Are laterality effects present in novel object responses of calves?

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Chenyu Zhang, Molly Kindell, Rebecca Meagher This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4462883/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Many animals exhibit preferential viewing of fear-inducing stimuli with their left eyes, reflecting cerebral lateralisation in emotion processing. In novel object tests, often used to assess fear, spatial positioning of objects relative to the animal can vary. This study aimed to investigate visual lateralisation in fear processing in novel object tests, evaluate its effectiveness as an indicator of fear, and examine how initial monocular presentation of fear-inducing stimuli impacts fear responses and their reliability. Thirty-six calves underwent a novel object test at six weeks of age and a second at seven weeks of age using a different object, with behavioural responses towards the novel objects recorded. There was no correlation in latency to first contact with novel objects between tests. Proportion of time viewing novel objects with the left eye did not significantly differ from the right eye, nor did it correlate with latency to first contact with novel objects. The probability of contact with novel objects and latency to first contact with novel objects did not differ based on initial eye contact with novel objects. Overall, fear responses in calves during repeated novel object tests were inconsistent, but this inconsistency could not be explained by novel objects being randomly presented to calves’ different visual fields, which suggests potential absence of cerebral lateralisation in fear processing. Thus, laterality of visual response may not reliably indicate fear in calves at this age. Biological sciences/Neuroscience Biological sciences/Zoology dairy calf novel object test repeatability visual lateralisation fear Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Fear has been considered as a negative emotion resulting from a perception of danger 1 . It can be expressed through a series of physiological and behavioural reactions to a perceived threat 2 . In natural conditions, predators are an important source to stimulate fear. Although most domestic animals do not commonly experience predation from natural predators, inappropriate housing and management procedures, such as dehorning, tail docking, castration, herding and transportation, can elicit fear-related responses 3 , 4 , which may reduce animals’ productivity, lead to chronic stress and thus alter fundamental behaviours 2 . For example, fear-related reactions affect sexual and maternal behaviours and social dominance in cattle and sheep 5 , 6 . Therefore, fear may be a severe welfare issue. although animals often feel fear when they have a perception of danger, finding effective ways to assess their fear levels and tendency to express fear (the personality trait of fearfulness 3 ) is an important step to improve their welfare. In calves, fear is often assessed through their responses to novelty and to humans 2 , 7 . Reliability has been considered as a criterion to assess the effectiveness of novelty tests, meaning that an effective fear test would get the same result in repeated measures of the same construct 2 . However, Meagher, et al. 7 reported poor test-retest reliability in novel object tests for calves, which indicated that it is difficult to draw strong inferences from a single test. Similar results are also found in older heifers and adult cows when tested using avoidance 8 , reactivity 9 , number of interactions and time in proximity 9 . Although the poor test-retest reliability in novel object tests has been widely reported, the mechanisms underlying this result are rarely studied. Growing numbers of studies have focused on non-human vertebrates’ cerebral lateralisation in emotional processing. In domestic species, many researchers have determined that animals dominate the processing of fear and anxiety through a right-hemisphere 10 . This is expressed by a left visual preference because the high degree of decussation of optical fibres ensures that input from the used eye is predominantly processed in the contra-lateral hemisphere 11 . For instance, chicks preferentially use the left eye to observe aerial predators 12 and adult chickens were found to scan the air for predators preferentially with the left eye after hearing a conspecific alarm call 13 . Studies in horses and cattle also showed evidence of a right-hemisphere dominance for the visual processing of fear. Austin and Rogers 14 reported that when a person walked toward a domestic horse while opening an umbrella, the horse reacted more, by moving further away, when approached from its left-side than they do when approached from its right-side. Robins and Phillips 15 investigated whether dairy and beef cattle exhibit preferences to monitor challenging and found that cattle had the preference to use the left monocular field viewing an experimenter walking to repeatedly split the herd through its centre. For altricial species, their brain is mainly developed after birth 16 and their specialisations of the left and right hemispheres are slowly established during infancy 17 . For instance, in chicks, hemispheric laterality in charge of behaviour occurs quite precisely during early development post-hatching 17 . In contrast, the brain development of precocial species mainly takes place before birth 16 and thus their hemispheric laterality is assumed to be well developed after parturition. For example, neonatal lambs are reported to have strong behavioural biases 18 . Since dairy calves are precocial, it is worth studying their development of emotional laterality. Austin and Rogers 14 reported that when a horse was presented with the novel object first on its right-side, it lowered the horse’s initial fear responses and lowered fear responses to subsequent presentations of the object compared to those following an initial left-side presentation. Since the majority of evidence suggests that the left hemisphere is dominant in processing positive emotions 19 , Austin and Rogers 14 suggested that the initial right-side presentation might inhibit the fear response and allowed horses to learn that the stimulus posed no threat, and this information could be transferred to the right-hemisphere. Therefore, animals’ responses to fear-inducing stimuli may be affected by the initial presentation of the stimuli. The first aim of the present study was to assess the test-retest reliability of calves’ responses in novel object tests. It was hypothesised that calves’ latency to make first contact with the novel objects in the first novel object test and the repeated novel object test would be inconsistent as in repeated tests, novel objects randomly presented to calves’ left, right, or both eyes initially, which might result in varying degrees of fear responses during repeated tests. The second aim of the present study was to determine if calves had visual lateralisation (cerebral lateralisation) in processing fear and whether such lateralisation could then be considered an effective indicator of fear. It was hypothesised that 1) due to brain development before birth, in novel object tests calves would prefer to view the novel objects with the left eye; 2) there would be a positive relationship between calves’ proportion of time viewing the novel objects with the left eye and their latency to make first contact with the novel objects, both reflecting increased fear. The third aim of the present study was to determine if calves’ initial monocular presentation toward fear-inducing stimuli would affect calves’ fear responses. It was hypothesised that in novel object tests, 1) fewer calves would make contact with the novel objects when they first viewed the novel objects with the left eye; and 2) calves would have longer latencies to make first contact with the novel objects when they first viewed the novel objects with the left eye. Results There was a poor correlation between the first novel object test and the repeated novel object test for calves’ latency to make first contact with the novel objects (r s = -0.148, p = 0.403; Fig. 1 ). In the first novel object test and repeated novel object test, there were no statistically significant differences in the proportion of time viewing the novel objects with left and right eyes (F 1,99 = 0.045, p = 0.832; Fig. 2 ). In addition, the object used, whether it was the first or repeated test, and social housing did not have significant effects on calves’ proportion of time viewing the novel objects with the left eye (objects: F 1,32 = 0.041, p = 0.842; tests: F 1,32 = 1.537, p = 0.224; social housing: F 1,31 = 0.437, p = 0.513), whilst calves from physically enriched pens spent longer durations viewing the novel objects with the left eye than those from non-physically-enriched pens (F 1,31 = 6.864, p = 0.013). In individual level, 15 calves exhibited a preference for utilising one eye over the other in both tests (Fig. 3 ). Among these calves, 9 displayed a preference for the left eye, while 6 favoured the right eye, however, the left and right eye preference did not have significant difference (two-tailed binomial p = 0.607). There was a poor correlation between calves’ proportion of time viewing the novel objects with the left eye and calves’ latency to make first contact with the novel objects (r s = -0.002, p = 0.503; Fig. 4 ). Probability of making contact with the novel objects in the