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Effect of pre-weaning temporary isolation within different age periods on the early post-weaning behaviour of juvenile lambs
Applied Animal Behaviour Science 141 (2012) 43–48
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Applied Animal Behaviour Science journal homepage: www.elsevier.com/locate/applanim
Effect of pre-weaning temporary isolation within different age periods on the early post-weaning behaviour of juvenile lambs Panagiotis Simitzis ∗ , Miria Petrou, Nikos Demiris, Stelios Deligeorgis Department of Animal Breeding and Husbandry, Faculty of Animal Science and Aquaculture, Agricultural University of Athens, 75 Iera Odos, 118 55 Athens, Greece
a r t i c l e
i n f o
Article history: Accepted 9 July 2012 Available online 25 July 2012 Keywords: Lamb Temporary maternal deprivation Escape behaviour Laterality Novelty
a b s t r a c t Temporary maternal separation is a stressful situation typical under modern husbandry practices and may have an undesirable impact on lamb welfare. However, at present there is insufficient evidence on temporary maternal separation effects on the behaviour of juvenile lambs. The aim of the present study was to assess if and how partial isolation during different periods at the early age can induce differences in the expression of juvenile lamb behaviour at the age of 2 and 3 months old. Twenty-seven male Chios lambs were assigned to 3 experimental groups: C (control), S1 (lamb daily isolation for 1 h between the 13th and 20th day of age) and S2 (lamb daily isolation for 1 h between the 20th and 27th day of age). At the age of 2 and 3 months old, the behaviour of lambs was tested by the implementation of 2 types of test: isolation and novel object (Y-test). There were no statistical differences between lamb treatments in the number of jumps, vocalizations and heart rates during isolation test and the latency to approach a novel or a familiar object in the novel object test conducted at 2 and 3 months of age. However, lambs showed a tendency of turning to the right-hand side of the test pen, irrespective of treatment group, at the age of 2 and 3 months old during the implementation of novel object test. It is concluded that no discernible effects of maternal temporary deprivation on the display of stress induced behavioural components of juvenile lambs at the age of 2 and 3 months old were found. © 2012 Elsevier B.V. All rights reserved.
1. Introduction The quality of mother–infant relationships and the juvenile’s subsequent social milieu are considered to be crucially formative for adjustment and social competence in adulthood (Ferdman et al., 2007). However, maternal deprivation is nowadays widespread among commercially reared animals, particularly in the form of separation from the mother earlier than would happen naturally. In contemporary sheep farming, lambs are often separated from their mothers, with the intention to increase the amount of
∗ Corresponding author. Tel.: +30 2105294449; fax: +30 2105294442. E-mail address: [email protected] (P. Simitzis). 0168-1591/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.applanim.2012.07.004
milk available to be processed into dairy products, resulting in an early disruption of the mother–young relationship (Napolitano et al., 2008). There is a large body of evidence that stressful early life experiences can affect brain development and subsequent behaviour (Hall, 1998). Adverse early life experiences in humans during childhood have lasting effects on the neuroendocrine stress axis, which are thought to increase the risk for the development of mood and anxiety disorders at adulthood (Nemeroff, 2004). In the rat, separation from the mother has been shown to have potentially deleterious effects, since the separated rat pups became hyperresponsive to stressors during behavioural development by exhibiting neophobia at the age of 2 months old (Hall et al., 1997a,b) or increased locomotor activity during
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stress-inducing tests at the age of 3 and 6 months old (Rebouc¸as and Schmidek, 1997) and as adults are unable to appropriately process environmental stimuli (Fone and Porkess, 2008), display reduced cognitive performance (Ladd et al., 2000) and aggression (Hatch et al., 1965). On the other hand, rat pups exposed to a positive postnatal experience (i.e. mothers that show high levels of maternal care) are less behaviourally and physiologically responsive to fear and stress-inducing stimuli when tested at the age of 3 months old, since they have shown alterations in the neural circuitry controlling fear behaviour (Caldji et al., 1998). Therefore, increased maternal care is beneficial for the development of infants, while on the other hand a prolonged maternal absence or neglect can be unfavourable or even harmful (Caldji et al., 1998). Immature animals are very sensitive to abnormal experiences and early life experiences can cause significant changes in the stress response system and emotionality, since the developing brain is suddenly exposed to a variety of neurotransmitters, neuromodulators and transcription factors, which are normally not present, or at least not in that quantity (Anisman et al., 1998). In lambs, exposure to stressful events such as isolation or a novel environment during the first days of their life disrupts neural development, activates hypothalamic-pituitary-adrenal axis, increases corticosteroid concentrations (serotonin, dopamine and the neuropeptide cortico-releasing factor) and may induce immune-suppression (Cockram et al., 1994). Sheep, like most domestic ungulates, are characterised by a strong social tendency. Due to this gregarious behaviour, they react very strongly to social isolation with high-pitch vocalisations and increased locomotion (Price and Thos, 1980; Romeyer and Bouissou, 1992). Both ewes and lambs express their separation distress by an increase in bleating and locomotion activity which indicates their effort to re-establish their contact (Zito et al., 1977). Lambs that are separated from the dam at an early age show reduced levels of vocalisation, are slower to initiate movements, spend less time in ambulatory behaviour and display an increased response compared to non-separated animals when exposed to open field tests (Napolitano et al., 2008). In addition, reactivity appears to be strongly associated with learning and cognitive function, since low levels of learning performance can be a result of a high level of stress behaviour (Boissy and Le Neindre, 1990), which it is strongly associated with the function of the hypothalamicpituitary-adrenal (HPA) axis and hippocampus. Specific behavioural elements that are exhibited during isolation in sheep include high rates of jumping and bleating (Simitzis et al., 2009). Vocalizations and locomotory activity are regarded as reliable indicators to estimate distress response in sheep. The behaviour of sheep can provide clues to the types of situations that might cause suffering or distress and indicate welfare or management problems associated with stress due to husbandry procedures (Le Neindre et al., 1993; Boivin and Braastad, 1996). These behaviours are aimed at maintaining contact with the other lambs and help the animals in localising, finding and identifying them. High locomotive activity and number of vocalizations observed in lambs (Degabriele and Fell,
