICINA VETERINÁRIA E ZOOTECNIA CAMPUS DE BOTUCATU DOCTORAL THESIS BEHAVIOR AND PRODUCTIVE INDICATORS FOR BROILER CHICKENS: IS ENVIRONMENTAL ENRICHMENT ALWAYS POSITIVE? MARCONI ITALO LOURENÇO DA SILVA Advisor: Assoc. Prof. Dr. Ibiara Correia de Lima Almeida Paz Co-advisor: Assoc. Prof. Dr. Leonie Jacobs BOTUCATU, SP August, 2023 Thesis submitted to the Graduate Program in Animal Sciences in fulfillment of the requirements for the degree of Doctor of Philosophy in Animal Sciences III IV “There is no such thing as a neutral education process. Education either functions as an instrument that is used to facilitate the integration of generations into the logic of the present system and bring about conformity to it, or it becomes the "practice of freedom", the means by which men and women deal critically and creatively with reality and discover how to participate in the transformation of their world.” (Paulo Freire) V BIOGRAPHY Marconi Italo Lourenço da Silva is an Animal Scientist from Federal Rural University of Pernambuco, Brazil (2017). Master in Animal Science with an emphasis on poultry production, animal welfare, and ethology from Sao Paulo State University, campus in Botucatu, Brazil (2020). In His Ph.D. in Animal Science at the same institution, he developed studies related to the use of environmental enrichment aiming to increase the quality of life of chickens. Still during his Ph.D., Marconi performed a sandwich doctorate in a 15-month period at Virginia Tech under the supervision of Dr. Leonie Jacobs. At Dr. Jacobs’ lab, Marconi performed studies on cognitive bias tests for broiler chickens. VI DEDICATION To my parents, Sandra Maria Santana da Silva and Ivanildo Lourenço da Silva (in memoriam), for their support at any time, unconditionally. I am now finishing this graduate degree due to their effort to provide me with conditions and for always believing in me. To my siblings Marcelo and Simone, uncles, cousins, and nephews, for also supporting me in all different ways, always. I thank my maternal grandparents Henrique (in memoriam) and Beatriz (in memoriam), for always giving me advice and unconditional love, which contributed and still contribute to my education. My paternal grandmother Irene (in memoriam) for all the teaching and advice. I love you! VII ACKNOWLEDGEMENTS My research advisor and chair, Dr. Ibiara Correia de Lima Almeida Paz, for the opportunity, guidance, support, patience, and inspiration. Thank you very much for your indelible contributions to my education! My co-advisor, Dr. Leonie Jacobs, for her support and teachings during my exchange period at Virginia Tech, USA. To the Coordination for the Improvement of Higher Education Personnel, Brazil for granting a Doctorate scholarship. The Latin American Poultry Association and the Brazilian Animal Protein Association for funding the research project. To all graduate colleagues, friends, scientific initiation students, and interns in Brazil who passed through the poultry barn, in particular, Evelyn, Iasmin, Lucas, Pedro, Pequi, Alejandro, Ana Júlia, Gabriel, Andressa, Ingrid, Francine, Gilson, and Walter, who helped a lot for my project to be developed and carried out; also for moments of relaxation, laughter, games, and coffee. To all my colleagues and friends in the USA who helped me a lot during my exchange, in particular, Ally, Bidur, Jess, Drew, Sam, Jéssica, Gustavo, Kenan, Rafael, Mariane, Sabrina, Alexis, Bronson, Letícia, Mateus, Beth, and Maria Helena. The Supervision of the Teaching, Research, and Extension Farms (FEPE) and the FMVZ Feed Mill for their support before, during, and after carrying out my experiment. To the professors of the Department of Animal Production and Preventive Veterinary Medicine, for always helping me promptly and for all the teachings. To the Committee members for their availability and contribution to my study. This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001. Thank you so much! VIII COMPORTAMENTO E INDICADORES PRODUTIVOS PARA FRANGOS DE CORTE: O ENRIQUECIMENTO AMBIENTAL É SEMPRE POSITIVO? RESUMO – Com o consenso que os animais são seres sencientes as pesquisas sobre o bem-estar animal estão focadas no indivíduo e suas experiências. Dois experimentos foram conduzidos, objetivou-se no Exp. 1, avaliar o uso de enriquecimento ambiental e a produtividade, comportamento, prevalência de espondilolistese em frangos de corte de crescimento rápido. No Exp. 2, objetivou-se desenvolver um teste de viés de julgamento (JBT) para frangos de corte de crescimento lento, utilizando os aspectos sociais da espécie, para avaliar os efeitos do medo, ansiedade e estresse crônico e mensurar os estados afetivos dos animais. No Exp. 1 foram utilizadas 2400 aves Ross® AP95, machos, com um dia de idade. Os animais foram alocados em 4 tratamentos distribuídos em delineamento experimental casualizado com 4 repetições cada. Os tratamentos foram: controle (C) - ambiente similar ao comercial sem enriquecimento; ambientes enriquecidos com feno (HB), plataformas com degraus (SP), ou projetores de luz (LL). No Exp. 2 foram utilizadas 600 aves, Hubbard Redbro, machos, com um dia de idade. Os animais foram alocados em 2 tratamentos distribuídos em delineamento em blocos casualizados, com 6 repetições cada. Os tratamentos foram: controle - baixa complexidade, similar aos padrões comerciais, ou alta complexidade, adição de enriquecimentos permanentes e temporários. No Exp. 1, quando criados com acesso a SP e LL, houve menor frequência de espondilolistese quando comparados aos frangos criados sem enriquecimento ou com acesso a HB. Frangos com acesso a SP apresentaram maior rendimento de asas e menos gordura abdominal comparados com o grupo C. Frangos com LL e HB exploraram mais e descansaram menos que os animais dos tratamentos C e SP. No Exp. 2, medo, ansiedade e estresse crônico não afetaram o JBT. Frangos aproximaram e bicaram mais as pistas ambíguas comparado com as pistas não reforçadas. Frangos do tratamento controle aproximaram mais rápido das pistas ambíguas do que os frangos do tratamento alta complexidade, sugerindo que eles estão em estados afetivos mais positivo. Frangos submetidos à alta complexidade foram mais estressados. Em conclusão, enriquecimento ambiental reduz a prevalência de espondilolistese e melhora o comportamento exploratório. O hiper enriquecimento ambiental do Exp. 2 não foi apropriado para frangos de corte de crescimento lento. Palavras-chave: Avicultura, comportamento, cognição, espondilolistese, estados afetivos, performance. IX BEHAVIOR AND PRODUCTIVE INDICATORS FOR BROILER CHICKENS: IS ENVIRONMENTAL ENRICHMENT ALWAYS POSITIVE? ABSTRACT - With the consensus that animals are sentient beings, research on animal welfare is focused on the individual and its experiences. Two experiments were conducted, the objective of Exp. 1 was to evaluate the use of environmental enrichment on the productivity, behavior, and prevalence of spondylolisthesis in fast-growing broilers. In Exp. 2, the objective was to develop a judgment bias test (JBT) for slow- growing broilers, using the social aspects of the species, to assess the effects of fear, anxiety, and chronic stress and to measure the affective states of the broilers. In Exp. 1, 2400 one-day-old, male, Ross® AP95 chicks were used. The animals were allocated into 4 treatments distributed in a randomized experimental design with 4 replications each. The treatments were: control (C) - environment similar to the commercial one without enrichment; environments enriched with hay (HB), step platforms (SP), or laser lights (LL). In Exp. 2, 600 one-day-old, male, Hubbard Redbro chicks were used. The animals were allocated into 2 treatments distributed in a randomized block design, with 6 replications each. Treatments were: low complexity, environment similar to commercial settings, or high complexity, a combination of permanent and temporary enrichments. In Exp. 1, when raised with access to SP and LL, there was a lower frequency of subclinical spondylolisthesis when compared to chickens raised without enrichment or with access to HB. Chickens with access to SP had a higher wing yield and less abdominal fat compared to the C group. Chickens with LL and HB explored more and rested less than the animals in the C and SP treatments. In Exp. 2, fear, anxiety, and chronic stress did not affect JBT training and testing performance. Chickens approached and pecked more the ambiguous cues near the reward cue than those near the neutral cue. Chickens in the control treatment approached the ambiguous cues faster than chickens in the high complexity treatment, suggesting that they are in more positive affective states. Chickens kept in high-complexity treatment were more stressed. In conclusion, environmental enrichment reduces the prevalence of spondylolisthesis and improves exploratory behavior. The enrichment strategy in Exp. 2 was not suitable for slow-growing broilers. Keywords: Affective states, behavior, cognition, spondylolisthesis, performance, poultry. X LIST OF TABLES CHAPTER 2 Table 1. Experimental ethogram of recorded broiler chicken behaviors, based on [44]…… .......................................................................................................................... 