first novel object tests and repeated novel object tests was 70.80% if the calf could view the object first with the left eye vs. 71.40% if the right, but this difference was not significant (Chi-squared p = 0.965). In both the first novel object test and repeated novel object test, there were no statistically significant differences in the latency to make first contact with the novel objects among calves’ first view of the novel objects with the left, right or both eyes (F 2,31 = 0.611, p = 0.549; Fig. 5 ). Discussion Since fear has been considered a welfare concern for many animals 20 , it is important to have methods, such as novelty tests, to assess its levels 2 . However, the results in this study show poor test-retest reliability of calves’ response to novel objects, which may make it difficult to draw inferences about welfare or personality from a single novel object test. The finding is consistent with some other studies on cattle 8 , 21 . For example, Gibbons, et al. 9 studied the consistency of cattle’s reactivity responses to three novel stimuli and found low reliability among tests. Thereafter, we tried to find out the potential mechanisms resulting in the poor test-retest reliability. Vertebrates typically prefer to view fear-inducing stimuli using the left eye 15 , and the eye that first sees a stimulus can influence fear related behaviours 14 . Inconsistency in the head orientation of calves relative to fear-inducing stimuli may explain differences in responses on repeated tests. We therefore studied whether calves had a preference to view fear-inducing stimuli using their left eyes, and if having their first view of the object with the left, right, or both eyes affected their fear responses. We assumed that if calves had cerebral lateralisation in processing fear resulting from the novel objects, initially presenting the fear-inducing stimuli into calves’ left visual field could stimulate increased fear responses. Since in the novel object tests, objects are randomly presented to calves’ left, right or both eyes initially, it may lead to different levels of fear responses for every calf in repeated tests. However, our findings were inconsistent with the predictions. No cerebral lateralisation in processing fear resulting from novel objects was found in this study, whilst the opposite findings have previously been reported in cattle and other species. For example, many bony fish species show a right hemisphere dominance of processing of predator stimuli, evidenced by lateralised motor responses 22 and lateralised visual perception 23 . In reptiles, a left eye preference during predator inspection exists for wall lizard 24 . In domestic chickens, Evans, et al. 13 found that adult hens prefer to scan the air for predators with the left eye after hearing a conspecific alarm call. Robins and Phillips 15 found that dairy and beef cattle had significant left eye preferences for viewing an experimenter walking to split the herd through its centre. The difference between this and other studies may result from the development of hemispheric specialisation. In this study, calves attended the tests when they were six or seven weeks of age. Although the brain development for ungulate livestock mainly takes place before birth 16 , it can be hypothesised that hemispheric specialisation may not be well developed in the neonatal period. The finding of a poor correlation between calves’ proportion of time viewing the novel objects with the left eye and calves’ latency to make first contact with the novel objects in this study supports this hypothesis and indicates that left eye observation towards fear-inducing stimuli may not be considered an effective fear indicator at this age. Since calves may not have had visual lateralisation in processing novel stimuli in this study, it is not surprising that they did not show a significant difference in fear-related responses to novel stimuli initially perceived in the left compared to the right monocular field. However, this finding is in contrast to Austin and Rogers 14 . They studied whether horses display greater reactivity to a novel stimulus initially presented in the left compared to the right monocular visual field by testing horses in a scenario of a person opening an umbrella five metres away and then approaching. The measured reaction was the distance each horse moved away before stopping. They found that horses tested initially on the left-side exhibited greater reactivity for the left approach, whereas horses tested on the right-side first displayed no side difference in reactivity. This greater reactivity to the left-side approach, therefore, depended on the side of the initial monocular presentation. When the initial monocular presentation was in the left visual field, the horses responded with greater left-side reactivity, but when the stimulus was presented in the right monocular field first, subsequent reactivity to the stimulus in the left monocular field was similar to that of the right eye. It may suggest that due to interocular transfer 25 , which occurs from the left hemisphere to the right hemisphere 26 , habituation to the stimulus occurs when the first presentation is to the right eye but not when it is to the left eye. However, for neonatal calves, their hemispheric specialisation may not be well developed and thus, they may not have the interocular transfer. Therefore, calves’ poor test-retest reliability may not result from cerebral lateralisation. In addition to calves not having well-developed hemispheric specialisation in processing fear at an early age, there is another potential explanation for the lack of lateralised responses seen in this study. When we conducted the tests, some calves lay down in the sawdust on the ground several minutes after entering the test arena, and showed limited behavioural responses to the objects. This may indicate that calves were not frightened enough in the novel object tests and thus, this test environment could not stimulate their lateralised responses. Future research needs to adjust the test environments to further stimulate calves’ fear (e.g. providing a more frightening object to calves) and confirm there is a true fear response to draw strong conclusions regarding lateralisation. Animals’ early experience is suggested to affect their development of hemispheric specialisation 27 . Neonatal stimulation can have profound effects on the specialisation of their brain in mammalian species 28 . For instance, neonatal handling of rats can provide stimulation to give rise to right hemispheric dominance, which increases animals’ reactivity, emotionality and the aggressive behaviour of muricide 28 . Exposure of neonatal rats to novelty, meanwhile, modulates hand preference towards control by the right hemisphere 29 and increases the right hippocampal volumetric dominance 30 . Moreover, Galuret et al. 31 found that chicks lacking maternal care showed stronger lateralisation, enhanced sociability, and reduced emotional responsiveness when compared to maternally raised chicks. In the current study, calves experienced differential levels of stimulation (i.e. social housing, physical enrichment) before attending the first and repeated novel object tests. Social housing seems unlikely to have affected calves’ cerebral lateralisation in processing fear, evidenced by calves from individual pens and pair pens spending similar amounts of time viewing novel objects with their left eyes. Although physical enrichment was found to reduce calves’ time spent viewing novel objects with the left eye, this does not necessarily mean this stimulus promotes the development of cerebral lateralisation in processing fear. Animals with environmental enrichment may be less likely to approach novel stimuli 32 , 33 . Since physically enriched calves had more experience with novel objects (i.e. stationary brushes, plastic chains, dry teats and nets filled with scented hay) in their home pens before attending the novel object tests, the objects in the novel object tests might be less attractive to them, and thus they spent less time viewing the objects without necessarily being less fearful of the objects. The findings in Zhang, et al. 34 may also support this point of view, which indicated physical enrichment had no effects on calves’ fear responses (e.g. latency to make first contact with novel objects) in novel object tests. At the individual level, this study found that certain calves exhibited left or right eye laterality when viewing novel objects. This variance could be attributed to the subjective nature of emotion among individuals, leading to variable reactions to stimuli 35 . This study classified novel objects as stressors owing to their unpredictable and uncontrollable characteristics. Nonetheless, the varying subjective perception of stimuli among individual animals may influence the degree to which they perceive them as stressors and how approach them 36 . Furthermore, this study revealed that the majority of calves exhibited an absence of eye laterality, potentially attributed to the prolonged testing duration for each individual calf. Baragli et al. 37 has reported that on horses, the strength of their eye preference to view a novel stimulus (an inflated balloon) decreased over time during a 5-minute test. As the test progressed, horses tended to increasingly use their non-preferred eye to observe the stimulus 37 . In this study, each calf underwent a 10-minute testing session. The extended duration of testing may have contributed to the elimination