2001) and kids (Bergamasco et al., 2005; Siebert et al., 2011) in isolation could be interpreted as an indication of fear, but also an attempt to rejoin conspecifics. The results vary depending on the time and the duration of the isolation as well as the gender and genetic background of the animals (Alexander, 1977). Ewe–lamb separation represents a more stressful management procedure for the younger than the older lambs, since younger lambs appear to exhibit their distress in a greater extent when compared with older lambs, possibly due to the stronger mother–young attachment at the early stages of life (Napolitano et al., 2008). It has been observed that ewe–lamb separation soon after lamb birth can cause impaired immune functions, enhanced cortisol response to isolation and altered behavioural reaction to open-field testing (Sevi et al., 1999; Napolitano et al., 1995, 2003). However, little is known on the effect of repeated temporary ewe–lamb separation on lamb early life and subsequent behavioural development. The current interest in early life experience stems from the observation that specific early events and specific time periods before weaning can apparently programme the “set-point” of the HPA axis by altering the effective feedback. At the same time, methods of behavioural measurements appear to be more sensitive for the detection of stressful conditions than the other commonly used endocrine or immune indicators of welfare (Napolitano et al., 2008). To our knowledge, no behavioural studies have been implemented for examining the impact of temporary isolation within different ages on the behaviour of juvenile lambs. Therefore, the aim of the present study was to assess if and how partial isolation induce differences in the expression of lamb behaviour and compare the effects of temporary hourly isolation either within the 13th–20th or the 20th–27th day of age on the behavioural components of juvenile lambs at the age of 2 and 3 months old. 2. Materials and methods 2.1. Pre-weaning period A total of 27 male Chios lambs were used in the present study. Lambs were delivered naturally after oestrus synchronisation of their mothers with intravaginal progestagen sponges (Chronogest, Intervet, The Netherlands). After delivery they were ear tagged, weighed and the date of birth was recorded. All lambs were born as singles within 3 days. They were kept all together in one group with their mothers (2 m2 /ewe and lamb). All lambs had ad libitum access to hay and water. Lambs were then randomly assigned to 3 experimental groups (C: control, S1: lamb isolation for 1 h between the 13th and 20th day of age, and S2: lamb isolation for 1 h between the 20th and 27th day of age). According to their experimental group (lamb isolation between 13th and 20th or 20th and 27th day of age; 1 h/day on every day from day 13 to day 20 and 1 h/day on every day from day 20 to day 27), lambs were daily separated from their mothers and isolated for an hour by moving them to a different barn with pens (1 m2 /lamb), prohibiting visual contact and vocal communication with the ewes, but permitting vocal
P. Simitzis et al. / Applied Animal Behaviour Science 141 (2012) 43–48
communication between the examined lambs. The two different lamb ages (13th–20th or the 20th–27th day of age) were chosen in the absence of any relevant information and in order to represent the early life of the lambs. After the hourly isolation, lambs returned to the pen with their mothers. Methods used in the present experiment were approved by the bioethical committee of the Agricultural University of Athens under the guidelines of “Council Directive 86/609/EEC regarding the protection of animals used for experimental and other scientific purposes”.
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2m
Novel or familiar object
4m
2.2. Post-weaning period After weaning (45th day), lambs were subjected to two behavioural tests carried out on three consecutive days at two different ages (2 and 3 months). Tests were conducted between 8:30 a.m. and 12:00 p.m. during each experimental day. Before testing, groups of lambs were moved to a holding pen, where they stayed for 30 min. After this adaptational period, each individual was separated from the group and moved to the entrance of the test pens (ca. 7 m distance). The procedure was designed to ensure that the handling protocol applied to the animals between the holding and test pen was standardised as far as possible. Lambs from different treatment groups were tested alternately, so that the treatment factor was not confounded with the order of testing. Test 1 (isolation test) consisted of social isolation in a novel environment (1 min), without any additional fearinducing stimuli (Erhard et al., 2004; Simitzis et al., 2009; Hernandez et al., 2009). The test pen represented a novel environment in which the tested animal was visually isolated from other members of the group. Each pen (2 m × 2 m) had solid walls and floor was covered with sawdust. Average, minimum and maximum heart rate of lambs (beats per minute – bpm) was measured by the POLAR S180i heart rate monitor system adapted to sheep, at the beginning of the adaptational period (30 min) in the holding pen. The system consisted of the transmitter and the receiver, which detected and displayed the heart rate, respectively. The transmitter was fitted with an elastic strap which held it firmly in the heart girth of each lamb. The frequency – number of jumps (when all four legs left the ground at the walls of the test pen – escape attempts) and vocalizations were the variables recorded during the isolation test. Test 2 (novel object test) was designed to examine the initial reaction of lamb when exposed together to a novel or familiar object (1 min) (Erhard et al., 2004; Simitzis et al., 2009). A Y test pen was implemented with a coloured ball representing the novel object and a feeder representing the familiar object placed in the two front sides of the test pen (Fig. 1). Lambs were exposed to the familiar object (feeder) between the 1st and the 45th day of their life. The novel object test consisted of two subtests, varying according to the position of novel and familiar object, which was shifted between the two subtests, without any further changes in the experimental design. During Test 2, laterality of initial preference for one side over the other or direction first turned upon entry and response to novel or familiar object, expressed as time spent to approach within approximately
2m
2m Fig. 1. Layout of the experimental pen during novel object test (Test 2).