36 Table 2. Performance (mean ± SEM) of broiler chickens between days 1-21, days 1-35, and days 1-42 of rearing, n = 16 ...................................................................................... 45 Table 3. Yield (mean % ± SEM) of carcass, breast, wings, legs, back, breast fillet, boneless legs, and abdominal fat of broiler chickens slaughtered at day 43 of age, n = 16…. ................................................................................................................................ 46 CHAPTER 3 Table S1. Mean estimate responses and proportions (%) of broiler chickens characterized as fearful or fearless in the tonic immobility test (s ± SEM; 0-300 s) and broiler chickens characterized as anxious or calm in the attention bias test (s ± SEM; 0-300 s) .............. 73 Table 1. Statistical approaches per measurement, with response variable tested, distribution of data residuals, statistical test used, predictors that were assessed, and random variables that were included in the model .......................................................... 75 Table S2. Number of successful chickens (n) and JBT training rounds (mean ± SD) needed to meet the learning criteria by complexity treatment, behavioral and chronic stress categorization ......................................................................................................... 77 Table S3. Least squares mean estimates (± SEM) and proportions (%) of broiler chickens from low- or high-complexity treatments in the tonic immobility test (0-300 s) and the attention bias test (0-300 s) .............................................................................................. 67 XI LIST OF FIGURES CHAPTER 1 Figure 1. Three animal welfare conceptions. Adapted from Fraser (2008).................... 05 CHAPTER 2 Figure 1. Broiler chickens housed in four environments. (A) Control (B) Hay bale, (C) Step platform, and (D) Laser lights ................................................................................. 35 Figure 2. Presence or not of subclinical spondylolisthesis in broiler chickens’ backs at 43 days of age. (A) Score 0: vertebrae found on their normal axis without compressing the bone marrow, (B) Score 1: vertebrae compressing the bone marrow ............................. 38 Figure 3. Frequency of observations (mean % ± SEM) of each behavioral category by treatment (C = control, HB = hay bales, SP = step platforms, LL = laser lights) across 2 time periods (09:00 and 15:00 h), n = 192 observations. Frequencies of (A) resting behavior, (B) exploratory behavior, (C) comfort behavior, and (D) consummatory behavior. Means within behavioral category without a common superscript (a–c) differed at P < 0.05 ........................................................................................................................ 40 Figure 4. Frequency of observations (mean % ± SEM) of each behavioral category by chicken age (in weeks) across two time periods (09:00 and 15:00 h), n = 192 observations. Frequencies of (A) resting behavior, (B) comfort behavior, (C) exploratory behavior, and (D) consummatory behavior. Means within a behavioral category without a common superscript (a–e) differed at P < 0.05 ................................................................................ 41 Figure 5. Frequency of observations (mean % ± SEM) of behaviors associated with the provided hay bale by age (in weeks) across two time periods (09:00 and 15:00 h). Sitting or resting by bale (n = 24), Pecking (n = 24), and Resting on top (n = 24). Frequencies without a common superscript (a-c) differed at P < 0.05 .................................................. 42 Figure 6. Frequency of observations (mean % ± SEM) of behaviors associated with the provided laser lights by age (in weeks) across two time periods (09:00 and 15:00 h), n = 24. Frequencies without a common superscript (a-c) differed at P < 0.05 ....................... 42 XII Figure 7. Frequencies (%) of broiler chickens by gait score (GS) at 21, 28, 35, and 42 days of age, n = 16. GS 0: Healthy chickens that exhibited no abnormality when walking, GS 1: Chickens that exhibited difficulty walking in a way that was easily identifiable through observation, and GS 2: Chickens exhibiting severe issues walking [45]. Ages without an uncommon superscript (a–d) differed at P < 0.05 ........................................... 43 Figure 8. Proportion (%) of broiler chickens with subclinical spondylolisthesis score 1 (vertebrae compressed the bone marrow) at 43 days of age (n = 192). C = control, HB = hay bales, SP = step platforms, LL = laser lights. Bars without a common superscript (a– c) differed at P < 0.05 ....................................................................................................... 44 CHAPTER 3 Figure 1. Chickens housed in two complexity environments. (A) Low-complexity, similar to commercial standards with feed, water and shaving; and (B) High-complexity, with permanent and temporary enrichments ................................................................... 63 Figure S1. Judgment bias test arena. Chickens were placed in the start box prior to all training or testing sessions. Colored cues and associated containers (black or white) were placed at either reinforced location (left or right). During testing, three additional ambiguous-colored cues were placed at intermediate locations (near positive, middle, near neutral). During all training and testing phases, cues were individually presented…… .................................................................................................................. 64 Figure S2. Diagram of the judgment bias habituation phase protocol (n = 24 chickens). All chicks undergo 4 habituation rounds. Blue boxes represent habituation phase methods, white boxes represent bird responses. Adapted from59 .................................... 65 Figure S3. Diagram of the judgment bias training phase 1A protocol for round 1 (n = 24 chickens). Blue boxes represent training phase 1A methods, white boxes represent bird responses. Adapted from59 ............................................................................................... 67 Figure S4. Diagram of the judgment bias training phase 1B protocol (shaping) (n = 24 chickens). Blue boxes represent training phase 1B methods, white boxes represent bird responses. Adapted from59 ............................................................................................... 68 XIII Figure S5. Diagram of the shaping procedure that was applied in each round of training phase 1B (n = 24 chickens). Learning criteria were orient (round 1), approach (round 2, or peck cue (round 3). Chicks will remain in this phase until successful, or the birds will be excluded from the trial after 7 rounds. Blue boxes represent shaping procedure methods, white boxes represent bird responses ............................................................... 69 Figure S6. Diagram of the judgment bias training phase 2 protocol (n = 22 chickens). Blue boxes represent training phase 2 methods, white boxes represent bird responses. (A) Chicks are presented with a reward cue. (B) Chicks are presented with a neutral cue. Adapted from59 ................................................................................................................ 70 Figure S7. Diagram of the judgment bias testing phase protocol (n = 20 chickens). Blue boxes represent testing phase methods, white boxes represent bird responses. Each round has seven 1-minute attempts with reference cues (reward and neutral) and ambiguous cues (Near-positive [NP]; Middle; Near-neutral [NN]) being presented individually ............ 71 Figure 2. Mean (± SEM) latency to approach (s) all five cues (Reward; Near-positive [NP]; Middle; Near-neutral [NN]; and Neutral) in 4 rounds of the judgment bias test (n = 11 social pairs). Means with uncommon superscripts (a–c ) differ at P < 0.001 ............ 78 Figure 3. Proportion (%) of chickens that pecked all five cues (Reward; Near-positive [NP]; Middle; Near-neutral [NN]; and Neutral) in 4 rounds of the judgment bias test (n = 11 social pairs). Proportions with uncommon superscripts (a–c ) differ at P < 0.001 .... 