of any eye preference in viewing novel objects for most calves. Future research should consider investigating calf eye laterality within a shorter testing duration to provide further insights. When we study the effects of cerebral lateralisation on calves’ test-retest reliability, we need to be careful to control for other factors that may affect the results. Meagher, et al. 7 reported that a long test-retest interval, combined with important management changes between tests such as weaning from milk, might have negative effects on test-retest reliability. They also found that reducing test-retest intervals from 20 days to 7 days with more consistent management of milk provision between tests might improve the reliability. In addition, increasing the test duration is suggested to improve the reliability of the latency measures because longer test durations can avoid an artificial upper limit in measures of latency 7 , 38 . Moreover, animals’ health status may be another factor to affect the reliability. For instance, Cramer and Stanton 39 found that in calves, respiratory illness and fever decreased the probability of calves approaching novel objects and stationary humans. Meagher, et al. 7 reported that excluding calves who had had a cough on the day of testing could increase the reliability of calves’ responses to humans. In this study, the test-retest interval for every calf was one week; milk was provided two times a week; the test duration for every calf was 10-min; calves’ health status during the test periods was well managed. Therefore, these factors may have limited effects on test-retest reliability in this study. Conclusion There was a poor test-retest reliability of calves’ response in terms of latency to make first contact with the novel objects in novel object tests. This could not be explained by the hypothesis that fear responses would be stronger if calves first viewed the novel object with the left eye, as calves’ initial monocular presentation towards novel objects did not affect their expression of latency to make first contact with the novel objects. Since the present study found no evidence that calves had visual lateralisation in processing fear, it might suggest that calves of this age did not develop cerebral lateralisation in processing fear, and thus monocular presentation was unlikely to have an effect on test repeatability. The absence of visual lateralisation also suggests that using the left eye to observe potentially fear-inducing stimuli is unlikely to be a useful measure of mild fear in calves at this age. Methods Ethical statement The study was conducted at the Centre for Dairy Research, University of Reading (CEDAR), Reading, UK. It was approved by the ethics administrator at the university and the departmental ethics coordinator. All procedures in this study complied with the guidelines for the Ethical Treatment of Animals in Applied Animal Behaviour and Welfare Research 40 . Additionally, all procedures are reported in accordance with ARRIVE guidelines . Animal, housing and feeding Thirty-six pure registered male Holsteins calves with birth weights (mean ± SD) of 44.67 ± 4.43 kg were included from 2 days of age to 49 days of age. At 2 days of age, calves were assigned into six groups (six calves in each) according to their date of birth. Within groups, calves were randomly allocated to one of two social housing treatments: either individual (two calves per group) or pair (four calves per group) pens. Half of the calves in each housing treatment were provided with physical enrichment items: one stationary brush, one plastic chain, one rubber teat and one haynet filled with strawberry-scented ryegrass hay for an individual pen; one haynet filled with strawberry-scented ryegrass hay and two of all other items for a pair pen. The remaining calves received no additional physical enrichment items. More details related to housing and feeding can be found in Zhang, et al. 34 . Novel object tests The first novel object tests were conducted one calf at a time when they were 6 weeks of age. A wheeled scale was used to move each test calf between its home pen and the test arena (4.0 × 4.0 m 2 ). When arriving at the entry of the test arena, the test calf was lightly tapped on the hindquarters to encourage him to enter the test arena, in which the test calf could not see any other calves. Once entry into the test arena the test calf was allowed to habituate for 5 min. Following the period of habituation, a novel object (a white bucket or a traffic cone, used for alternate groups of calves) was lowered to the centre of the test arena on a pulley. The test calf remained in the pen with the novel object for 10 min. The test was recorded by either CCTV (Transit-PTZ, Revader Security Ltd, UK) or webcam (C525, Logitech International S.A, Switzerland) fixed to the ceiling. Video recordings were observed continuously to record the behaviours as defined in Table 1. Latency to make first contact with the novel object has been suggested as an indicator of fear in calves because of its high correlation with other fear-related measures indicating physiological arousal 41 . Table 1. Ethogram of the recorded behaviours in the first novel object test and the repeated novel object test. Behaviour Definition First view Which eye (left eye, right eye or both eyes) would have had a clear line of sight to the object when it came into the test calf's field of vision as it started moving towards the floor. Left eye a The object was to the left-side of the head’s orientation of the test calf; the line of vision of the right eye was obscured; the object was not posterior to the abdomen. Right eye a The object was to the right-side of the head’s orientation of the test calf; the line of vision of the left eye was obscured; the object was not posterior to the abdomen. Both eyes a The calf’s head was orientated so that line of vision between both eyes and the object was unobstructed. Latency to first contact with the novel object a Time interval from the object been lowering to the floor to the test calf touched the object. a The time duration of the behaviour was recorded. Repeated novel object tests were conducted with the same procedure one week later for every calf. For every test calf, the novel object (a white bucket or a traffic cone) that had not been used in its first novel object test was used in its repeated novel object test. Statistical analysis All data were analysed using the SPSS Statistics (version 27.0.1.0, IBM). Significant differences were declared at p ≤ 0.05. Due to non-normality of the data, a Spearman correlation was used to analyse the relationship between calves’ latency to make first contact with the novel objects in the first novel object test and in the repeated novel object test. The proportion of time viewing the novel objects with left or right eyes in the first and repeated novel object tests for every calf was calculated (= duration to view the novel objects with left or right eye / total duration to view the novel objects with left, right and both eyes). A generalised linear mixed model was used to compare calves’ proportion of time viewing the novel objects with left and right eyes. The subjects were calves’ ID number and left or right eye; the repeated measure was first novel object test or repeated novel object test. The fixed factors in the model included left or right eye, object, first novel object test or repeated novel object test, social housing and physical enrichment. The random factor was calves’ ID number. If calves at this age had the preference to view novel objects with the left eye, based on the design of this experiment, it was worth studying the effects of different novel objects, first or repeated novel object tests, and housing environments on calves’ proportion of time viewing the novel objects with left eye. A generalised linear mixed model was used to analyse the data. The subject was calves’ ID number; the repeated measure was first novel object test or repeated novel object test. The fixed factors in the model included object, first novel object test or repeated novel object test, social housing and physical enrichment. The random factor was calves’ ID number. For each calf, a laterality index (LI) was derived using the formula LI = (L - R) / (L + R), where L denotes the duration of viewing the novel objects with the left eye and R denotes the duration of viewing the novel objects with the right eye. Calves with a laterality index surpassing 0.5 were classified as favouring the left eye, while those with a laterality index below -0.5 were classified as favouring the right eye 37 . After classifying the calves as either left or right lateralised according to the laterality index, a binomial test was used to compare the number of left-lateralised calves with that of right-lateralised ones. Due to non-normality of the data, a Spearman correlation was used to analyse the relationship between calves’ proportion of time viewing the novel objects with left eye and their latency to make first contact with the novel objects to test whether left eye use correlated with an established indicator of fear. Calves that did not make contact with the novel objects were excluded from the analysis, as those calves’ fear levels could not be assessed through the indicator of latency to make first contact with the novel object. A Chi-squared test was used to analyse the relationship between calves’ first view towards the novel objects with the left or right eye and whether they made contact with