10 cm for novel or familiar object were recorded, respectively. When lambs did not move in the test pen or they did not choose one side over the other, their behaviour was characterised as “indifference”. Behavioural patterns in the two tests were recorded using a Panasonic PV120 video camera fitted with a 3.6 mm lens and mounted 3 m above the pen floor. Video recordings were stored in a digital video recorder equipped with a hard disk (TX168, Telexper Inc., USA) and behavioural components (number of jumps, number of vocalizations, laterality and time spent with novel or familiar object) were measured. 2.3. Statistical analysis Data from the first test were the counts of jumps and vocalizations as well as the heart rate mean and standard error. As is common with count data we assumed that the number of jumps and vocalizations followed a Poisson distribution, which appeared to be a reasonable assumption. A normal distribution was adequate for the heart measurements. Furthermore, the potential correlation in measuring these three variables on the same lambs was taken into account by including (correlated) random effects. In particular, we used a trivariate generalised linear model where the log-rates of the jump and vocalisation distributions were connected to the heart rate via normally distributed random effects. The data from the second test were analysed using a combination of multinomial and exponential distributions. Specifically, we used a trinomial distribution in order to describe the animal behaviour in terms of choosing the right, the left direction or being indifferent. The times spent with the novel or the familiar object were assumed to follow distinct exponential distributions. Subsequently, we regressed the logit transformation of the probabilities and the log-rates of the exponential distributions upon the potential explanatory variables which included the
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Table 1 Live weights (kg) of lambs at birth and the ages of testing (L.S. mean ± S.E.). Age (months)
Birth 2 3
Treatmenta C
S1
S2
3.57 ± 0.25 14.9 ± 1.16 19.8 ± 1.56
4.06 ± 0.25 16.3 ± 1.16 20.0 ± 1.56
3.83 ± 0.25 15.0 ± 1.16 19.7 ± 1.56
a C: control, S1: lamb isolation for 1 h between the 13th and 20th day of age, S2: lamb isolation for 1 h between the 20th and 27th day of age, no significant differences were found among the treatment groups.
treatment, age and their interaction. All the statistical analyses in this paper were performed using the Winbugs (Lunn et al., 2000) software. 3. Results Lack of differences in body weight from birth till the age of 3 months old between treatments and control sheep was demonstrated (Table 1). Lamb pre-weaning temporary isolation seemed not to affect lambs’ growth. Moreover, as it was illustrated during the isolation test, no significant differences were observed among the lambs’ experimental groups, although the number of jumps and vocalizations tended to be higher for S2 lambs (Table 2). At the same time, rates for number of vocalizations tended to decrease as the age of lambs increased. No discernible differences were also illustrated among treatment groups regarding the average heart rate of lambs during the isolation test. Maximum and minimum heart rates also appeared not to vary significantly among lamb treatments (Table 2). As it was illustrated in the novel object test (Test 2), there were no differences in latency to approach a novel or a familiar object among treatments of lambs at the age of 2 and 3 months old (Table 3). Moreover, lambs preweaning temporary isolation did not significantly influence the behavioural laterality of lambs during Test 2, although lambs showed a tendency of turning to the right-hand side of the test pen, irrespective of lamb treatment (Table 3). 4. Discussion The results of behavioural measurements in the present study revealed that lambs pre-weaning temporary isolation did not significantly influence the reactivity of lambs at the early stages of their lives. Number of jumps and vocalizations during the isolation test were not significantly different among lamb treatments (Table 2). The same conclusions have been reached by Simitzis et al. (2009) and Hernandez et al. (2009) in experiments implemented in juvenile lambs at the age of 2, 3, 4 and 5 months old. No significant differences were also found concerning heart rates (Table 2). Fear can be evoked by social isolation and novel environment (Baldock and Silby, 1990) and heart rates can provide a useful measure of stress in sheep (Syme and Elphick, 1982). Average heart rates in the present study varied between 160 and 200 bpm, in accordance with previous implemented stress inducing experiments in 2 and 3 years old sheep (Baldock and Silby, 1990) and 2, 3, 4 and 5 months old lambs (Simitzis et al., 2009). The negative