78 Figure 4. Mean (± SEM) of latency to approach (s) all five cues (Reward; Near-positive [NP]; Middle; Near-neutral [NN]; and Neutral) in 4 rounds of the judgment bias test by complexity treatment (n = 11 social pairs). Means with uncommon superscripts (a–d ) differ at P < 0.05 .............................................................................................................. 79 Figure 5. Least square mean estimates (± SEM) of feather corticosterone concentrations for chickens from low- or high-complexity treatments (n = 22 chickens). Means with uncommon superscripts (A-B ) differ at P < 0.1 ............................................................. 81 XIV TABLE OF CONTENTS Page CHAPTER 1 ..................................................................................................................... 1 1. Initial considerations ..................................................................................................... 2 2. Literature review............................................................................................................ 4 2.1. Animal welfare .................................................................................................... 4 2.2. Husbandry conditions and broiler welfare concerns ........................................... 5 2.3. Impacts of lameness on welfare .......................................................................... 7 2.4. Environmental enrichment .................................................................................. 9 2.5. Cognition, emotions, and affective states.......................................................... 10 2.6. Judgment bias test ............................................................................................. 12 3. References ................................................................................................................... 15 CHAPTER 2 ................................................................................................................... 29 Providing environmental enrichments can reduce subclinical spondylolisthesis prevalence without affecting performance in broiler chickens. ............................... 30 Abstract .................................................................................................................... 30 Introduction .............................................................................................................. 31 Materials and methods ............................................................................................. 33 Results ...................................................................................................................... 39 Discussion ................................................................................................................ 46 References ................................................................................................................ 50 CHAPTER 3 ................................................................................................................... 58 Social-pair judgment bias testing in slow-growing broiler chickens raised in low- or high-complexity environments ................................................................................. 59 Abstract .................................................................................................................... 59 Introduction ............................................................................................................. 59 Methods .................................................................................................................... 61 Results ...................................................................................................................... 76 Discussion ................................................................................................................ 81 References ............................................................................................................... 85 XV CHAPTER 4 ................................................................................................................... 94 Implications .............................................................................................................. 95 1 CHAPTER 1 2 1. INITIAL CONSIDERATIONS Broiler chicken production in Brazil and the world has significantly evolved over recent years. With investments in technological innovations in genetic improvement, ambiance, nutrition, and health, broilers today are more efficient and productive (ABREU; ABREU, 2011). Brazilian production stands out on the world stage, being the largest exporter and third-largest chicken meat producer (ABPA, 2022). Such Brazilian importance in the world poultry market makes discussions related to the welfare of these chickens more relevant for maintaining the country's potential. Lameness is the main welfare problem encountered in poultry production. Spondylolisthesis, for example, is a deformity in the sixth thoracic vertebra of chickens that compresses the spinal cord, causing pain and difficulties in walking, which can progress to paralysis of the pelvic limbs in clinical cases. (DINEV, 2013). The subclinical form of spondylolisthesis can affect 15 to 47% of the flock, silently compromising the welfare. With the advancement of animal welfare science and the consensus that animals are sentient beings, research on animal welfare is increasingly focused on the individual and their experiences throughout raising (CARENZI; VERGA, 2009). Based on this, making the environment more complex and dynamic using environmental enrichment resources is a strategy adopted by researchers and industry to improve birds' quality of life in production systems. Environmental enrichment is a management strategy in poultry barns that increases the expression of natural behaviors, meeting the natural living sphere from the animal welfare conceptualization (FRASER, 2008). Additionally, increased locomotion due to increased exploratory activity promoted by the environmental enrichment use strengthens the locomotor system and reduces the lameness prevalence (LOURENÇO DA SILVA et al., 2021). Thus, meeting the basic health and functioning sphere from the animal welfare conceptualization (FRASER, 2008). Even with a limited number of studies on broiler chickens, the use of environmental enrichment is associated with an increase in positive emotional experiences, improving the animals' affective states (ANDERSON et al., 2021a, 2021b) and, in turn, meeting the affective state sphere from the animal welfare conceptualization (FRASER, 2008). The development and application of cognitive bias tests, such as the judgment bias test, by ethologists, has been shown to be a valuable tool to assess animal’s affective 3 states (LAGISZ et al., 2020). However, they have limitations in the applicability for chickens, with long training phases and varied success rates, which may be related to how the test is designed and applied for the species. Therefore, studies focused on improving the test are necessary for the viability of this tool in animal welfare assessments. The effects of environmental enrichment on the prevalence of subclinical spondylolisthesis and a social-pair judgment bias test for broiler chickens are currently knowledge gaps in the literature. This doctoral thesis evaluates the current understanding of the use of environmental enrichment in the production and welfare of broiler chickens. The literature review in chapter 1 focuses on animal welfare parameters and how husbandry conditions, lameness, and environmental enrichment impact broiler welfare. In addition, the literature review dives into the current understanding of cognition, emotions, and affective states in broiler chickens. Additionally, two more chapters were included, which were published in high-impact scientific journals. The study described in chapter 2 entitled “Providing environmental enrichments can reduce subclinical spondylolisthesis prevalence without affecting performance in broiler chickens” investigates the environmental enrichment effects on the prevalence of subclinical spondylolisthesis and the performance of broiler chickens. This chapter is formatted according to the guide for authors of the journal PLoS ONE, where it was published. https://doi.org/10.1371/journal.pone.0284087 The study described in chapter 3 entitled “Social-pair judgment bias testing in slow-growing broiler chickens raised in low- or high-complexity environments” investigates the environmental enrichment effects on the