LYU Jing ,Wang Chao ,ZHAO Xun-wu ,MlAO Er-yu ,WANG Zhi-peng ,XU Yuan ,BAl Xiu-juan#,BAO Jun#

1College of Animal Science and Technology,Northeast Agricultural University,Harbin 150030,P.R.China

2College of Wildlife and Protected Area,Northeast Forestry University,Harbin 150040,P.R.China

Abstract Mixing or regrouping of calves from different pens is a common animal management practice on the farm,which frequently occurs after weaning and has a negative effect on calve welfare. Social integration before regrouping may relieve stresses,but more evidences are needed to verify this hypothesis. The present study aimed to investigate acute physiological and behavioral variations of individually-or group-housed calves after being introduced into a mixed group. A total of 132 postnatal calves were randomly divided into groups of 1,3,6 and 12 animals (S,G3,G6,and G12;6 replicates in each group) until 59 days of age. At 60 days of age,every two replicates from different groups (S,G3,G6 and G12)were introduced in a larger pen which containing 44 of the aboved experimental calves. Before and after regrouping,physiological parameters of stress,including heart rate (HR),saliva cortisol (S-CORT),saliva secretory immunoglobulin A (SIgA),interleukin-2 (IL-2),interleukin-6 (IL-6),tumor necrosis factor-α (TNF-α) levels,and behavioral responses were recorded. After regrouping,HR and S-CORT increased immediately (P＜0.05),and higher (P＜0.05) levels of such molecules were found in S calves compared to those in group-housed calves. Levels of SIgA and IL-2 were decreased(P＜0.05),and the lowest (P＜0.05) IL-2 values were found in S calves compared to those in group-housed calves. In addition,the introduced calves displayed a distinct behavior,including altered active and rest time,which was associated with negative emotions triggered by the novel surroundings. Allogrooming,play,exploration behaviors and lying time were increased significantly (P＜0.05) in group-housed calves than those in S calves. Conversely,self-grooming,aggressive behaviors,standing and walking time were increased (P＜0.05) in S calves than those in group-housed calves. These findings suggest that individually-housed calves may be more susceptible to stressors arising from regrouping than grouphoused calves,which consequently negatively affected behavioral and neuroendocrine responses. Furthermore,moving calves with previous social experience may help mitigate regrouping stress.

Keywords: calf,regrouping,group size,behavior,stress,welfare

1.lntroduction

For the healthy growth of newborn calves,it is crucial to provide adequate feeding and management conditions that support thermal,physiological and behavioral needs. However,it is usually difficult to meet the above requirements in modern pastures,especially considering psychological needs and behavioral expressions (Stull and Reynolds 2008). Early socialization of dairy calves with the dam or other newborns during the milk-feeding phase (commonly the first 6 to 8 weeks of age) has been demonstrated to improve responses ability and foster expression of play behavior under pressure which has been associated with introduction to a new breeding house (Duve and Jensen 2012;de Paulaet al.2012). Moreover,due to the high motivation for social contact,isolation can impair calves’ social and learning abilities (Louiseet al.2002;Costaet al.2016). Furthermore,social isolation tends to cause calves to be less active,more anxious and emotional,therefore,calves are less likely to respond appropriately to new environmental stimuli compared to calves raised around their peers (Jensen and Budde 2006). The EU acknowledges that calves over 8-week old must be housed in groups (Council Directive 97/2/EC). For calves under 8-week old,the regulations only provide for visual and tactile contact with conspecifics,and calves are reared separately from dams in most countries. The consequences of the appropriate scales and benefits of social contact during early calf rearing are not fully understood.