effects of lamb pre-weaning temporary isolation are not evident at the early stages of lamb life, though disruption of ewe–lamb relationship could be a source of poor welfare for the lamb and could disturb the acquisition of its social behaviour (Dwyer, 2008). Normally, ewe–lamb separation results in an increase in plasma cortisol (Mears and Brown, 1997; Orgeur et al., 1999), increased behavioural and vocal activity, disrupted cicardian rhythms and altered social interactions (Orgeur et al., 1998). This increased emotional stress is a result of the expectation of lamb being rejoined after the temporary separation from its mother or the other lambs (Sevi et al., 2003). Moreover, in contrast to the present study, where no significant differences in latency to approach a novel or a familiar object among treatments of lambs were found (Table 3), partially isolated lambs appear to respond to isolation in a novel environment less actively than those of the control group (no separation), showing withdrawal behaviours after introduction, expressed as longer latency times to move, shorter ambulatory activity and less frequent jumping attempts compared to the control group of lambs between 12 and 32 days of age (Sevi et al., 2003). Moberg and Wood (1982) have stated that isolation-reared lambs show behavioural withdrawal in stressful situations such as a novel environment when tested at 12 and 42 days of age. At the same time, lambs also bleated less in reaction to final weaning when they had been subjected to a dairy, temporary separation from their mothers, as compared to lambs that had not been habituated to short term separations (Orgeur et al., 1998). Lambs’ exposure to stressful events such as isolation or a novel environment disrupts neural development, activates hypothalamic-pituitary-adrenal axis, increases corticosteroid concentrations and may induce immune-suppression (Cockram et al., 1994). However, no display of these effects in the present study could be partially explained by the hypothesis that at the early stages of lamb age, the evoked effects on the hypothalamus–pituitary–adrenal axis are not so evident in terms of behaviour. Additionally, these different findings in relation to the display of heightened anxiety states may result from varying adversities inherent in the different test paradigms, differing isolation onset and duration, or may represent differences between different species or breeds (Versace et al., 2007). Behavioural lateralisation has gained considerable attention as an indirect measure of the effects of adverse early life environments on brain development. In mammals, laterality in behaviour and asymmetry in morphological characteristics are accentuated by neonatal stress (Versace et al., 2007). However, no significant differences concerning side choice among treatments were found in the present study, although lambs showed a tendency of turning to the right-hand side of the test pen, irrespective of lamb treatment (Table 3). This side choice is similar to that reported in lambs of Scottish Blackface (Erhard et al., 2004), Chios (Simitzis et al., 2009) and Sarda (Versace et al., 2007) sheep, Scottish Cashmere goats (Erhard et al., 2002), dairy calves (Arave et al., 1992) and rats (Alonso et al., 1991). However, no solid conclusions can be reached concerning laterality, since no statistical significances were found in differences.
P. Simitzis et al. / Applied Animal Behaviour Science 141 (2012) 43–48
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Table 2 Effect of treatment on maximum, minimum and average heart rate (beats per min – bpm) presented as L.S. mean ± S.E. and on number of vocalizations and jumps, presented as medians (lower upper quartile, Q1–Q3, respectively), during the isolation test (Test 1). Age (months)
2
Treatmenta
Parameters
Heart rates
Average Max Min
Jumps Vocalizations
3
Heart rates
Average Max Min
Jumps Vocalizations
C
S1
S2
172.8 ± 9.70 211.4 ± 7.28 153.2 ± 12.53 0 (0,0) 15 (12,16) 167.9 ± 8.42 213.3 ± 8.92 123.7 ± 5.93 0 (0,1) 11 (9,16)
178.7 ± 9.70 225.7 ± 7.28 134.8 ± 12.53 0 (0,1) 14 (12,15) 166.6 ± 8.42 211.2 ± 8.92 118.8 ± 5.93 0 (0,6) 10 (9,13)
175.3 ± 9.70 210.1 ± 7.28 147.7 ± 12.53 1 (0,3) 17 (13,18) 167.1 ± 8.42 210.9 ± 8.92 132.2 ± 5.93 1 (0,3) 12 (6,16)
a C: control, S1: lamb isolation for 1 h between the 13th and 20th day of age, S2: lamb isolation for 1 h between the 20th and 27th day of age, no significant differences were found among the treatment groups.
Table 3 Effect of treatment on side choice (%) and the time (s) spent with familiar or novel object during novelty Y maze test (Test 2), presented as medians (lower upper quartile, Q1–Q3, respectively). Age (months)
Treatmenta
Parameters
Side choice (%) 2 Time spent (s)
Side choice (%) 3 Time spent (s)
C
S1
S2
Right Left Known Novel None
61.1 38.9 1.5 (0,5) 0 (0,3.75) 56.5 (50.75,60)
55.6 44.4 0 (0,12.75) 3.5 (1.25,10.75) 49 (37.75,58.5)
77.8 22.2 1(0,14.75) 3 (0,9.75) 51 (21.5,59.5)
Right Left Known Novel None
50 50 16.5 (0,32.25) 1 (0,8.75) 32 (15,53)
61.1 38.9 3.5 (1,30.75) 2 (0,10) 39.5 (20.25,55.75)
55.6 44.4 32 (1.75,49.25) 2.5 (0.25,7) 14.2 (7.5,53)
a C: control, S1: lamb isolation for 1 h between the 13th and 20th day of age, S2: lamb isolation for 1 h between the 20th and 27th day of age, no significant differences were found among the treatment groups.
A wealth of information shows that exposure of mammals to early life adversity, such as maternal separation and isolation adversely affects long-term brain development and adult behaviour. However, as it was illustrated in the present study no discernible effects of maternal temporary deprivation in 1 h a day on the display of behavioural components of juvenile lambs at the age of 2 and 3 months old were found.