the novel objects. Variables in the model included calves’ first view towards the novel objects with left, right or both eyes and if calves made contact with the novel objects during the testing period (yes/no). A generalised linear mixed model was used to compare calves’ latency to make first contact with the novel objects when they first viewed the novel objects with left, right or both eyes. The subject was calves’ ID number; the repeated measure was first novel object test or repeated novel object test. The fixed factors in the model included calves’ first view towards the novel objects with left, right or both eyes, object, first novel object test or repeated novel object test, social housing and physical enrichment. The random factor was calves’ ID number. A test of least significant difference was carried out to identify differences among first viewing the novel object with the left, right, or both eyes. Before conducting the general linear model, calves that did not make contact with the novel objects were excluded from the analysis. The data relating to two calves were discarded from all tests before analysis because one calf was familiar with the white bucket and traffic cone, and the other one caught his head in the handle of the white bucket in the repeated novel object test and could not remove it. Declarations Competing Interests Statement The author(s) declare no competing interests. Author Contribution R.M. designed the experiments. C.Z. and M.K. performed the experiments, C.Z. analysed and interpreted the experimental data, and wrote the manuscript. R.M. reviewed and revised the manuscript. All the authors have read and approved the final version. Acknowledgement The authors express their gratitude for the support from the Department of Animal Sciences, University of Reading, and acknowledge the assistance and animal care provided by the staff at CEDAR. Data Availability The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. References Ennaceur, A. Tests of unconditioned anxiety—pitfalls and disappointments. Physiol. Behav. 135, 55–71 (2014). Forkman, B., Boissy, A., Meunier-Salaün, M.-C., Canali, E. & Jones, R. A critical review of fear tests used on cattle, pigs, sheep, poultry and horses. Physiol. Behav. 92, 340–374 (2007). Wohlt, J., Allyn, M., Zajac, P. & Katz, L. 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Effects of physical enrichment items and social housing on calves’ growth, behaviour and response to novelty. Appl. Anim. Behav. Sci. 237, 105295 (2021). Panksepp J. Cross-species affective neuroscience decoding of the primal affective experiences of humans and related animals. PLoS One 6, e21236 (2011). Cohen H, Zohar J, Matar MA, Zeev K, Loewenthal U & Richter-Levin G. Setting apart the affected: the use of behavioral criteria in animal models of post traumatic stress disorder. Neuropsychopharmacol. 29, 1962–1970 (2004). Baragli, P., Scopa, C., Felici, M. & Reddon, A.R. Horses show individual level lateralisation when inspecting an unfamiliar and unexpected stimulus. PLoS One 16, e0255688 (2021). Fonio, E., Benjamini, Y. & Golani, I. Short and long term measures of anxiety exhibit opposite results. PLoS One 7, e48414 (2012). Cramer, M. & Stanton, A. Associations between health status and the probability of approaching a novel object or stationary human in preweaned group-housed dairy calves. J. Dairy Sci. 98, 7298–7308 (2015). Sherwin, C. et al. Ethical treatment of animals in applied animal behavior research. Int. Soc. Appl. Ethol. (2017). Van Reenen, C. G. et al. Responses of calves to acute stress: individual consistency and relations between behavioral and physiological measures. Physiol. Behav. 85, 557–570 (2005). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4462883","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":315509497,"identity":"5f3e407e-bf9f-494d-9d95-c4766e684a25","order_by":0,"name":"Chenyu Zhang","email":"","orcid":"","institution":"University of Reading","correspondingAuthor":false,"prefix":"","firstName":"Chenyu","middleName":"","lastName":"Zhang","suffix":""},{"id":315509501,"identity":"c4584a69-c2a8-4690-af2f-8fb26b9a0593","order_by":1,"name":"Molly Kindell","email":"","orcid":"","institution":"University of Reading","correspondingAuthor":false,"prefix":"","firstName":"Molly","middleName":"","lastName":"Kindell","suffix":""},{"id":315509503,"identity":"c33fed42-9b44-4f3d-a652-b417e0adec99","order_by":2,"name":"Rebecca Meagher","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABD0lEQVRIiWNgGAWjYBAC+wYeCMPgAAMDM5CWYzjA3IBXi8EBqBZLqBZjhgOMRGqxh2pJbCCo5XjvwQc/auwYzI43P35dUHEvve/4wQaGHzV4/NJzLtmw51gyg9mZY2bWM84U5848k9jA2HMMtxY7iRwzacYGZgazGwlmxrxtCbkbDiQCuWy4tRjLvzH/zdhQz2Bw//k3Y95/CekG5x8CtfzDrcVwBo8ZM2PDYQaDGzzGj3kbEhIMbgBtYWzD4/0zecmSPceO8xicySlj5jmWYDjzxsOGg719eLQcP3vww4+aajmD48c3f+apSZDnO58MDMNvuLXAACh22CRgvAOENUAA8wdiVY6CUTAKRsHIAgDp8Vy8Q94JPwAAAABJRU5ErkJggg==","orcid":"","institution":"Dalhousie University","correspondingAuthor":true,"prefix":"","firstName":"Rebecca","middleName":"","lastName":"Meagher","suffix":""}],"badges":[],"createdAt":"2024-05-22 19:53:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4462883/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4462883/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":58546160,"identity":"8465b913-f15b-4575-9ae1-2c4265878b90","added_by":"auto","created_at":"2024-06-18 05:44:52","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2378116,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation between calves’ latency to touch the novel object across the first novel object tests (FNOT) and repeated novel object tests (RNOT).\u003c/p\u003e","description":"","filename":"OnlineFigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4462883/v1/40568ac78f3aa30e9b09a31f.png"},{"id":58546164,"identity":"de792dde-b7cd-48dd-af65-2fc4653a8810","added_by":"auto","created_at":"2024-06-18 05:44:52","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1489251,"visible":true,"origin":"","legend":"\u003cp\u003eLSM (±SE) of the proportion of total viewing time using the left and right eyes (n=34 calves).\u003c/p\u003e","description":"","filename":"OnlineFigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4462883/v1/6f9626957c788581213b250e.png"},{"id":58546854,"identity":"80183282-94ba-4eac-94bd-aeaa04fdf15d","added_by":"auto","created_at":"2024-06-18 05:52:52","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":193531,"visible":true,"origin":"","legend":"\u003cp\u003eNumber of calves having left eye preference (LEP, 1.0 \u0026gt;= laterality index \u0026gt;= 0.5), absent eye preference (AEP, 0.5 \u0026gt; laterality index \u0026gt; -0.5) and right eye preference (REP, -0.5 \u0026gt;= laterality index \u0026gt;= -1.0) in viewing novel objects.\u003c/p\u003e","description":"","filename":"OnlineFigure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4462883/v1/3871508e650858631e7169a5.png"},{"id":58546161,"identity":"ea9fdb06-a404-47d7-8984-f616292882ec","added_by":"auto","created_at":"2024-06-18 05:44:52","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2323622,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation between calves’ proportion of time viewing the novel objects with the left eye and calves’ latency to touch the novel objects.\u003c/p\u003e","description":"","filename":"OnlineFigure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4462883/v1/a9a4e65b99e045ec595b79e8.png"},{"id":58546163,"identity":"ce2f87cf-0421-4300-acb8-4780215a4076","added_by":"auto","created_at":"2024-06-18 05:44:52","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3167362,"visible":true,"origin":"","legend":"\u003cp\u003eLSM (±SE) of the latency to touch the novel objects among calves’ first view towards the novel objects with left, right and both eyes (n=34 calves).\u003c/p\u003e","description":"","filename":"OnlineFigure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4462883/v1/1e047aa0188267fa261fa24e.png"},{"id":62877654,"identity":"4ec23b80-d3fc-4c06-8210-4b81db5e5786","added_by":"auto","created_at":"2024-08-20 14:14:01","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2027677,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4462883/v1/773edf9a-7717-4fb8-9850-34de34528a83.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Are laterality effects present in novel object responses of calves?","fulltext":[{"header":"Introduction","content":"\u003cp\u003eFear has been considered as a negative emotion resulting from a perception of danger\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. It can be expressed through a series of physiological and behavioural reactions to a perceived threat\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. In natural conditions, predators are an important source to stimulate fear. Although most domestic animals do not commonly experience predation from natural predators, inappropriate housing and management procedures, such as dehorning, tail docking, castration, herding and transportation, can elicit fear-related responses\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e, which may reduce animals\u0026rsquo; productivity, lead to chronic stress and thus alter fundamental behaviours\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. For example, fear-related reactions affect sexual and maternal behaviours and social dominance in cattle and sheep\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Therefore, fear may be a severe welfare issue. although animals often feel fear when they have a perception of danger, finding effective ways to assess their fear levels and tendency to express fear (the personality trait of fearfulness\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e) is an important step to improve their welfare.