After birth,dairy calves will experience a new physical and social environment. Regrouping is one of the main social and environmental problems that calves need to be solved. During regrouping,new dominance relationships are established,which is caused by introduction of a newcomer to a new fold with new penmates. Common management techniques,such as moving cattle among groups or introducing newcomers into an established group can lead to competition,fighting,stress and altered circadian rhythm (B?e and F?revik 2003). This situation is specially challenging to calves in terms of access to feed,which may result in aggressive interactions and distress among penmates (Millsonet al.1979),thus leading to reduced feed consumption (Schirmannet al.2011). When weaned heifers are fed under high stocking densities,behaviors such as social licking and agonistic interactions was increased (Kerr and Wood-Gush 1987),which result in strong fear reaction to the perceived stress and retarded physical growth (de Paulaet al.2010;Cobbet al.2014).Therefore,perceiving fear in a new environment may cause problems for animals and breeders. In stressful situations,the presence of another calf produces a calming effect on the behavioral response that lasts for 1-2 weeks (Grignardet al.2000;Takedaet al.2003;F?reviket al.2006). The ability to adapt to a new environment and social unrest may have important effects on animal performance and welfare,but behavioral responses during this critical transition have not been well characterized.

Thus,it could be hypothesized that regrouping in a new pen of calves who were individually grouped before weaning would negatively affect behavior and physiology compared to calves with previous social experience.Therefore,the purpose of this study was to characterize the development of activity patterns,social behaviors,and stress indices in regrouping of individually-or sociallyreared calves. The findings of this study might provide new insight for transition management of calves.

2.Materials and methods

2.1.Experimental design and treatments

The experiment was carried out on Yuanshengtai Dairy Farm Co.,Ltd.,Heilongjiang,China (usually 6-10 calves in each pen). The barn was mechanically and naturally ventilated interactively;ambient temperature was during 15-25°C;and relative humidity was maintained during 20-80%.

The experiment used a completely random design involving a 4 (differnet group sizes)×2 (before regrouping and after regrouping). Calves were allocated randomly in experimental group size: S (individually-housed calves),G3 (calves housed in groups of the three individuals),G6 (calves housed in groups of 6 individuals),and G12(calves housed in groups of 12 individuals);6 repetitions(pens) in each group. All the calves were regrouped into a larger pen at the end of the experiment.

2.2.Animals,diet and feeding

A total of 132 female dairy calves ((40.96±3.49) kg) were used after deprivation from the dams and colostrum feeding. Age difference between the youngest and the oldest calves in each experimental block was (2.7±0.89)days. One replicate of each treatment made up a block and there were 6 blocks in total,calves born earliest in the 1st block one and latest in the 6th block.

Pens for each treatment with different sizes: 3.0 m(L)×1.2 m (W) for S group;6.0 m (L)×1.8 m (W) for G3 group;6.0 m (L)×3.6 m (W) for G6 group;and 7.2 m(L)×6.0 m (W) for G12 group,thus allowing equal space for each calf. Pens consisted of rice hull bedding (85%)and solid concrete feeding area (15%). Sight and smell contacts were allowed,but no cross-sucking through movable fences separating pens. Suckling calves were fed twice daily at approximately 07:30 and 16:30 h as the followings: 0-2 weeks of age,6 L;3-4 weeks of age,8 L;and 5-7 weeks of age,10 L. Calves were weaned from milk at the 8th week of age. Starter were fedadlibitumfrom 4 days of age (Table 1). Routine management practices and inspection for health problems were conducted daily. Milk stealing from other buckets was prevented by barriers and wire mesh covers as to guarantee that no antagonistic behaviors were observed before weaning.

The experiment period was 60 days,all the calves were regrouped into a larger pen between 14:00 and 16:00 h on the last day,and this regrouping period was the focus of the present study. Regrouping was conducted following dairy herd’s standard operating practices for calve management. Every 2 repetitions of different groups (S,G3,G6 and G12) were introduced to form an experiment unit which was a larger pen containing 44 calves (2 calves from S,6 calves from G3,12 calves from G6,and 24 calves from G12),the 3 regroup pens in total. During regrouping,3 calves from G3,6 calves from G6,12 calves from G12 had prior experience with each other. Replicate (pen) acted as elements of regrouping,and every focus animal came from each replicate,so the focus animals were equally divided in the 3 regroup pens.