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Applied Animal Behaviour Science journal homepage: www.elsevier.com/locate/applanim
Effect of pre-weaning temporary isolation within different age periods on the early post-weaning behaviour of juvenile lambs Panagiotis Simitzis ∗ , Miria Petrou, Nikos Demiris, Stelios Deligeorgis Department of Animal Breeding and Husbandry, Faculty of Animal Science and Aquaculture, Agricultural University of Athens, 75 Iera Odos, 118 55 Athens, Greece
a r t i c l e
i n f o
Article history: Accepted 9 July 2012 Available online 25 July 2012 Keywords: Lamb Temporary maternal deprivation Escape behaviour Laterality Novelty
a b s t r a c t Temporary maternal separation is a stressful situation typical under modern husbandry practices and may have an undesirable impact on lamb welfare. However, at present there is insufficient evidence on temporary maternal separation effects on the behaviour of juvenile lambs. The aim of the present study was to assess if and how partial isolation during different periods at the early age can induce differences in the expression of juvenile lamb behaviour at the age of 2 and 3 months old. Twenty-seven male Chios lambs were assigned to 3 experimental groups: C (control), S1 (lamb daily isolation for 1 h between the 13th and 20th day of age) and S2 (lamb daily isolation for 1 h between the 20th and 27th day of age). At the age of 2 and 3 months old, the behaviour of lambs was tested by the implementation of 2 types of test: isolation and novel object (Y-test). There were no statistical differences between lamb treatments in the number of jumps, vocalizations and heart rates during isolation test and the latency to approach a novel or a familiar object in the novel object test conducted at 2 and 3 months of age. However, lambs showed a tendency of turning to the right-hand side of the test pen, irrespective of treatment group, at the age of 2 and 3 months old during the implementation of novel object test. It is concluded that no discernible effects of maternal temporary deprivation on the display of stress induced behavioural components of juvenile lambs at the age of 2 and 3 months old were found. © 2012 Elsevier B.V. All rights reserved.
1. Introduction The quality of mother–infant relationships and the juvenile’s subsequent social milieu are considered to be crucially formative for adjustment and social competence in adulthood (Ferdman et al., 2007). However, maternal deprivation is nowadays widespread among commercially reared animals, particularly in the form of separation from the mother earlier than would happen naturally. In contemporary sheep farming, lambs are often separated from their mothers, with the intention to increase the amount of
∗ Corresponding author. Tel.: +30 2105294449; fax: +30 2105294442. E-mail address: [email protected] (P. Simitzis). 0168-1591/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.applanim.2012.07.004
milk available to be processed into dairy products, resulting in an early disruption of the mother–young relationship (Napolitano et al., 2008). There is a large body of evidence that stressful early life experiences can affect brain development and subsequent behaviour (Hall, 1998). Adverse early life experiences in humans during childhood have lasting effects on the neuroendocrine stress axis, which are thought to increase the risk for the development of mood and anxiety disorders at adulthood (Nemeroff, 2004). In the rat, separation from the mother has been shown to have potentially deleterious effects, since the separated rat pups became hyperresponsive to stressors during behavioural development by exhibiting neophobia at the age of 2 months old (Hall et al., 1997a,b) or increased locomotor activity during
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stress-inducing tests at the age of 3 and 6 months old (Rebouc¸as and Schmidek, 1997) and as adults are unable to appropriately process environmental stimuli (Fone and Porkess, 2008), display reduced cognitive performance (Ladd et al., 2000) and aggression (Hatch et al., 1965). On the other hand, rat pups exposed to a positive postnatal experience (i.e. mothers that show high levels of maternal care) are less behaviourally and physiologically responsive to fear and stress-inducing stimuli when tested at the age of 3 months old, since they have shown alterations in the neural circuitry controlling fear behaviour (Caldji et al., 1998). Therefore, increased maternal care is beneficial for the development of infants, while on the other hand a prolonged maternal absence or neglect can be unfavourable or even harmful (Caldji et al., 1998). Immature animals are very sensitive to abnormal experiences and early life experiences can cause significant changes in the stress response system and emotionality, since the developing brain is suddenly exposed to a variety of neurotransmitters, neuromodulators and transcription factors, which are normally not present, or at least not in that quantity (Anisman et al., 1998). In lambs, exposure to stressful events such as isolation or a novel environment during the first days of their life disrupts neural development, activates hypothalamic-pituitary-adrenal axis, increases corticosteroid concentrations (serotonin, dopamine and the neuropeptide cortico-releasing factor) and may induce immune-suppression (Cockram et al., 1994). Sheep, like most domestic ungulates, are characterised by a strong social tendency. Due to this gregarious behaviour, they react very strongly to social isolation with high-pitch vocalisations and increased locomotion (Price and Thos, 1980; Romeyer and Bouissou, 1992). Both ewes and lambs express their separation distress by an increase in bleating and locomotion activity which indicates their effort to re-establish their contact (Zito et al., 1977). Lambs that are separated from the dam at an early age show reduced levels of vocalisation, are slower to initiate movements, spend less time in ambulatory behaviour and display an increased response compared to non-separated animals when exposed to open field tests (Napolitano et al., 2008). In addition, reactivity appears to be strongly associated with learning and cognitive function, since low levels of learning performance can be a result of a high level of stress behaviour (Boissy and Le Neindre, 1990), which it is strongly associated with the function of the hypothalamicpituitary-adrenal (HPA) axis and hippocampus. Specific behavioural elements that are exhibited during isolation in sheep include high rates of jumping and bleating (Simitzis et al., 2009). Vocalizations and locomotory activity are regarded as reliable indicators to estimate distress response in sheep. The behaviour of sheep can provide clues to the types of situations that might cause suffering or distress and indicate welfare or management problems associated with stress due to husbandry procedures (Le Neindre et al., 1993; Boivin and Braastad, 1996). These behaviours are aimed at maintaining contact with the other lambs and help the animals in localising, finding and identifying them. High locomotive activity and number of vocalizations observed in lambs (Degabriele and Fell,