\u003c/p\u003e \u003cp\u003eIn calves, fear is often assessed through their responses to novelty and to humans\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Reliability has been considered as a criterion to assess the effectiveness of novelty tests, meaning that an effective fear test would get the same result in repeated measures of the same construct\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. However, Meagher, \u003cem\u003eet al.\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e reported poor test-retest reliability in novel object tests for calves, which indicated that it is difficult to draw strong inferences from a single test. Similar results are also found in older heifers and adult cows when tested using avoidance\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e, reactivity\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, number of interactions and time in proximity\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Although the poor test-retest reliability in novel object tests has been widely reported, the mechanisms underlying this result are rarely studied.\u003c/p\u003e \u003cp\u003eGrowing numbers of studies have focused on non-human vertebrates\u0026rsquo; cerebral lateralisation in emotional processing. In domestic species, many researchers have determined that animals dominate the processing of fear and anxiety through a right-hemisphere\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. This is expressed by a left visual preference because the high degree of decussation of optical fibres ensures that input from the used eye is predominantly processed in the contra-lateral hemisphere\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. For instance, chicks preferentially use the left eye to observe aerial predators\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e and adult chickens were found to scan the air for predators preferentially with the left eye after hearing a conspecific alarm call\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Studies in horses and cattle also showed evidence of a right-hemisphere dominance for the visual processing of fear. Austin and Rogers \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e reported that when a person walked toward a domestic horse while opening an umbrella, the horse reacted more, by moving further away, when approached from its left-side than they do when approached from its right-side. Robins and Phillips \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e investigated whether dairy and beef cattle exhibit preferences to monitor challenging and found that cattle had the preference to use the left monocular field viewing an experimenter walking to repeatedly split the herd through its centre. For altricial species, their brain is mainly developed after birth\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e and their specialisations of the left and right hemispheres are slowly established during infancy\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. For instance, in chicks, hemispheric laterality in charge of behaviour occurs quite precisely during early development post-hatching\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. In contrast, the brain development of precocial species mainly takes place before birth\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e and thus their hemispheric laterality is assumed to be well developed after parturition. For example, neonatal lambs are reported to have strong behavioural biases\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Since dairy calves are precocial, it is worth studying their development of emotional laterality.\u003c/p\u003e \u003cp\u003eAustin and Rogers\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e reported that when a horse was presented with the novel object first on its right-side, it lowered the horse\u0026rsquo;s initial fear responses and lowered fear responses to subsequent presentations of the object compared to those following an initial left-side presentation. Since the majority of evidence suggests that the left hemisphere is dominant in processing positive emotions\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, Austin and Rogers \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e suggested that the initial right-side presentation might inhibit the fear response and allowed horses to learn that the stimulus posed no threat, and this information could be transferred to the right-hemisphere. Therefore, animals\u0026rsquo; responses to fear-inducing stimuli may be affected by the initial presentation of the stimuli.\u003c/p\u003e \u003cp\u003eThe first aim of the present study was to assess the test-retest reliability of calves\u0026rsquo; responses in novel object tests. It was hypothesised that calves\u0026rsquo; latency to make first contact with the novel objects in the first novel object test and the repeated novel object test would be inconsistent as in repeated tests, novel objects randomly presented to calves\u0026rsquo; left, right, or both eyes initially, which might result in varying degrees of fear responses during repeated tests. The second aim of the present study was to determine if calves had visual lateralisation (cerebral lateralisation) in processing fear and whether such lateralisation could then be considered an effective indicator of fear. It was hypothesised that 1) due to brain development before birth, in novel object tests calves would prefer to view the novel objects with the left eye; 2) there would be a positive relationship between calves\u0026rsquo; proportion of time viewing the novel objects with the left eye and their latency to make first contact with the novel objects, both reflecting increased fear. The third aim of the present study was to determine if calves\u0026rsquo; initial monocular presentation toward fear-inducing stimuli would affect calves\u0026rsquo; fear responses. It was hypothesised that in novel object tests, 1) fewer calves would make contact with the novel objects when they first viewed the novel objects with the left eye; and 2) calves would have longer latencies to make first contact with the novel objects when they first viewed the novel objects with the left eye.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eThere was a poor correlation between the first novel object test and the repeated novel object test for calves\u0026rsquo; latency to make first contact with the novel objects (r\u003csub\u003es\u003c/sub\u003e = -0.148, p\u0026thinsp;=\u0026thinsp;0.403; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the first novel object test and repeated novel object test, there were no statistically significant differences in the proportion of time viewing the novel objects with left and right eyes (F\u003csub\u003e1,99\u003c/sub\u003e = 0.045, p\u0026thinsp;=\u0026thinsp;0.832; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In addition, the object used, whether it was the first or repeated test, and social housing did not have significant effects on calves\u0026rsquo; proportion of time viewing the novel objects with the left eye (objects: F\u003csub\u003e1,32\u003c/sub\u003e = 0.041, p\u0026thinsp;=\u0026thinsp;0.842; tests: F\u003csub\u003e1,32\u003c/sub\u003e = 1.537, p\u0026thinsp;=\u0026thinsp;0.224; social housing: F\u003csub\u003e1,31\u003c/sub\u003e = 0.437, p\u0026thinsp;=\u0026thinsp;0.513), whilst calves from physically enriched pens spent longer durations viewing the novel objects with the left eye than those from non-physically-enriched pens (F\u003csub\u003e1,31\u003c/sub\u003e = 6.864, p\u0026thinsp;=\u0026thinsp;0.013). In individual level, 15 calves exhibited a preference for utilising one eye over the other in both tests (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Among these calves, 9 displayed a preference for the left eye, while 6 favoured the right eye, however, the left and right eye preference did not have significant difference (two-tailed binomial p\u0026thinsp;=\u0026thinsp;0.607).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThere was a poor correlation between calves\u0026rsquo; proportion of time viewing the novel objects with the left eye and calves\u0026rsquo; latency to make first contact with the novel objects (r\u003csub\u003es\u003c/sub\u003e = -0.002, p\u0026thinsp;=\u0026thinsp;0.503; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eProbability of making contact with the novel objects in the first novel object tests and repeated novel object tests was 70.80% if the calf could view the object first with the left eye vs. 71.40% if the right, but this difference was not significant (Chi-squared p\u0026thinsp;=\u0026thinsp;0.965). In both the first novel object test and repeated novel object test, there were no statistically significant differences in the latency to make first contact with the novel objects among calves\u0026rsquo; first view of the novel objects with the left, right or both eyes (F\u003csub\u003e2,31\u003c/sub\u003e = 0.611, p\u0026thinsp;=\u0026thinsp;0.549; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eSince fear has been considered a welfare concern for many animals\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, it is important to have methods, such as novelty tests, to assess its levels\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. However, the results in this study show poor test-retest reliability of calves\u0026rsquo; response to novel objects, which may make it difficult to draw inferences about welfare or personality from a single novel object test. The finding is consistent with some other studies on cattle\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. For example, Gibbons, \u003cem\u003eet al.