2.3.Measurement of behavioral parameters

Behavioral parameters of focal animals were determined by image recording using a digital camera (model No.JX-8702;Jixingweishi,Chinese Taibei) placed over each pen to record behaviors for 15 min before and after regrouping. Event behaviors included exploration(Duve and Jensen 2012),and playing (F?reviket al.2007),aggression (Bouissouet al.2001),self-grooming(Duve and Jensen 2012),allogrooming (Duve andJensen 2012) which were calculated considering the number of occurrences (no.) using the one-zero sampling method through continuous observations of focal animals. Postural behaviors included lying (Abdelfattahet al.2013),standing (Duve and Jensen 2012),and walking (Abdelfattahet al.2013),which were calculated considering the percentage of observations (%) using the instantaneous recording method through scan sampling,each observation period lasted 2 s at 1-min intervals.When the postural behavior performed for more than 10 s,it was considered an independent event. However,a new event was counted if the behavior was suspended at least for 5 s and then represented. The ethogram of the behaviors was listed in Table 2.

Table 1 Ingredients and nutrient levels of the starter (%,DM basis)

Table 2 The ethogram of the behaviors

2.4.Sample collections and preparations

Before and after the mixing,one calf from each replicate of S,G3,G6,and G12 groups was randomly selected as the focal animal,immediately. And all the behavioral and physiological samples were completed in 1 h. Parameters were obtained in the following order: heart rate (HR),collection of saliva and blood samples,measurement of behavioral parameters;interval between each operation was minimum 5 min. Detailed description regarding sampling was previously reported by Lvet al.(2018).Calves were fed and processed by the same technician familiar to the animals in order to avoid possible interferences in experimental data caused by temporary pressure during measurements/sample collection.

2.5.Sample analyses

HR was measured by a CS010 Heart Rate Sensor(Infineon Technology Co.,Ltd.,Tongxiang,Zhejiang Province,China,297 mm×32 mm×12 mm,41.4 g) which was constituted of a Bluetooth belt tied to the foreleg.The average of measurements which were obtained 5 min before and after regrouping were considered as the final HR values.

Collection of focused calves’ saliva and blood samples was conducted as described by Chacónet al.(2004) and Lvet al.(2021). Contents of saliva cortisol (S-CORT) and saliva secretory immunoglobulin A (SIgA) in saliva and contents of interleukin-2 (IL-2),interleukin-6 (IL-6) and tumor necrosis factor-α(TNF-α) in serum were determined using commercial ELISA test kits (Shanghai Jinma Laboratory Equipment Corporation.,Ltd.,Shanghai,China,LOT201410) following manufacturer’s instructions.Optical density was determined at 450 nm in a microplate reader (Bio-Tek Instrument Inc.,USA). Concentration was calculated through linear regression based on a standardcurve which was generated by standards provided in the microplate reader software.

2.6.Statistical analyses

Data were processed in SPSS version 17.0 (IBM Corp.,Armonk,NY,USA). Physiological and behavioral parameters were analyzed by generalized linear models with repeated measurements. Each calve served as the experimental unit for analyses. All the parameters were examined for the normality of data,and no data transformation was needed. The analysis model was as the followings:yijk=μ+ti+bj+ti×bj+eijk,whereywas the phenotypic observation,μwas the population mean,tiwas the treatment effect (4 levels),bjwas the regrouping effect (2 levels),ti×bjwas the treatment and stress interaction effect,andeijkwas the random residual effect.The Duncan test was used to put up multiple comparison.Differences were statistically significant ifP＜0.05.