2001) and kids (Bergamasco et al., 2005; Siebert et al., 2011) in isolation could be interpreted as an indication of fear, but also an attempt to rejoin conspecifics. The results vary depending on the time and the duration of the isolation as well as the gender and genetic background of the animals (Alexander, 1977). Ewe–lamb separation represents a more stressful management procedure for the younger than the older lambs, since younger lambs appear to exhibit their distress in a greater extent when compared with older lambs, possibly due to the stronger mother–young attachment at the early stages of life (Napolitano et al., 2008). It has been observed that ewe–lamb separation soon after lamb birth can cause impaired immune functions, enhanced cortisol response to isolation and altered behavioural reaction to open-field testing (Sevi et al., 1999; Napolitano et al., 1995, 2003). However, little is known on the effect of repeated temporary ewe–lamb separation on lamb early life and subsequent behavioural development. The current interest in early life experience stems from the observation that specific early events and specific time periods before weaning can apparently programme the “set-point” of the HPA axis by altering the effective feedback. At the same time, methods of behavioural measurements appear to be more sensitive for the detection of stressful conditions than the other commonly used endocrine or immune indicators of welfare (Napolitano et al., 2008). To our knowledge, no behavioural studies have been implemented for examining the impact of temporary isolation within different ages on the behaviour of juvenile lambs. Therefore, the aim of the present study was to assess if and how partial isolation induce differences in the expression of lamb behaviour and compare the effects of temporary hourly isolation either within the 13th–20th or the 20th–27th day of age on the behavioural components of juvenile lambs at the age of 2 and 3 months old. 2. Materials and methods 2.1. Pre-weaning period A total of 27 male Chios lambs were used in the present study. Lambs were delivered naturally after oestrus synchronisation of their mothers with intravaginal progestagen sponges (Chronogest, Intervet, The Netherlands). After delivery they were ear tagged, weighed and the date of birth was recorded. All lambs were born as singles within 3 days. They were kept all together in one group with their mothers (2 m2 /ewe and lamb). All lambs had ad libitum access to hay and water. Lambs were then randomly assigned to 3 experimental groups (C: control, S1: lamb isolation for 1 h between the 13th and 20th day of age, and S2: lamb isolation for 1 h between the 20th and 27th day of age). According to their experimental group (lamb isolation between 13th and 20th or 20th and 27th day of age; 1 h/day on every day from day 13 to day 20 and 1 h/day on every day from day 20 to day 27), lambs were daily separated from their mothers and isolated for an hour by moving them to a different barn with pens (1 m2 /lamb), prohibiting visual contact and vocal communication with the ewes, but permitting vocal
P. Simitzis et al. / Applied Animal Behaviour Science 141 (2012) 43–48
communication between the examined lambs. The two different lamb ages (13th–20th or the 20th–27th day of age) were chosen in the absence of any relevant information and in order to represent the early life of the lambs. After the hourly isolation, lambs returned to the pen with their mothers. Methods used in the present experiment were approved by the bioethical committee of the Agricultural University of Athens under the guidelines of “Council Directive 86/609/EEC regarding the protection of animals used for experimental and other scientific purposes”.
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2m
Novel or familiar object
4m
2.2. Post-weaning period After weaning (45th day), lambs were subjected to two behavioural tests carried out on three consecutive days at two different ages (2 and 3 months). Tests were conducted between 8:30 a.m. and 12:00 p.m. during each experimental day. Before testing, groups of lambs were moved to a holding pen, where they stayed for 30 min. After this adaptational period, each individual was separated from the group and moved to the entrance of the test pens (ca. 7 m distance). The procedure was designed to ensure that the handling protocol applied to the animals between the holding and test pen was standardised as far as possible. Lambs from different treatment groups were tested alternately, so that the treatment factor was not confounded with the order of testing. Test 1 (isolation test) consisted of social isolation in a novel environment (1 min), without any additional fearinducing stimuli (Erhard et al., 2004; Simitzis et al., 2009; Hernandez et al., 2009). The test pen represented a novel environment in which the tested animal was visually isolated from other members of the group. Each pen (2 m × 2 m) had solid walls and floor was covered with sawdust. Average, minimum and maximum heart rate of lambs (beats per minute – bpm) was measured by the POLAR S180i heart rate monitor system adapted to sheep, at the beginning of the adaptational period (30 min) in the holding pen. The system consisted of the transmitter and the receiver, which detected and displayed the heart rate, respectively. The transmitter was fitted with an elastic strap which held it firmly in the heart girth of each lamb. The frequency – number of jumps (when all four legs left the ground at the walls of the test pen – escape attempts) and vocalizations were the variables recorded during the isolation test. Test 2 (novel object test) was designed to examine the initial reaction of lamb when exposed together to a novel or familiar object (1 min) (Erhard et al., 2004; Simitzis et al., 2009). A Y test pen was implemented with a coloured ball representing the novel object and a feeder representing the familiar object placed in the two front sides of the test pen (Fig. 1). Lambs were exposed to the familiar object (feeder) between the 1st and the 45th day of their life. The novel object test consisted of two subtests, varying according to the position of novel and familiar object, which was shifted between the two subtests, without any further changes in the experimental design. During Test 2, laterality of initial preference for one side over the other or direction first turned upon entry and response to novel or familiar object, expressed as time spent to approach within approximately
2m
2m Fig. 1. Layout of the experimental pen during novel object test (Test 2).