\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e studied the consistency of cattle\u0026rsquo;s reactivity responses to three novel stimuli and found low reliability among tests. Thereafter, we tried to find out the potential mechanisms resulting in the poor test-retest reliability.\u003c/p\u003e \u003cp\u003eVertebrates typically prefer to view fear-inducing stimuli using the left eye\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, and the eye that first sees a stimulus can influence fear related behaviours\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Inconsistency in the head orientation of calves relative to fear-inducing stimuli may explain differences in responses on repeated tests. We therefore studied whether calves had a preference to view fear-inducing stimuli using their left eyes, and if having their first view of the object with the left, right, or both eyes affected their fear responses. We assumed that if calves had cerebral lateralisation in processing fear resulting from the novel objects, initially presenting the fear-inducing stimuli into calves\u0026rsquo; left visual field could stimulate increased fear responses. Since in the novel object tests, objects are randomly presented to calves\u0026rsquo; left, right or both eyes initially, it may lead to different levels of fear responses for every calf in repeated tests. However, our findings were inconsistent with the predictions.\u003c/p\u003e \u003cp\u003eNo cerebral lateralisation in processing fear resulting from novel objects was found in this study, whilst the opposite findings have previously been reported in cattle and other species. For example, many bony fish species show a right hemisphere dominance of processing of predator stimuli, evidenced by lateralised motor responses\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e and lateralised visual perception\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. In reptiles, a left eye preference during predator inspection exists for wall lizard\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. In domestic chickens, Evans, \u003cem\u003eet al.\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e found that adult hens prefer to scan the air for predators with the left eye after hearing a conspecific alarm call. Robins and Phillips \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e found that dairy and beef cattle had significant left eye preferences for viewing an experimenter walking to split the herd through its centre. The difference between this and other studies may result from the development of hemispheric specialisation. In this study, calves attended the tests when they were six or seven weeks of age. Although the brain development for ungulate livestock mainly takes place before birth\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, it can be hypothesised that hemispheric specialisation may not be well developed in the neonatal period. The finding of a poor correlation between calves\u0026rsquo; proportion of time viewing the novel objects with the left eye and calves\u0026rsquo; latency to make first contact with the novel objects in this study supports this hypothesis and indicates that left eye observation towards fear-inducing stimuli may not be considered an effective fear indicator at this age.\u003c/p\u003e \u003cp\u003eSince calves may not have had visual lateralisation in processing novel stimuli in this study, it is not surprising that they did not show a significant difference in fear-related responses to novel stimuli initially perceived in the left compared to the right monocular field. However, this finding is in contrast to Austin and Rogers \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. They studied whether horses display greater reactivity to a novel stimulus initially presented in the left compared to the right monocular visual field by testing horses in a scenario of a person opening an umbrella five metres away and then approaching. The measured reaction was the distance each horse moved away before stopping. They found that horses tested initially on the left-side exhibited greater reactivity for the left approach, whereas horses tested on the right-side first displayed no side difference in reactivity. This greater reactivity to the left-side approach, therefore, depended on the side of the initial monocular presentation. When the initial monocular presentation was in the left visual field, the horses responded with greater left-side reactivity, but when the stimulus was presented in the right monocular field first, subsequent reactivity to the stimulus in the left monocular field was similar to that of the right eye. It may suggest that due to interocular transfer\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, which occurs from the left hemisphere to the right hemisphere\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, habituation to the stimulus occurs when the first presentation is to the right eye but not when it is to the left eye. However, for neonatal calves, their hemispheric specialisation may not be well developed and thus, they may not have the interocular transfer. Therefore, calves\u0026rsquo; poor test-retest reliability may not result from cerebral lateralisation.\u003c/p\u003e \u003cp\u003eIn addition to calves not having well-developed hemispheric specialisation in processing fear at an early age, there is another potential explanation for the lack of lateralised responses seen in this study. When we conducted the tests, some calves lay down in the sawdust on the ground several minutes after entering the test arena, and showed limited behavioural responses to the objects. This may indicate that calves were not frightened enough in the novel object tests and thus, this test environment could not stimulate their lateralised responses. Future research needs to adjust the test environments to further stimulate calves\u0026rsquo; fear (e.g. providing a more frightening object to calves) and confirm there is a true fear response to draw strong conclusions regarding lateralisation.\u003c/p\u003e \u003cp\u003eAnimals\u0026rsquo; early experience is suggested to affect their development of hemispheric specialisation\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Neonatal stimulation can have profound effects on the specialisation of their brain in mammalian species\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. For instance, neonatal handling of rats can provide stimulation to give rise to right hemispheric dominance, which increases animals\u0026rsquo; reactivity, emotionality and the aggressive behaviour of muricide\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Exposure of neonatal rats to novelty, meanwhile, modulates hand preference towards control by the right hemisphere\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e and increases the right hippocampal volumetric dominance\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Moreover, Galuret \u003cem\u003eet al.\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e found that chicks lacking maternal care showed stronger lateralisation, enhanced sociability, and reduced emotional responsiveness when compared to maternally raised chicks. In the current study, calves experienced differential levels of stimulation (i.e. social housing, physical enrichment) before attending the first and repeated novel object tests. Social housing seems unlikely to have affected calves\u0026rsquo; cerebral lateralisation in processing fear, evidenced by calves from individual pens and pair pens spending similar amounts of time viewing novel objects with their left eyes. Although physical enrichment was found to reduce calves\u0026rsquo; time spent viewing novel objects with the left eye, this does not necessarily mean this stimulus promotes the development of cerebral lateralisation in processing fear. Animals with environmental enrichment may be less likely to approach novel stimuli\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Since physically enriched calves had more experience with novel objects (i.e. stationary brushes, plastic chains, dry teats and nets filled with scented hay) in their home pens before attending the novel object tests, the objects in the novel object tests might be less attractive to them, and thus they spent less time viewing the objects without necessarily being less fearful of the objects. The findings in Zhang, \u003cem\u003eet al.\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e may also support this point of view, which indicated physical enrichment had no effects on calves\u0026rsquo; fear responses (e.g. latency to make first contact with novel objects) in novel object tests.