affective states of slow-growing broiler chickens. In addition, this study developed a new judgment bias test considering the social aspects of the species. This chapter is formatted according to the guide for authors of the journal Scientific Reports, where it was published. https://doi.org/10.1038/s41598-023-36275-1 Finally, the general implications of the studies developed in this thesis were included in chapter 4. https://doi.org/10.1371/journal.pone.0284087 4 2. LITERATURE REVIEW 2.1 Animal welfare The understanding of animal welfare has evolved over the years; animal welfare is related to the individual and their experiences throughout life (CARENZI; VERGA, 2009). Although multiple animals are raised under the same conditions, each individual may have a different level of welfare due to their life experience in the breeding environment, their health, and their genetics (BROWNING, 2022). Widely used since it was mentioned in the Brambell report (1965) and improved by Farm Animal Welfare Council (FAWC) in 1993, the concept of the five freedoms guided the building of legislation, policy statements on the subject, and standards related to human responsibility to ensure animal welfare (MELLOR, 2016), being them: (1) Freedom from hunger and thirst (2) Freedom from discomfort (3) Freedom from pain, injury, or disease (4) Freedom to express normal behavior (5) Freedom from fear and distress This concept has been internationally recognized as a fundamental principle of animal welfare (VAPNEK et al., 2010). With the consensus that animals are sentient beings, research on animal welfare is focused on the individual and its experiences, researchers began to understand that animal welfare goes far beyond physical issues and involves the animal's willingness (DAWKINS, 2017). Thus, researchers began to consider the positive emotions of animals, also known as positive welfare (MELLOR, 2016; LAWRENCE; VIGORS; SANDØE, 2019). Then comes a multifactorial and comprehensive approach to assessing the animal’s mental welfare, where the animal has a life worth living. (GREEN; MELLOR, 2011). The animal welfare conceptualization involves 3 spheres that are interconnected (Figure 1), namely basic health and biological functioning, natural living, and affective states (FRASER, 2008; MOLENTO, 2012). The basic health and biological functioning sphere seeks to ensure the proper functioning of the body, focused on aspects of health, nutrition, reproduction, and growth (BARNETT et al., 2001). Welfare is the “state of the organism during its attempts to adjust to its environment” (BROOM, 1986). The natural 5 living sphere focuses on the importance of the animal living as if it were in its natural habitat, in this case, the breeding environment must provide conditions for the animals to express their natural behavior as if they were in the wild (DUNCAN; FRASER, 1997). The affective state sphere seeks to ensure positive experiences for animals (comfort, contentment, and positive social interactions) and reduce negative experiences (frustration, fear, pain, and suffering). The focus is on feelings, emotions, and affective states (DUNCAN, 2005). Figure 1. Three animal welfare conceptions. Adapted from Fraser (2008) For broiler chickens, several factors affect animal welfare, such as husbandry conditions, walking ability, bird health, the expression of natural behaviors through a more complex environment, and positive experiences affecting the affective states throughout rearing (MELUZZI; SIRRI, 2009; JACOBS et al., 2023). 2.2 Husbandry conditions and broiler welfare concerns The production of chickens on an industrial scale is the most adopted husbandry system in the world poultry chain, as it meets the needs of poultry farmers and guarantees good profitability. Intensive husbandry meets the demands of the national market, stands out in the international market, and meets the farmers’ goals with more return (BASSI; SILVA, 2017). Breeding is usually done in large poultry barns equipped with feeders, 6 drinkers, and bedding material with varying technology levels. It presents a stocking density between 31 and 42 kg/m², varying according to the region weather, the year season, the barn ambiance, and the slaughter age (FAIRCHILD, 2005). Industrial breeding stands out for its high productivity and high investments in genetics, nutrition, health, and management that contribute to the evolution of breeding (ABREU; ABREU, 2011). Other factors, such as legislation, council recommendations, or farmer associations, influence the stocking density used in poultry barns. In Brazil, the welfare protocol of the Animal Protein Brazilian Association suggests a maximum breeding density of 39 kg/m² (ABPA, 2016). In the United States of America, the National Chicken Council (2017) recommends that the maximum stocking density be related to the slaughter weight, for example, 31.7 kg/m², 36.6 kg/m², 41.5 kg/m², and 43.9 kg/m² related to the slaughter weights of 2.0 kg, between 2.0 kg and 2.5 kg, between 2.5 kg and 3.4 kg, and greater than 3.4 kg live weight, respectively. The European Union allows a maximum of 39 kg/m², which may exceed up to 3 kg/m² if specific criteria are met. In Germany, the maximum density is also 39 kg/m² (LOUTON et al., 2018), while in Norway, the legislation only allows 36 kg/m² (VASDAL et al., 2019). When breeding on an industrial scale is analyzed, it is observed that there have been significant advances in the animals’ comfort through improvements in the ambiance of the poultry houses to meet the requirements of the birds' thermoneutral zone (ABREU; ABREU, 2011). Additionally, there have been advances in sanitary aspects, animal nutrition, and reduction of lameness with genetic improvement (ABREU; ABREU, 2011). However, this husbandry model still receives criticism, and improvements are needed to improve the quality of life of the animals (DUNCAN, 2006; CORNISH; RAUBENHEIMER; MCGREEVY, 2016). From a practical point of view, environments such as poultry barns lack resources that stimulate the expression of natural behaviors such as foraging, dust bathing, and perching, which restricts animals to environmental and behavioral stimuli (COSTA et al., 2012; TAHAMTANI et al., 2018; LOURENÇO DA SILVA et al., 2021). The husbandry environments have food, water, and shelter easily available through well-distributed feeders and drinkers, in addition to the high stocking density that reduces the willingness to explore and, consequently, makes it impossible for the animal to express its natural behavior, causing a negative impact on the animal’s quality of life and leading to higher incidences of lameness (HAYE; SIMONS, 1978; COSTA, 2002; SANS et al., 2014). 7 2.3 Impacts of lameness on welfare At the beginning of the 21st century, farms recorded a high lameness prevalence causing economic losses of up to 40% (MENDES et al., 2016). These problems are multifactorial and are related to genetic factors (WEEKS et al., 2000; ALVES et al., 2016a), nutritional imbalance (COOK, 2000; WALDENSTEDT, 2006), husbandry conditions (BIZERAY et al., 2002; CORDEIRO; NÄÄS; SALGADO, 2009), age (BILGILI et al., 2006), stocking density (SORENSEN; SU; KESTIN, 2000), incubation conditions (OVIEDO-RONDÓN et al., 2009) and materials used as bedding (ALMEIDA PAZ et al., 2010). Genetic improvement has managed to reduce to acceptable levels the prevalence of these disturbances in the musculoskeletal system of birds. Currently, birds first develop a bone structure and then gain muscle mass (HARTCHER; LUM, 2020). However, the environment where birds are reared still does not favor increased exploratory activity, reducing locomotor activity, consequently registering lameness and restricting the expression of natural behaviors (LOURENÇO DA SILVA et al., 2021). Lame birds remain seated on the tibiotarsometatarsal joints most of the time, with a greater predisposition to the development of wounds on the feet, burns in the tarsal region (contact dermatitis), and blisters on the chest that can therefore depreciate the carcass (KESTIN; SU; SØRENSEN, 1999; SANOTRA et al., 2001). Due to the painful experience caused by locomotor disorders, modern broilers spend more time lying down and resting, between 53 to 86%, compared to their ancestor, the red junglefowl, approximately 10% (MURPHY; PRESTON, 1988; DAWKINS, 1989; WEEKS et al., 2000; ALVINO; ARCHER; MENCH, 2009; RIBER, 2015). Walking ability is the main indicator used by importing countries, which have stricter legislation, to assess animal welfare (CORDEIRO; NÄÄS; SALGADO, 2009). The gait score, initially developed by Kestin et al. (1992), is a practical method that evaluates the gait of birds along a