10 cm for novel or familiar object were recorded, respectively. When lambs did not move in the test pen or they did not choose one side over the other, their behaviour was characterised as “indifference”. Behavioural patterns in the two tests were recorded using a Panasonic PV120 video camera fitted with a 3.6 mm lens and mounted 3 m above the pen floor. Video recordings were stored in a digital video recorder equipped with a hard disk (TX168, Telexper Inc., USA) and behavioural components (number of jumps, number of vocalizations, laterality and time spent with novel or familiar object) were measured. 2.3. Statistical analysis Data from the first test were the counts of jumps and vocalizations as well as the heart rate mean and standard error. As is common with count data we assumed that the number of jumps and vocalizations followed a Poisson distribution, which appeared to be a reasonable assumption. A normal distribution was adequate for the heart measurements. Furthermore, the potential correlation in measuring these three variables on the same lambs was taken into account by including (correlated) random effects. In particular, we used a trivariate generalised linear model where the log-rates of the jump and vocalisation distributions were connected to the heart rate via normally distributed random effects. The data from the second test were analysed using a combination of multinomial and exponential distributions. Specifically, we used a trinomial distribution in order to describe the animal behaviour in terms of choosing the right, the left direction or being indifferent. The times spent with the novel or the familiar object were assumed to follow distinct exponential distributions. Subsequently, we regressed the logit transformation of the probabilities and the log-rates of the exponential distributions upon the potential explanatory variables which included the
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Table 1 Live weights (kg) of lambs at birth and the ages of testing (L.S. mean ± S.E.). Age (months)
Birth 2 3
Treatmenta C
S1
S2
3.57 ± 0.25 14.9 ± 1.16 19.8 ± 1.56
4.06 ± 0.25 16.3 ± 1.16 20.0 ± 1.56
3.83 ± 0.25 15.0 ± 1.16 19.7 ± 1.56
a C: control, S1: lamb isolation for 1 h between the 13th and 20th day of age, S2: lamb isolation for 1 h between the 20th and 27th day of age, no significant differences were found among the treatment groups.
treatment, age and their interaction. All the statistical analyses in this paper were performed using the Winbugs (Lunn et al., 2000) software. 3. Results Lack of differences in body weight from birth till the age of 3 months old between treatments and control sheep was demonstrated (Table 1). Lamb pre-weaning temporary isolation seemed not to affect lambs’ growth. Moreover, as it was illustrated during the isolation test, no significant differences were observed among the lambs’ experimental groups, although the number of jumps and vocalizations tended to be higher for S2 lambs (Table 2). At the same time, rates for number of vocalizations tended to decrease as the age of lambs increased. No discernible differences were also illustrated among treatment groups regarding the average heart rate of lambs during the isolation test. Maximum and minimum heart rates also appeared not to vary significantly among lamb treatments (Table 2). As it was illustrated in the novel object test (Test 2), there were no differences in latency to approach a novel or a familiar object among treatments of lambs at the age of 2 and 3 months old (Table 3). Moreover, lambs preweaning temporary isolation did not significantly influence the behavioural laterality of lambs during Test 2, although lambs showed a tendency of turning to the right-hand side of the test pen, irrespective of lamb treatment (Table 3). 4. Discussion The results of behavioural measurements in the present study revealed that lambs pre-weaning temporary isolation did not significantly influence the reactivity of lambs at the early stages of their lives. Number of jumps and vocalizations during the isolation test were not significantly different among lamb treatments (Table 2). The same conclusions have been reached by Simitzis et al. (2009) and Hernandez et al. (2009) in experiments implemented in juvenile lambs at the age of 2, 3, 4 and 5 months old. No significant differences were also found concerning heart rates (Table 2). Fear can be evoked by social isolation and novel environment (Baldock and Silby, 1990) and heart rates can provide a useful measure of stress in sheep (Syme and Elphick, 1982). Average heart rates in the present study varied between 160 and 200 bpm, in accordance with previous implemented stress inducing experiments in 2 and 3 years old sheep (Baldock and Silby, 1990) and 2, 3, 4 and 5 months old lambs (Simitzis et al., 2009). The negative