3.Results

3.1.Effect of group size and regrouping on calves’physiology

Physiological measurements in different group sizes before or after regrouping are shown in Table 3. The interaction effect of group sizes (P=0.56)×regrouping(P＜0.05) on HR was significant (P＜0.05). The interaction effect of group sizes (P＜0.05)×regrouping (P＜0.05) on S-CORT was significant (P＜0.05). HR and S-CORT values in the regrouping progress showed an significantly increasing in S group,and HR and S-CORT values in S group were the highest (P＜0.05) among those in the group-housed calves after regrouping. The significant increase of above indexes were exacerbated by the regrouping treatment and earlier social environment.The interaction effect of group sizes and regrouping on SIgA and IL-2 was not significant (P=0.15;P=0.38),and the serum IL-2 was lower (P＜0.05) in the S group,but showed an increasing (P＜0.05) trend in all the groups after regrouping. The interaction effect of group sizes and regrouping on IL-6 and TNF-αwere not significant (P=0.24;P=0.11). Serum IL-6 and TNF-α values in S group were significantly lower (P＜0.05;P＜0.05) than those in G12 calves,and significant increasing was found in all the groups after regrouping (P＜0.05;P＜0.05).

3.2.Effect of group size and regrouping on calves’behavior

Parameters of observed behaviors in different group sizes before or after regrouping are shown in Table 4.Calves displayed more exploring and playing than group housed calves (P＜0.05). After regrouping,the exploring increased (P＜0.05) and the play behavior decreased(P＜0.05) in all the groups. The interaction effect of group sizes (P＜0.05)×regrouping (P＜0.05) on exploring and playing was not significant (P=0.72;P=0.37). Calves displayed no fighting events before regrouping,but the number of aggression events in the S calves was higher after regrouping. The interaction effect of group sizes×regrouping on aggression was not significant(P=0.08).

Table 3 Effect of group sizes and regrouping on the measurements of heart rate (HR) and levels of SIgA,S-CORT,serum IL-2,IL-6,and TNF-α of calves1)

Significant effect on self-grooming (P＜0.05) between regrouping (P＜0.05) and treatment (P=0.91) was detected. Self-grooming was reduced by the interaction between group rearing and mixed treatment. However,there was an opposite trend in single housed rearing and mixed treatment interaction. Self-grooming was higher(P＜0.05) in G6 group before regrouping,and the decrease of it after regrouping was the most (P＜0.05). Calves in S group had higher (P＜0.05) self-grooming compared to G6 and G12 calves after regrouping. Additionally,the effect ofregrouping (P＜0.05)×treatment (P＜0.05) on allogrooming was significant (P＜0.05). The number of allogrooming events in S group was lower (P＜0.05) than that of the group-housed calves,whereas the allogrooming was the highest in G12 calves after regrouping (P＜0.05).

The interaction effect of group sizes (P=0.56)×regrouping (P＜0.05) on standing was significant (P＜0.05).No significant difference was found in standing time among different groups before regrouping (P=0.44).However,the significant increase of standing was exacerbated by the regrouping treatment and singlehoused environment. The increasing of standing were observable after regrouping,which were the highest in S calves (P＜0.05).

The interaction effect of group sizes (P＜0.05)×regrouping (P＜0.05) on walking was not significant(P=0.06). No significant difference was found in walking and lying among different groups (P=0.05;P=0.29)before regrouping. Walking was lower in grouphoused calves than that of the S calves (P＜0.05),and significantly increased in all the groups through after regrouping (P＜0.05). The interaction effect of group sizes(P=0.38)×regrouping (P＜0.05) on lying was significant(P＜0.05). Lying time was significantly decreased in S group through regrouping progress (P＜0.05),and was noticeably higher in grouped rearing calves than that in single rearing calves after regrouping (P＜0.05).