\u003c/p\u003e \u003cp\u003eAt the individual level, this study found that certain calves exhibited left or right eye laterality when viewing novel objects. This variance could be attributed to the subjective nature of emotion among individuals, leading to variable reactions to stimuli\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. This study classified novel objects as stressors owing to their unpredictable and uncontrollable characteristics. Nonetheless, the varying subjective perception of stimuli among individual animals may influence the degree to which they perceive them as stressors and how approach them\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Furthermore, this study revealed that the majority of calves exhibited an absence of eye laterality, potentially attributed to the prolonged testing duration for each individual calf. Baragli \u003cem\u003eet al.\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e has reported that on horses, the strength of their eye preference to view a novel stimulus (an inflated balloon) decreased over time during a 5-minute test. As the test progressed, horses tended to increasingly use their non-preferred eye to observe the stimulus\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. In this study, each calf underwent a 10-minute testing session. The extended duration of testing may have contributed to the elimination of any eye preference in viewing novel objects for most calves. Future research should consider investigating calf eye laterality within a shorter testing duration to provide further insights.\u003c/p\u003e \u003cp\u003eWhen we study the effects of cerebral lateralisation on calves\u0026rsquo; test-retest reliability, we need to be careful to control for other factors that may affect the results. Meagher, \u003cem\u003eet al.\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e reported that a long test-retest interval, combined with important management changes between tests such as weaning from milk, might have negative effects on test-retest reliability. They also found that reducing test-retest intervals from 20 days to 7 days with more consistent management of milk provision between tests might improve the reliability. In addition, increasing the test duration is suggested to improve the reliability of the latency measures because longer test durations can avoid an artificial upper limit in measures of latency\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Moreover, animals\u0026rsquo; health status may be another factor to affect the reliability. For instance, Cramer and Stanton \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e found that in calves, respiratory illness and fever decreased the probability of calves approaching novel objects and stationary humans. Meagher, \u003cem\u003eet al.\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e reported that excluding calves who had had a cough on the day of testing could increase the reliability of calves\u0026rsquo; responses to humans. In this study, the test-retest interval for every calf was one week; milk was provided two times a week; the test duration for every calf was 10-min; calves\u0026rsquo; health status during the test periods was well managed. Therefore, these factors may have limited effects on test-retest reliability in this study.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThere was a poor test-retest reliability of calves\u0026rsquo; response in terms of latency to make first contact with the novel objects in novel object tests. This could not be explained by the hypothesis that fear responses would be stronger if calves first viewed the novel object with the left eye, as calves\u0026rsquo; initial monocular presentation towards novel objects did not affect their expression of latency to make first contact with the novel objects. Since the present study found no evidence that calves had visual lateralisation in processing fear, it might suggest that calves of this age did not develop cerebral lateralisation in processing fear, and thus monocular presentation was unlikely to have an effect on test repeatability. The absence of visual lateralisation also suggests that using the left eye to observe potentially fear-inducing stimuli is unlikely to be a useful measure of mild fear in calves at this age.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cem\u003eEthical statement\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe study was conducted at the Centre for Dairy Research, University of Reading (CEDAR), Reading, UK. It was approved by the ethics administrator at the university and the departmental ethics coordinator. All procedures in this study complied with the guidelines for \u003cem\u003ethe Ethical Treatment of Animals in Applied Animal Behaviour and Welfare Research\u003c/em\u003e\u003csup\u003e40\u003c/sup\u003e. Additionally, all procedures are reported in accordance with \u003cem\u003eARRIVE guidelines\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAnimal, housing and feeding\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThirty-six pure registered male Holsteins calves with birth weights (mean \u0026plusmn; SD) of 44.67 \u0026plusmn; 4.43 kg were included from 2 days of age to 49 days of age. At 2 days of age, calves were assigned into six groups (six calves in each) according to their date of birth. Within groups, calves were randomly allocated to one of two social housing treatments: either individual (two calves per group) or pair (four calves per group) pens. Half of the calves in each housing treatment were provided with physical enrichment items: one stationary brush, one plastic chain, one rubber teat and one haynet filled with strawberry-scented ryegrass hay for an individual pen; one haynet filled with strawberry-scented ryegrass hay and two of all other items for a pair pen. The remaining calves received no additional physical enrichment items. More details related to housing and feeding can be found in Zhang, \u003cem\u003eet al.\u003c/em\u003e\u003csup\u003e34\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eNovel object tests\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe first novel object tests were conducted one calf at a time when they were 6 weeks of age. A wheeled scale was used to move each test calf between its home pen and the test arena (4.0 \u0026times; 4.0 m\u003csup\u003e2\u003c/sup\u003e). When arriving at the entry of the test arena, the test calf was lightly tapped on the hindquarters to encourage him to enter the test arena, in which the test calf could not see any other calves. Once entry into the test arena the test calf was allowed to habituate for 5 min. Following the period of habituation, a novel object (a white bucket or a traffic cone, used for alternate groups of calves) was lowered to the centre of the test arena on a pulley. The test calf remained in the pen with the novel object for 10 min. The test was recorded by either CCTV (Transit-PTZ, Revader Security Ltd, UK) or webcam (C525, Logitech International S.A, Switzerland) fixed to the ceiling. Video recordings were observed continuously to record the behaviours as defined in Table 1. Latency to make first contact with the novel object\u003csup\u003e\u0026nbsp;\u003c/sup\u003ehas been suggested as an indicator of fear in calves because of\u0026nbsp;its high correlation with other fear-related measures indicating\u0026nbsp;physiological arousal\u003csup\u003e41\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eTable 1.\u003c/strong\u003e Ethogram of the recorded behaviours in the first novel object test and the repeated novel object test.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"28.286189683860233%\" valign=\"top\"\u003e\n \u003cp\u003eBehaviour\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"71.71381031613977%\" valign=\"top\"\u003e\n \u003cp\u003eDefinition\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"28.286189683860233%\" valign=\"top\"\u003e\n \u003cp\u003eFirst view\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"71.71381031613977%\" valign=\"top\"\u003e\n \u003cp\u003eWhich eye (left eye, right eye or both eyes) would have had a clear line of sight to the object when it came into the test calf\u0026apos;s field of vision as it started moving towards the floor.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"28.286189683860233%\" valign=\"top\"\u003e\n \u003cp\u003eLeft eye\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"71.71381031613977%\" valign=\"top\"\u003e\n \u003cp\u003eThe object was to the left-side of the head\u0026rsquo;s orientation of the test calf; the line of vision of the right eye was obscured; the object was not posterior to the abdomen.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"28.286189683860233%\" valign=\"top\"\u003e\n \u003cp\u003eRight eye\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"71.71381031613977%\" valign=\"top\"\u003e\n \u003cp\u003eThe object was to the right-side of the head\u0026rsquo;s orientation of the test calf; the line of vision of the left eye was obscured; the object was not posterior to the abdomen.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"28.286189683860233%\" valign=\"top\"\u003e\n \u003cp\u003eBoth eyes\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"71.71381031613977%\" valign=\"top\"\u003e\n \u003cp\u003eThe calf\u0026rsquo;s head was orientated so that line of vision between both eyes and the object was unobstructed.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"28.286189683860233%\" valign=\"top\"\u003e\n \u003cp\u003eLatency to first contact with the novel object\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"71.71381031613977%\" valign=\"top\"\u003e\n \u003cp\u003eTime interval from the object been lowering to the floor to the test calf touched the object.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003csup\u003ea\u003c/sup\u003e The time duration of the behaviour was recorded.