effects of lamb pre-weaning temporary isolation are not evident at the early stages of lamb life, though disruption of ewe–lamb relationship could be a source of poor welfare for the lamb and could disturb the acquisition of its social behaviour (Dwyer, 2008). Normally, ewe–lamb separation results in an increase in plasma cortisol (Mears and Brown, 1997; Orgeur et al., 1999), increased behavioural and vocal activity, disrupted cicardian rhythms and altered social interactions (Orgeur et al., 1998). This increased emotional stress is a result of the expectation of lamb being rejoined after the temporary separation from its mother or the other lambs (Sevi et al., 2003). Moreover, in contrast to the present study, where no significant differences in latency to approach a novel or a familiar object among treatments of lambs were found (Table 3), partially isolated lambs appear to respond to isolation in a novel environment less actively than those of the control group (no separation), showing withdrawal behaviours after introduction, expressed as longer latency times to move, shorter ambulatory activity and less frequent jumping attempts compared to the control group of lambs between 12 and 32 days of age (Sevi et al., 2003). Moberg and Wood (1982) have stated that isolation-reared lambs show behavioural withdrawal in stressful situations such as a novel environment when tested at 12 and 42 days of age. At the same time, lambs also bleated less in reaction to final weaning when they had been subjected to a dairy, temporary separation from their mothers, as compared to lambs that had not been habituated to short term separations (Orgeur et al., 1998). Lambs’ exposure to stressful events such as isolation or a novel environment disrupts neural development, activates hypothalamic-pituitary-adrenal axis, increases corticosteroid concentrations and may induce immune-suppression (Cockram et al., 1994). However, no display of these effects in the present study could be partially explained by the hypothesis that at the early stages of lamb age, the evoked effects on the hypothalamus–pituitary–adrenal axis are not so evident in terms of behaviour. Additionally, these different findings in relation to the display of heightened anxiety states may result from varying adversities inherent in the different test paradigms, differing isolation onset and duration, or may represent differences between different species or breeds (Versace et al., 2007). Behavioural lateralisation has gained considerable attention as an indirect measure of the effects of adverse early life environments on brain development. In mammals, laterality in behaviour and asymmetry in morphological characteristics are accentuated by neonatal stress (Versace et al., 2007). However, no significant differences concerning side choice among treatments were found in the present study, although lambs showed a tendency of turning to the right-hand side of the test pen, irrespective of lamb treatment (Table 3). This side choice is similar to that reported in lambs of Scottish Blackface (Erhard et al., 2004), Chios (Simitzis et al., 2009) and Sarda (Versace et al., 2007) sheep, Scottish Cashmere goats (Erhard et al., 2002), dairy calves (Arave et al., 1992) and rats (Alonso et al., 1991). However, no solid conclusions can be reached concerning laterality, since no statistical significances were found in differences.
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Table 2 Effect of treatment on maximum, minimum and average heart rate (beats per min – bpm) presented as L.S. mean ± S.E. and on number of vocalizations and jumps, presented as medians (lower upper quartile, Q1–Q3, respectively), during the isolation test (Test 1). Age (months)
2
Treatmenta
Parameters
Heart rates
Average Max Min
Jumps Vocalizations
3
Heart rates
Average Max Min
Jumps Vocalizations
C
S1
S2
172.8 ± 9.70 211.4 ± 7.28 153.2 ± 12.53 0 (0,0) 15 (12,16) 167.9 ± 8.42 213.3 ± 8.92 123.7 ± 5.93 0 (0,1) 11 (9,16)
178.7 ± 9.70 225.7 ± 7.28 134.8 ± 12.53 0 (0,1) 14 (12,15) 166.6 ± 8.42 211.2 ± 8.92 118.8 ± 5.93 0 (0,6) 10 (9,13)
175.3 ± 9.70 210.1 ± 7.28 147.7 ± 12.53 1 (0,3) 17 (13,18) 167.1 ± 8.42 210.9 ± 8.92 132.2 ± 5.93 1 (0,3) 12 (6,16)
a C: control, S1: lamb isolation for 1 h between the 13th and 20th day of age, S2: lamb isolation for 1 h between the 20th and 27th day of age, no significant differences were found among the treatment groups.
Table 3 Effect of treatment on side choice (%) and the time (s) spent with familiar or novel object during novelty Y maze test (Test 2), presented as medians (lower upper quartile, Q1–Q3, respectively). Age (months)
Treatmenta
Parameters
Side choice (%) 2 Time spent (s)
Side choice (%) 3 Time spent (s)
C
S1
S2
Right Left Known Novel None
61.1 38.9 1.5 (0,5) 0 (0,3.75) 56.5 (50.75,60)
55.6 44.4 0 (0,12.75) 3.5 (1.25,10.75) 49 (37.75,58.5)
77.8 22.2 1(0,14.75) 3 (0,9.75) 51 (21.5,59.5)
Right Left Known Novel None
50 50 16.5 (0,32.25) 1 (0,8.75) 32 (15,53)
61.1 38.9 3.5 (1,30.75) 2 (0,10) 39.5 (20.25,55.75)
55.6 44.4 32 (1.75,49.25) 2.5 (0.25,7) 14.2 (7.5,53)
a C: control, S1: lamb isolation for 1 h between the 13th and 20th day of age, S2: lamb isolation for 1 h between the 20th and 27th day of age, no significant differences were found among the treatment groups.
A wealth of information shows that exposure of mammals to early life adversity, such as maternal separation and isolation adversely affects long-term brain development and adult behaviour. However, as it was illustrated in the present study no discernible effects of maternal temporary deprivation in 1 h a day on the display of behavioural components of juvenile lambs at the age of 2 and 3 months old were found.
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