path of one linear meter. Garner et al. (2002) improved the method by removing intermediate scores and assigning scores ranging from 0 to 2, with 0 being a bird that walks normally and takes at least ten uninterrupted steps, 1 - a bird that has difficulty walking and takes between six and ten uninterrupted steps; and 2 - a bird that walks with great difficulty and takes less than six uninterrupted steps or does not walk at all (GARNER et al., 2002). The main locomotor problems commonly found that affect the gait score and, consequently, broiler welfare are: spine and joint deviations (spondylolisthesis, valgus 8 and varus), tibial dyschondroplasia, femoral degeneration, pododermatitis, and dorsal cranial myopathy (BERNARDI, 2011; ZIMERMANN et al., 2011; AMARAL et al., 2017). Hereinafter, spondylolisthesis stands out in this study, which presents a subclinical phase silently compromising broiler welfare (DINEV, 2013). Spondylolisthesis, also called kinky back, is a deformity caused by broilers' disturbed growth of the thoracic vertebral arch, with a higher prevalence in the sixth vertebra (T6). It compresses the spinal cord bringing locomotor difficulties and leading to paralysis of the pelvic limbs in severe cases (KELLY, 1971; ABBASABADI; GOLSHAHI; SEIFI, 2021). Commonly, this condition affects birds between the 3rd and 6th week of age before progressing to clinical cases. (DINEV, 2013). Between 15 to 47% of the flock is affected by the subclinical form of spondylolisthesis, while about 2% of the animals show clinical signs such as lameness and the behavior of sitting with the feet outstretched or falling on one side (HOWLETT; WOOD, 1984; JULIAN, 2004; CRESPO; SHIVAPRASAD, 2008; ALVES et al., 2016a). Previous studies have suggested that the main cause of this condition is a genetic predisposition (OSBALDISTON, 1967; WISE, 1970; KELLY, 1971). According to Alves et al. (2016a), indigenous chickens (slow-growing) have better postural balance conditions, better gait scores, and no prevalence of subclinical spondylolisthesis compared to commercial strains (fast-growing). However, other studies have not confirmed this hypothesis (ALMEIDA PAZ et al., 2010; DINEV, 2012). Likely, a combination of genetic predisposition and environment (rearing and nutrition conditions) may cause the high prevalence of subclinical spondylolisthesis (ABBASABADI; GOLSHAHI; SEIFI, 2021). Due to the locomotor system immaturity, modern chicken strains have weak tendons and bones to support the body weight caused by rapid muscle growth and exacerbated development of the Pectoralis major muscle (ALVES et al., 2016a). This condition changes the center of gravity and posture of the birds, leading to an overload on the locomotor system, which affects walking ability and increases the prevalence of lameness (WEEKS et al., 2000; ALVES et al., 2016b). In another study evaluating the provision of sawdust fiber-diluted diets in the first week of life, Wise (1970) found a lower prevalence of subclinical spondylolisthesis for birds that received high fiber concentrations, but daily gain was also stunted. Lameness is the main factor affecting broiler welfare, recent research has found evidence that lameness can be minimized due to the positive effects of skeletal and 9 muscular development in broiler chickens with increased locomotor activity (REITER; BESSEI, 2009; RUIZ-FERIA et al., 2014; LOURENÇO DA SILVA et al., 2021; JACOBS et al., 2023). Thus, researchers and industry are developing and adopting new technologies to increase the locomotor activity of animals in the barn and, consequently, reduce lameness prevalence, also known as environmental enrichment (KELLS; DAWKINS; CORTINA BORJA, 2001; BAILIE; BALL; O’CONNELL, 2013; DE JONG; GOËRTZ, 2017). 2.4 Environmental enrichment Environmental enrichment is a management strategy adopted by industry and researchers to increase the complexity of the environment for animals kept in captivity (BELZ et al., 2003). Modifications are made to the environment to increase behavioral possibilities, reduce abnormal behavior in the species, increase exploratory activity in the environment, and improve biological functions that allow animals to cope with behavioral and physiological challenges throughout their lives (NEWBERRY, 1995; RIBER et al., 2018). The economic success of implementing environmental enrichment in a production system depends on some criteria, namely: increasing species-specific behaviors, maintaining or improving animal health, maintaining or improving flock productivity, and being practical, easy to implement, and cleaning (VAN DE WEERD; DAY, 2009; RIBER et al., 2018). For broilers, objects such as hay bales, dust bath substrates, perches, pecking objects, and raised platforms are most commonly developed, implemented, and evaluated as environmental enrichment (RIBER et al., 2018). These resources are provided to broilers from different strategies, being used permanently and temporarily throughout each production cycle (JACOBS et al., 2023). There is a certain level of inconsistency in the literature about the impacts of each environmental enrichment resource on broiler performance, which may vary according to the type of resource and strategy used. For instance, studies evaluating hay bales, peat, perches, platforms, pecking objects (hanging metal chains), dust baths, balls, and mirrors for fast-growing chickens found no effects of these enrichments on final body weight, feed conversion ratio, mortality or percentage of animals rejected at the slaughterhouse (VENTURA; SIEWERDT; ESTEVEZ, 2010; YILDIRIM; TASKIN, 2017; JONG; GUNNINK, 2018; VASDAL et al., 2019). On the other hand, other studies have reported that animals with access to perches and ramps reduced final body weight and feed intake 10 compared to a control group, but no effect on feed conversion ratio was observed (MARTRENCHAR et al., 2000; RUIZ-FERIA et al., 2014). Ohara et al. (2015), assessing hay bales and perches for slow-growing broilers, found better average daily gain and final body weight, but worst feed conversion ratio and mortality compared to the control group. Due to these inconsistencies, more studies are needed to understand environmental enrichment effects on performance. Despite the inconsistencies about the environmental enrichment impacts on performance, its use for broiler chickens improves behavioral, physiological, health, and cognitive parameters (JACOBS et al., 2023). Previous studies have shown that access to perches, platforms, hay bales, light projectors, and pecking objects increased locomotor and exploratory activity and leg health compared to a control group (TABLANTE; ESTEVEZ; RUSSEK-COHEN, 2003; VENTURA; SIEWERDT; ESTEVEZ, 2010; ZHAO et al., 2013; BAILIE; O’CONNELL, 2015; BERGMANN et al., 2016; KAUKONEN; NORRING; VALROS, 2016; LOURENÇO DA SILVA et al., 2021). In addition to increased locomotor activity and improved leg health, the use of hay bales, platforms, light projectors, dust baths, vertical panels, mirrors, balls, and pecking objects reduces fear expression and promotes more behavioral opportunities (YILDIRIM; TASKIN, 2017; TAHAMTANI et al., 2018; ANDERSON et al., 2021a; LOURENÇO DA SILVA et al., 2021), besides promoting the development of cognitive functions, including learning and memory (TAHAMTANI et al., 2018). A complex and dynamic environment promoted by environmental enrichment stimulates positive emotions and behaviors and positively impacts the chickens’ affective states (JACOBS et al., 2023). Access to perches, dust baths, light projectors, balls, and pecking objects for broilers reduced negative affective states such as anxiety and pessimism compared to broilers raised in a barren environment (ANDERSON et al., 2021a, 2021b). 