\u003c/p\u003e\n\u003cp\u003eRepeated novel object tests were conducted with the same procedure one week later for every calf. For every test calf, the novel object (a white bucket or a traffic cone) that had not been used in its first novel object test was used in its repeated novel object test.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eStatistical analysis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAll data were analysed using the SPSS Statistics (version 27.0.1.0, IBM). Significant differences were declared at p \u0026le; 0.05.\u003c/p\u003e\n\u003cp\u003eDue to non-normality of the data, a Spearman correlation was used to analyse the relationship between calves\u0026rsquo; latency to make first contact with the novel objects in the first novel object test and in the repeated novel object test.\u003c/p\u003e\n\u003cp\u003eThe proportion of time viewing the novel objects with left or right eyes in the first and repeated novel object tests for every calf was calculated (= duration to view the novel objects with left or right eye / total duration to view the novel objects with left, right and both eyes). A generalised linear mixed model was used to compare calves\u0026rsquo; proportion of time viewing the novel objects with left and right eyes. The subjects were calves\u0026rsquo; ID number and left or right eye; the repeated measure was first novel object test or repeated novel object test. The fixed factors in the model included left or right eye, object, first novel object test or repeated novel object test, social housing and physical enrichment. The random factor was calves\u0026rsquo; ID number.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIf calves at this age had the preference to view novel objects with the left eye, based on the design of this experiment, it was worth studying the effects of different novel objects, first or repeated novel object tests, and housing environments on calves\u0026rsquo; proportion of time viewing the novel objects with left eye. A generalised linear mixed model was used to analyse the data. The subject was calves\u0026rsquo; ID number; the repeated measure was first novel object test or repeated novel object test. The fixed factors in the model included object, first novel object test or repeated novel object test, social housing and physical enrichment. The random factor was calves\u0026rsquo; ID number.\u003c/p\u003e\n\u003cp\u003eFor each calf, a laterality index (LI) was derived using the formula LI = (L - R) / (L + R), where L denotes the duration of viewing the novel objects with the left eye and R denotes the duration of viewing the novel objects with the right eye. Calves with a laterality index surpassing 0.5 were classified as favouring the left eye, while those with a laterality index below -0.5 were classified as favouring the right eye\u003csup\u003e37\u003c/sup\u003e. After classifying the calves as either left or right lateralised according to the laterality index, a binomial test was used to compare the number of left-lateralised calves with that of right-lateralised ones.\u003c/p\u003e\n\u003cp\u003eDue to non-normality of the data, a Spearman correlation was used to analyse the relationship between calves\u0026rsquo; proportion of time viewing the novel objects with left eye and their latency to make first contact with the novel objects to test whether left eye use correlated with an established indicator of fear. Calves that did not make contact with the novel objects were excluded from the analysis, as those calves\u0026rsquo; fear levels could not be assessed through the indicator of latency to make first contact with the novel object.\u003c/p\u003e\n\u003cp\u003eA Chi-squared test was used to analyse the relationship between calves\u0026rsquo; first view towards the novel objects with the left or right eye and whether they made contact with the novel objects. Variables in the model included calves\u0026rsquo; first view towards the novel objects with left, right or both eyes and if calves made contact with the novel objects during the testing period (yes/no).\u003c/p\u003e\n\u003cp\u003eA generalised linear mixed model was used to compare calves\u0026rsquo; latency to make first contact with the novel objects when they first viewed the novel objects with left, right or both eyes. The subject was calves\u0026rsquo; ID number; the repeated measure was first novel object test or repeated novel object test. The fixed factors in the model included calves\u0026rsquo; first view towards the novel objects with left, right or both eyes, object, first novel object test or repeated novel object test, social housing and physical enrichment. The random factor was calves\u0026rsquo; ID number. A test of least significant difference was carried out to identify differences among first viewing the novel object with the left, right, or both eyes. Before conducting the general linear model, calves that did not make contact with the novel objects were excluded from the analysis.\u003c/p\u003e\n\u003cp\u003eThe data relating to two calves were discarded from all tests before analysis because one calf was familiar with the white bucket and traffic cone, and the other one caught his head in the handle of the white bucket in the repeated novel object test and could not remove it.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting Interests Statement\u003c/h2\u003e\u003cp\u003eThe author(s) declare no competing interests. \u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eR.M. designed the experiments. C.Z. and M.K. performed the experiments, C.Z. analysed and interpreted the experimental data, and wrote the manuscript. R.M. reviewed and revised the manuscript. All the authors have read and approved the final version.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors express their gratitude for the support from the Department of Animal Sciences, University of Reading, and acknowledge the assistance and animal care provided by the staff at CEDAR.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eEnnaceur, A. Tests of unconditioned anxiety\u0026mdash;pitfalls and disappointments. Physiol. Behav. 135, 55\u0026ndash;71 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eForkman, B., Boissy, A., Meunier-Sala\u0026uuml;n, M.-C., Canali, E. \u0026amp; Jones, R. A critical review of fear tests used on cattle, pigs, sheep, poultry and horses. Physiol. 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Dairy Sci. 98, 7298\u0026ndash;7308 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSherwin, C. \u003cem\u003eet al.\u003c/em\u003e Ethical treatment of animals in applied animal behavior research. Int. Soc. Appl. Ethol. (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVan Reenen, C. G. \u003cem\u003eet al.\u003c/em\u003e Responses of calves to acute stress: individual consistency and relations between behavioral and physiological measures. Physiol. Behav. 85, 557\u0026ndash;570 (2005).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"dairy calf, novel object test, repeatability, visual lateralisation, fear","lastPublishedDoi":"10.21203/rs.3.rs-4462883/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4462883/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMany animals exhibit preferential viewing of fear-inducing stimuli with their left eyes, reflecting cerebral lateralisation in emotion processing. In novel object tests, often used to assess fear, spatial positioning of objects relative to the animal can vary. This study aimed to investigate visual lateralisation in fear processing in novel object tests, evaluate its effectiveness as an indicator of fear, and examine how initial monocular presentation of fear-inducing stimuli impacts fear responses and their reliability. Thirty-six calves underwent a novel object test at six weeks of age and a second at seven weeks of age using a different object, with behavioural responses towards the novel objects recorded. There was no correlation in latency to first contact with novel objects between tests. Proportion of time viewing novel objects with the left eye did not significantly differ from the right eye, nor did it correlate with latency to first contact with novel objects. The probability of contact with novel objects and latency to first contact with novel objects did not differ based on initial eye contact with novel objects. Overall, fear responses in calves during repeated novel object tests were inconsistent, but this inconsistency could not be explained by novel objects being randomly presented to calves\u0026rsquo; different visual fields, which suggests potential absence of cerebral lateralisation in fear processing. Thus, laterality of visual response may not reliably indicate fear in calves at this age.\u003c/p\u003e","manuscriptTitle":"Are laterality effects present in novel object responses of calves?","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-18 05:44:47","doi":"10.21203/rs.3.rs-4462883/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"750c4f4e-e33c-4d48-81d2-6c97f32fb631","owner":[],"postedDate":"June 18th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":33363731,"name":"Biological sciences/Neuroscience"},{"id":33363732,"name":"Biological sciences/Zoology"}],"tags":[],"updatedAt":"2024-08-20T14:05:48+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-18 05:44:47","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4462883","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4462883","identity":"rs-4462883","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}