4.Discussion

Social environment is a key factor affecting the ability of livestock to resist stresses. Therefore,investigating the changes of behavioral and physiological indexes of calves between the individually-or socially-rearing in recombination could represent the relationship between social environment and welfare. The results from the present study supported our hypothesis that regrouping in a new pen of calves who were individually grouped before weaning would negatively affect behavior and physiology compared to calves with previous social experience. The level of HR,S-CORT were higher and IL-2 was the lowest,and self-grooming,aggressive behaviors,standing and walking time were increased during the process of recombination in individually-reared calves. In addition,socially-rearing before weaning increased beneficial activities such as allogrooming,play,exploration behaviors,and rest time. Thus,these findings demonstrated the importance of early social environment for calf welfare and provided a basis data aimed at improving ruminant healthy.

HR is controlled by the vagal component of the autonomic nervous system and can be considered a relatively fast response to physiological changes,and whose increase is characteristic of negative emotion as shown in previous studies conducted by our laboratory group (Lvet al.2018). Therefore,stress from regrouping could contribute to high HR. In this study,calves in the individually-housed group had significantly increased HR than that of group-housed calves. These findings are in accordance with those found by Jensenet al.(2014) and Takedaet al.(2003) that grouped housing had a greater calming effect of HR in a new social environment.In addition,higher cortisol levels revealed that calves individually-housed had a stronger response to regrouping in our study. Lambs moved more slowly and had higher cortisol levels when tested in an open field compared to animals who were raised in a rich social environment(Gary and Valeria 1982;Napolitanoet al.2002,2008). Through increase in the hypothalamic-pituitary-adrenal axis HPA,cortisol could be a proxy for measuring the physiological effect of social separation (Hennessy 1997). Conditions known to be present during regrouping,such as reshaping of social relationships and competition between individuals,were more likely to be related to higher cortisol levels in cows (B?e and F?revik 2003;Silvaet al.2013). In the present study,saliva samples and HR were obtained within 15 min after regrouping,it was therefore likely that HPA arousal associated with negative emotion had been occurred.

Stress affects animals’ physiological response as well as alters immune responses due to the release of various immune modulating substances by sympathetic nerve fibers. As the most abundant antibody in mucosal secretions,SIgA has a crucial role in protecting against inhaled or ingested pathogens,and secretion of SIgA was known to be regulated by HPA and sympathetic adrenomedullary systems (Watanuki and Kim 2005). SIgA secretion could be inhibited by a variety of environmental and social stressors (Segerstrom and Miller 2004;Gourkowet al.2014). It has been demonstrated that decreased S-IgA levels in mice and dogs are associated with the contact with novel social partners (Guhad and Hau 1996;Kikkawaet al.2003). In the present study,saliva SIgA levels showed decreasing trend in S calves compared with group-housed calves upon regrouping,which might be resultant from the cumulative effect of the chronic stress caused by social separation from birth and an anxious state due to the lack of prior social experience arisen upon mixing with unfamiliar companions.

Stress has been reported to decrease IL-2 secretion(Mittwoch-Jaffeet al.1995). As mentioned above,elevated cortisol levels could reduce secretion of IL-2 by T-helper cells,leading to lower B-lymphocyte and cytotoxic T-cell activity (Dantzer 2001;Broom and Kirkden 2004). A stronger stress response might explain why the IL-2 level of S calves was significantly lower than that of group-housed calves after regrouping. TNF-αand IL-6,as pro-inflammatory cytokines,were depressogenic and anxiogenic,and could be induced under emotional stress(Berket al.2013). Secretion of inflammatory cytokines could be triggered in individual calves upon introduction to a new pen with unfamiliar individuals which could induce a passive emotion. In the present study,we found the above trend in the regrouping process through TNF-αand IL-6 value in S calves. Interestingly,Il-6 and TNF-αlevels in group rearing were significantly higher than those in single rearing group before regrouping,which might be associated with the increased immunoreactivity caused by social interaction among group members. Collectively,these findings suggested that individually-housed calves might be more susceptible to stressors arising from regrouping than group-housed calves. Moreover,moving of such calves with a familiar companion might help mitigate the physiological stress and bolster confidence upon regrouping.