2.5 Cognition, emotions, and affective states Throughout a broiler chicken life, the rearing environment provides a range of information, such as feeders, drinkers, litter, equipment noise, temperature, humidity, and conspecifics and human presence. The chicken then acquires, processes, and stores this information using information-processing mechanisms such as sensory perception and memory, called cognition (PAUL; HARDING; MENDL, 2005; SHETTLEWORTH, 2009; BETHELL, 2015; ROELOFS et al., 2016; MARINO, 2017). From this information 11 collected by cognitive mechanisms, the individual's behavioral response will be influenced by an emotional response and an affective state (HORBACK, 2019). Emotions are individual short-lived experiences that usually last seconds or minutes. They are object- or event-related and are more intense (KREMER et al., 2020). When a stimulus is presented to the chicken (i.e., a mealworm, an environmental enrichment, a predator presence, high temperature, and a conspecific playing or exploring something), the chicken will present an emotional response to that specific stimulus (PAUL; HARDING; MENDL, 2005). This emotional response is a discrete event that quickly dissipates when the stimulus is removed (JACOBS et al., 2023). According to Paul; Harding; Mendl (2005), emotional response refers to the physiological and neural processes that give animals the ability to avoid harm or punishment and seek valuable resources or rewards. An animal's emotions include fear, rage, panic, care, seeking, play, and lust (PANKSEPP, 1998). The animal's emotional response to a given stimulus involves subjective, physiological, behavioral, and cognitive components (PAUL; MENDL, 2018), and falls on a two-dimensional spectrum of valence and arousal (HORBACK, 2019). Valence is how the animal perceives a given stimulus, which can be positive (pleasant, rewarding) or negative (unpleasant, punitive) (DE WAAL, 2011; CRUMP et al., 2020). Arousal refers to the intensity of emotion that a given stimulus causes, which can be high or low (DE WAAL, 2011; CRUMP et al., 2020). Emotional response always has a valence level, can vary in arousal and persistence (duration) (PAUL; MENDL, 2018), and can be influenced by long diffuse states called mood or affective states (PAUL; HARDING; MENDL, 2005; MENDL; BURMAN; PAUL, 2010; KREMER et al., 2020). In contrast to emotion, affective states or moods are individual long-lived experiences that usually last for hours or days. (KREMER et al., 2020). They are not object- or event-related (free floating), are less intense, and can also fall into the valence spectrum (KREMER et al., 2020; JACOBS et al., 2023). For example, an anxious, bored, or pessimistic animal is in a negative affective state. A happy, excited, or optimistic animal is in a positive affective state (JACOBS et al., 2023). Affective states result from cumulative experience throughout the animal's life (RUSSELL, 2003; BARRETT et al., 2007; KREMER et al., 2020), which means they result from emotional experiences accumulated on scales of valence and arousal over time (MENDL; BURMAN; PAUL, 2010; NETTLE; BATESON, 2012; TRIMMER et al., 2013). As the affective state results from cumulative emotions triggered by the 12 environment, it is also indirectly affected by the environment (KREMER et al., 2020), which can provide us with a big picture of the individual's experience over time (JACOBS et al., 2023). According to Paul; Harding; Mendl (2005), there is ample evidence that both cognitive processes and affective states occur in a causal direction, thus affective states influence cognitive processes and vice versa (LAZARUS, 1982; MENDL et al., 2009; DOLCOS; DENKOVA, 2015). For example, an aspect of the cognitive process such as decision-making can be influenced by the affective state (BECHARA, 2000). In humans and also observed in animals, when the individual is in a negative affective state, it tends to judge an ambiguous stimulus in a negative way (pessimistic), expecting more for an unpleasant experience than for a pleasant, rewarding experience (MACLEOD; BYRNE, 1996; ROELOFS et al., 2016). This situation, when the affective state influences cognitive processes such as judgment, attention, and memory, is defined as cognitive bias. (PAUL; HARDING; MENDL, 2005; MENDL et al., 2009; BETHELL, 2015; FAUSTINO; OLIVEIRA; OLIVEIRA, 2015). Cognitive bias can be used as an indicator of the animals' affective states and can be measured using cognitive bias tests (PAUL; HARDING; MENDL, 2005; MENDL et al., 2009). Cognitive bias tests are new tools used by ethologists as indicators to measure the affective states of animals and, consequently, welfare. It provides information on how animals perceive their environment (MENDL et al., 2009; WHITTAKER; BARKER, 2020). For instance, the judgment bias test that provides information about the animal's optimism (ROELOFS et al., 2016). 2.6 Judgment bias test The goal of a judgment bias test is to infer whether the individual is in a positive affective state (optimistic) or is in a negative affective state (pessimistic) (MENDL et al., 2009). An ambiguous cue is presented to the individual, who in turn will use cognitive mechanisms, judge and act as a result of an emotional response biased by the affective state (MENDL et al., 2009; BETHELL, 2015; ROELOFS et al., 2016). Based on the animal's behavioral response, questions about how the animal perceives that ambiguous cue and its expected result (positive or negative) are answered. Thus, it is possible to infer the animal's optimism and pessimism levels (MENDL et al., 2009; BETHELL, 2015; ROELOFS et al., 2016). Initially developed and applied to humans (ROELOFS et al., 2016), the first judgment bias test used in non-human animals was developed by Harding; Paul; Mendl 13 (2004). The authors used mice kept in predictable and unpredictable environments. The animals were trained to perform two discrimination tasks between two conditioned stimuli, the first task being to press a lever when hearing a tone indicating a positive event (reward) and the second task being not to press the lever when hearing a tone indicating a negative event (punishment). After the rats met the training criteria, the judgment bias test was performed. The animals were exposed to three ambiguous tones without reinforcement (reward or punishment). As an animal behavioral response, the latency to approach the levers when each ambiguous tone was presented was recorded. Rats kept in the predictable environment were faster at pressing the levers when ambiguous tones were played than rats kept in the unpredictable environment, indicating that they expected more reward in an ambiguous situation and therefore were more optimistic (HARDING; PAUL; MENDL, 2004). Several judgment bias tests have been developed and applied to more than 22 species of non-human animals (LAGISZ et al., 2020). Different cues were applied in these tests, such as visual (SALMETO et al., 2011), olfactory (BOLEIJ et al., 2012), spatial (KIS et al., 2015), tactile (BRYDGES; HALL, 2017), auditory (MURPHY; NORDQUIST; VAN DER STAAY, 2013), and multimodal (combinations between two or more cues) (BETHELL, 2015; ANDERSON et al., 2021b). Animals are trained to discriminate which cue represents a positive event (reward) and which cue represents a negative, neutral, or less positive event (punishment, unrewarded, or low reward, respectively). For this, the test is designed in one of the three types of discrimination tasks developed in the literature for the judgment bias test, namely: (1) Go/No-Go, (2) active choice with positive reinforcement (Go/ Go +) and (3) active choice with negative reinforcement (Go/Go -) (BETHELL, 2015). In the judgment bias tests that adopted the Go/No-Go discrimination tasks, animals were trained to associate the positive (+) cue with a reward outcome and to associate the negative (-) or neutral (n) cue with a punishment or unrewarded outcomes, respectively (BETHELL, 2015). This type of discrimination task has already been adopted for several species such as dogs (MENDL et al., 2010; BURMAN et al., 2011; STARLING et al., 2014; KIS et al., 2015), swine (DOUGLAS et al., 2012; SCOLLO et al., 2014), goats (BRIEFER; MCELLIGOTT, 2013), sheep (DESTREZ et al., 2013; VERBEEK et al., 2014; GULDIMANN et al., 2015), cattle (NEAVE et al., 2013; DAROS et al., 2014), horses (BRIEFER FREYMOND et al., 2014), cats (TAMI et al., 2011), primates (BETHELL et al., 2012; GORDON; ROGERS, 2015), rats (BURMAN 14 et al., 2008; RICHTER et al., 2012), fish (LAUBU; LOUÂPRE; DECHAUME- MONCHARMONT, 2019; ROGERS et al., 2020) and chickens (SALMETO et al., 2011; HYMEL; SUFKA, 2012; SEEHUUS et al., 2013; DEAKIN et al., 2016; ZIDAR et al., 2018; ANDERSON et al., 2021b). In the active choice discrimination task with positive reinforcement (Go/Go +) applied in judgment bias tests, the animals were trained to make an active choice in both cues. In this situation, both cues are positive (+), but one has one high-value reward while the other has a low-value reward (MENDL et al., 2009; BETHELL, 2015). This discriminative task was developed to mitigate a possible lack of motivation behind the 'No-Go' response of the Go/No-Go discrimination task, which could lead to animals not approaching ambiguous cues due to lack of awareness, motivation, confusion, or distraction (MENDL et al., 2009; ROELOFS et al., 2016). However, this discrimination task also receives criticism due to the double reward aspect, which would limit the understanding of judgment biases related to negative experiences in the animal's life. (MENDL et al., 2009). This discrimination task has already been adopted in tests of judgment bias for several species such as swine (MURPHY et al., 2015), brown bears (KEEN et al., 2014), primates (POMERANTZ et al., 2012), rats (BRYDGES et al., 2012; CHABY et al., 2013; PARKER et al., 2014) and chickens (HERNANDEZ et al., 2015). In the judgment bias tests that adopt the active choice discrimination task with negative reinforcement (Go/Go -), animals are trained to associate the positive (+) cue with a reward and associate the negative cue (-) with the end or to avoid a negative stimulus (punishment) (BETHELL, 2015; ROELOFS et al., 2016). This approach is generally not feasible in studies involving animal welfare, being applied more frequently in studies involving pharmacological manipulation using rats (BETHELL, 2015). In chickens (Gallus gallus domesticus), the judgment bias test has been developed and applied to assess affective states using the Go/Go and Go/No-Go discrimination tasks. The effects of environmental conditions (WICHMAN; KEELING; FORKMAN, 2012; SEEHUUS et al., 2013; ROSS et al., 2019; ANDERSON et al., 2021b), feather pecking genetic selection (PICHOVÁ et al., 2021), corticosterone injections (IYASERE et al., 2017), pharmacological reversal in an anxiety-depression model (HYMEL; SUFKA, 2012), anxiety-depression model induction (SALMETO et al., 2011), temperature manipulation (DEAKIN et al., 2016) and acute stress (HERNANDEZ et al., 2015) were assessed on chickens’ affective states. The birds showed a negative affective state, being more pessimistic when subjected to a barren environment (ROSS et al., 2019; 15 ANDERSON et al., 2021b), when injected with high corticosterone levels (IYASERE et al., 2017), or when induced to be anxious and depressed (SALMETO et al., 2011; HYMEL; SUFKA, 2012). However, some studies have reported unexpected results, for example, high feather pecking strain or acutely stressed chickens being more optimistic. (HERNANDEZ et al., 2015; PICHOVÁ et al., 2021), or a high-complexity environment not inducing a more positive affective state compared to a control group (WICHMAN; KEELING; FORKMAN, 2012; ROSS et al., 2019). These unexpected results add to a series of limitations found in judgment bias tests, such as time-consuming during training phases (CRUMP; ARNOTT; BETHELL, 2018), which would make application unfeasible under commercial conditions; side bias due to stressful manipulations on the animal’s affective states caused by treatments (MENDL et al., 2009), which would reduce the animals' learning ability in discrimination tasks; and learning of ambiguous clues due to the need to repeat the test (DOYLE et al., 2010; WHITTAKER; BARKER, 2020). In chickens, the training phases are long and present low learning success rates (WICHMAN; KEELING; FORKMAN, 2012; SEEHUUS et al., 2013; HERNANDEZ et al., 2015; DEAKIN et al., 2016; IYASERE et al., 2017; ANDERSON et al., 2021b), which impairs the test practicality. This may be associated with how the test is developed and applied to the species, with all training phases and the test phase conducted individually (ROELOFS et al., 2016) for a species that is highly influenced and dependent on social factors for the development of cognitive processes such as learning and memory (NICOL, 2006; MARINO, 2017), which could influence performance during the training and testing phases. According to Marino (2017), chickens have more complex cognitive, emotional, and communicative processes besides social behavior. 3. REFERENCES ABBASABADI, B. M.; GOLSHAHI, H.; SEIFI, S. 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Avian Diseases, v. 56, n. 2, p. 418–421, 2011. 29 CHAPTER 2 30 Providing environmental enrichments can reduce subclinical spondylolisthesis prevalence without affecting performance in broiler chickens https://doi.org/10.1371/journal.pone.0284087 Abstract Environmental enrichment can increase the occurrence of natural behavior and improve leg health and other animal welfare outcomes in broiler chickens. This study aimed to assess the effects of three environmental enrichments, specifically hay bales, step platforms, and laser lights, on subclinical spondylolisthesis prevalence, productivity, behavior, and gait of broiler chickens (Gallus gallus domesticus). Twenty-four hundred day-old male Ross® AP95 chicks from a commercial hatchery were used in a completely randomized design with four treatments and four replicate pens per treatment. Pens contained either a Control (C) treatment, an environment similar to a commercial broiler chicken system without environmental enrichments, or an environment with either additional hay bales (HB), additional step platforms (SP), or additional laser lights (LL). Performance, yield, behavior (frequencies), gait score, and subclinical spondylolisthesis prevalences were assessed. When raised with SP or LL access, fewer chickens had subclinical spondylolisthesis than chickens without enrichments (C) or with HB access. Chickens with access to SP exhibited higher wing yield and less abdominal fat than animals from the C group. Chickens from the LL and HB treatments explored more and rested less frequently than animals from the C and SP treatments. As chickens aged, they became less active, exploring less and increasing resting and comfort behaviors. Treatments did not affect gait. Gait was not associated with subclinical spondylolisthesis prevalence. Environmental enrichments benefitted chicken health (subclinical spondylolisthesis) and behavior (exploration) without negative consequences for performance and yield. Keywords: Behavior, environmental complexity, leg health, gait score, poultry https://doi.org/10.1371/journal.pone.0284087 31 Introduction Locomotory activity is strongly associated with broiler chicken welfare; many behavioral patterns that depend on locomotion, such as exploration, seeking food, water, shelter, and escaping predators, are negatively affected by the poor walking ability in fast- growing broiler chickens [1]. Rapid muscle growth and exacerbated development of the Pectoralis major muscle in fast-growing broiler chickens change the chickens’ center of gravity, altering the chickens’ posture and load on the skeleton compared to slower- growing strains, leading to skeletal-biomechanical imbalances, in turn affecting walking ability and resulting in locomotor disorders [2,3]. Spondylolisthesis, also known as ‘kinky back’, is a deformity that affects the thoracic vertebral arch of chickens. It occurs in the sixth vertebra (T6), leading to spinal cord compression and locomotor difficulties and, in turn possibly resulting in paralysis of the pelvic limbs in severe cases [4,5]. This condition can often subclinically affect chickens between 3 and 6 weeks of age before it evolves into clinical cases [6]. Subclinical spondylolisthesis can affect 15 to 47% of the flock, while clinical spondylolisthesis can affect 2% of the flock [7–9]. Chickens with subclinical spondylolisthesis do not show symptoms, while broilers with clinical spondylolisthesis are lame and will sit with extended feet, show an imbalance, and fall on their side when attempting to stand [8,10–13]. A positive correlation between gait score and subclinical spondylolisthesis suggests this is a health and animal welfare concern [7]. The main cause of this condition is a genetic predisposition. Indigenous chickens (slow-growing) show a good balance, better gait, and no subclinical spondylolisthesis compared to fast-growing chickens [5,7,11,12]. However, recent studies did not confirm this [14,15]. It is probable that a combination of both genetic predisposition and environment (housing, nutrition) can lead to high prevalences of subclinical spondylolisthesis [4]. According to [7], tendons and bones in recent strains of broiler chickens are too weak to support the chickens’ body weight due to the immaturity of the musculoskeletal system. When providing diets diluted with wood sawdust fiber in the first week of life, prevalence of subclinical spondylolisthesis was reduced but daily gain was also stunted [16]. Locomotor disorders can to some extent be prevented by increasing animal activity, as locomotion can positively impact skeletal development [17,18]. Positive activity such as exploratory behavior and locomotion can be stimulated through 32 environmental enrichments, such as straw or hay bales, platforms, and moving laser lights [17–20]. Environmental enrichments increase the complexity of broiler chicken environments and enhance the expression of natural behaviors, i.e., foraging, perching, and playing [19,21]. Foraging is a highly motivated behavior associated with exploration and the appetitive phase of feeding behavior. Chickens