Social environment also has been associated with emotion which have effects on behaviors. For instance,calves raised alone show less exploratory and play behavior (Jensenet al.1997) and have a more strongly responsive reaction to novelty in the social environment compared with group-raised calves (de Paulaet al.2012). In the present study,exploratory and play behavior in individually-housed calves was significantly lower than in group-housed calves before regrouping.Additionally,calves that were separated from the group with a partner and participated in a novelty experiment together,the reaction to separation (e.g.,vocalization)was less frequent,but exploratory behavior was increased compared to individually-housed alone (F?reviket al.2006;Duve and Jensen 2011). Oostindjeret al.(2011a,b)found that piglets adopted increased exploratory behavior after stress from enriched environment before weaning,which is consistent with our findings. Play behaviour is regarded as a positive indicator of welfare (Jensenet al.1997). After regrouping,play behavior of each group was decreased,and it was always lower of single housed calves than that of group housed calves. The most probable causes of the presence of companions could reduce negative emotion arose from stressful events,and familiar animals can often provide better social buffering(F?reviket al.2006;Rault 2012).

Early social contact contributes to the development of normal social responses,so that more social calves exhibit less agonistic behaviors before and after regrouping (Jensenet al.1997;Veissieret al.1997).In the present study,calves that were raised together before regrouping showed less agonistic behaviors and were more motivated towards more friendly interactions with pen mates,such as social play and allogrooming.Neisenet al.(2009) found that heifers placed in a new herd in the company of a familiar heifer participated in fewer agonistic behaviors than heifers placed into the herd unescorted. This might be related to the fact that maintaining close physical proximity to a familiar partner bring positive effects by synchronizing their activity and therefore avoiding aggressive encounters.

There were behavioral changes of increased selfgrooming in isolated calves compared to group-housed calves after regrouping. However,the allogrooming behavior of group rearing calves was significantly higher than that of single rearing calves after regrouping. The high level of allogrooming behavior of calves which were group housed previously might be caused by familiar companion,so does play behavior. So it seems that it could be concluded that licking of own fur and skin frequently may be of related to the inability to perform and receive allogrooming from strange companions in isolation condition. This observation seemed to be explained by the fact that calves raised alone were more responsive to novelties in the social environment(de Paulaet al.2012),have a degree of cognitive impairment (Gaillardet al.2014),and discrepancy may be caused by poor reactivity.

Regrouping has been shown to increase walking time in our study,which is in agreement with prior studies (Raussiet al.2005;Gygaxet al.2009),which can be attributed to an increased motivation to explore their surroundings. Rest indicates comfort since it is associated with the property of high priority and inelastic behavioral needs in cows (Mattielloet al.2019). In the present study,lying times were diminished upon regrouping,but calves regrouped with a familiar partner had relatively more lying time. Previous studies have reported less time spent on lying (Hasegawaet al.1997;Gygaxet al.2009) after regrouping. The result also confirmed that the mixing brought stress to calves,and the stress response was stronger in single housed calves.

5.Conclusion

In this study,calves submitted to regrouping had higher levels of HR and S-CORT,decreased SIgA levels,higher frequencies of events related to aggressive behaviors and reduced play and lying time,thus indicating that regrouping negatively impacted calves’ welfare.Collectively,these results suggested that providing a more complex social environment in pre-production may improve the capacity of calves to cope with the novel environment found upon regrouping. More researches are needed to confirm the mechanism how social experience influences behavior and physiological stress responses following regrouping,as well as to assess the benefits of early social interaction for long-term animal welfare and production management.

Acknowledgements

The study was supported by the National Natural Science Foundation of China (2012BAD12B00).

Declaration of competing interest

The authors declare that they have no conflict of interest.

Ethical approval

Management of laboratory animals included in the experiments was previously approved by the Animal Ethic Committee of the Northeast Agricultural University,China(approval number: NEAU-[2011]-9).