peck and scratch the ground searching for food while collecting environmental information [22]. Perching is natural resting behavior; chickens seek an elevated place that allows them to perform surveillance behavior against predators [23,24]. Playing is another natural and social behavior; chickens run, jump, and interact with inanimate objects and conspecifics in a non- aggressive way [25,26]. Potentially beneficial enrichments are straw or hay bales, platforms, and moving laser lights. Hay bales can increase chicken activity by stimulating pecking, foraging, preening, worm running (play), and can provide a barrier for resting [27–29]. Platforms provide animals an elevated area for resting with fewer physical challenges compared to perches [17,23]. In addition, platforms allow chickens to perform natural locomotory behavior, such as jumping and walking up and down a ramp, which may improve chickens’ musculoskeletal strength and coordination, and in turn prevent skeletal- biomechanical imbalances [19,30]. Laser light enrichments have been used in previous research and seem a valuable resource to increase broilers' locomotion [19,31–33]. Laser lights can simulate the presence of an insect, stimulating chickens to approach the stimulus [34] and increasing locomotion during early life [19,34], which in turn may benefit the development of their musculoskeletal system. The effects of environmental enrichments on performance and yield depend on the type of resource used [35]. Studies assessing peat, bales, elevated platforms, perches, mirrors, balls, dust bathing substrates, and pecking objects (hanging metal chains) at 5 or 6 weeks of age for fast-growing broilers have reported no effects of these enrichments on final body weight, feed conversion ratio, mortality, or the percentage of animals rejected in the slaughterhouse [30,36–38]. In contrast, access to perches and ramps reduced body weights and feed intake compared to the control group, with no difference in feed conversion ratios [39,40]. Average daily gain and body weights improved in slow- growing chickens at 9 weeks of age when they had access to bales and perches, but feed conversion ratio and mortality were worsened compared to the control group [41]. Due 33 to these inconsistencies, more research is needed to understand the impact of environmental enrichments on productivity. Potential effects of environmental enrichments on subclinical spondylolisthesis have not previously been examined. Therefore, this study aimed to assess the effects of three environmental enrichments, specifically hay bales, step platforms, and laser lights, on subclinical spondylolisthesis prevalence, performance, behavior, and gait of broiler chickens. We hypothesized that each environmental enrichment would increase exploratory behaviors in broilers compared to an unenriched control. In turn, we hypothesized that enrichments would reduce the prevalence of subclinical spondylolisthesis (platforms>bales>laser lights) and improve gait scores without affecting productivity compared to an unenriched control. Materials and methods The experiment was carried out at the School of Veterinary Medicine and Animal Sciences (FMVZ) at São Paulo State University, Botucatu, SP, Brazil (22° 49’ 07” S and 48° 24’ 40” W). The experimental protocol was approved by the Animal Use Ethics Committee of FMVZ (number 0045/2020 CEUA). Chickens, facilities, and management Twenty-four hundred day-old male Ross® AP95 chicks from a commercial hatchery were used. The trial was carried out in a climate-controlled poultry barn featuring negative pressure ventilation. The barn contained sixteen pens (4 x 3 m) provided with 10 cm new wood shavings as bedding, three semi-automatic feeders (one feeder for 50 chickens), and a nipple drinker line (one nipple for 10 chickens). The litter was turned on days 17, 24, and 31. Each pen contained 150 chickens and a targeted maximum stocking density of 39 kg/m² [42]. Pens contained heat lamps in the first 10 days. House temperature was gradually decreased from 32°C on day 1 to 21°C on day 28 and remained 21°C until day 42. The chickens were maintained on an artificial lighting program of 24L:0D in the first 10 days due to the heat lamps and 16L:8D until the end of the trial. The corn- and soybean-meal-based diets were adapted from [43] and met the nutritional requirements in three rearing phases: starter (1-21 days, 24% CP and 3,000 kcal ME/kg), grower (22-35 days, 22.5% CP and 3,150 kcal ME/kg), and finisher (36-42 days, 19% CP and 3,250 kcal ME/kg). Both feed and water were provided ad libitum. 34 Experimental design The trial followed a completely randomized design with four treatments and four replicate pens per treatment. Pens contained either a Control (C) treatment (Fig. 1A), which consisted of an environment similar to commercial broiler chicken husbandry without environmental enrichments, or an environment with either additional hay bales (HB), step platforms (SP), or laser lights (LL). At 42 days of age, the stocking densities (mean±SE) calculated for each treatment were: C = 36.5±0.9 kg/m², HB = 36.0±0.9 kg/m², SP = 35.8±1.7 kg/m², and LL = 37.6±0.6 kg/m². Environmental enrichment All resources were introduced on the first day and remained available until day 42. One hay bale (75 x 42 x 30 cm, alfalfa hay) per HB pen (150 chickens) was provided and replaced on day 35. The bale was placed between the drinker line and the barn wall (Fig. 1B). Step platforms (60 × 60 × 7 cm for the lower base and 20 × 20 × 7 cm for the upper base) were made from MDF boards and one was provided in every SP pen (Figure 1C). When the litter was turned, the platforms were scraped to remove excreta. Step platforms were placed between the drinker line and the barn wall. One laser light projector per LL pen was placed at 1.5m height (Mini Stage Lighting XX-027, Spooboola, China). The projector emitted approximately 36 green and red laser lights simultaneously with wavelengths of 532nm (50mW) and 650nm (100mW; Figure 1D). These downwards projected lights moved across an area of approximately 12 m2 at a slow and steady pace. Projectors were turned on twice a day for 15 minutes, at 09:00 and 15:00 hours. 35 Fig 1. Broiler chickens housed in four environments. (A) Control (B) Hay bale, (C) Step platform, and (D) Laser lights. Measurements Behavior Sixteen high-resolution video cameras (Intelbras, São José, SC, Brazil) were installed at 1.5m height (angled down) to record behavior in each pen (total of 16 pens). Videos were recorded on days 6 (week 1), 13 (week 2), 20 (week 3), 27 (week 4), 34 (week 5), and 41 (week 6). Human disturbance was limited on recording days, and birds were only disturbed for health checks. Frequencies of all selected behavioral occurrences were recorded at pen-level by a single trained observer using 1-minute continuous scan sampling observation with 2 minutes inter-sampling intervals for two 15-minute time periods (starting at 09:00 and 15:00 hours). This resulted in 5 scans per time period and a targeted total of 960 behavioral entries (5 scans × 2 time points × 16 pens × 6 weeks). Behaviors were coded following the ethogram adapted from [44] (Table 1). After recording, behaviors were classified into four categories: consummatory, resting, exploratory, and comfort. Then, the frequencies of each behavioral category were calculated. In addition, the frequency of behaviors associated with the environmental enrichments was assessed individually. 36 Table 1. Experimental ethogram of recorded broiler chicken behaviors, based on [44]. Behavior Operational definition Consummatory Eating Chicken holds its head above the feeding trough or the surrounding area and actively taking in food Drinking Chicken is actively taking in water by pecking at nipple drinkers Resting Sitting/resting Chicken sits on the litter while the head rests on the ground or upright; eyes may be open or closed Sitting/resting by the bale (HB treatment) Chicken sits in immediate proximity to bale (within 10 cm) while the head rests on the ground or upright; eyes may be open or closed Rest on top of bale (HB treatment) Chicken stands or lies on top of a straw bale Rest on step platform (SP treatment) Chicken stands or lies on top of a step platform Exploratory Locomotion Moving using legs in a continuous forward motion (walking or running), chicken may be flapping wings Foraging Pecking and/or scratching at the flooring substrate Play After approach of another chicken at high speed, the chicken stops and faces the other briefly, without making physical contact. Pecking at hay bales (HB treatment) Chicken uses beak to manipulate hay in the bale Chasing and/or pecking at lig