Prenatal stress puzzle, the oxytocin piece: Prenatal stress alters the behaviour and autonomic regulation in piglets, insights from oxytocin

Applied Animal Behaviour Science 148 (2013) 99–107

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Applied Animal Behaviour Science journal homepage: www.elsevier.com/locate/applanim

Prenatal stress puzzle, the oxytocin piece: Prenatal stress alters the behaviour and autonomic regulation in piglets, insights from oxytocin Jean-Loup Rault a,∗ , Laurie A. Mack a , C. Sue Carter b , Joseph P. Garner a , Jeremy N. Marchant-Forde c , Brian T. Richert a , Donald C. Lay Jr. c a b c

Department of Animal Sciences, Purdue University, West Lafayette, IN 47907, USA Department of Psychiatry, University of Illinois at Chicago, IL 60607, USA United States Department of Agriculture-ARS, Livestock Behavior Research Unit, West Lafayette, IN 47907, USA

a r t i c l e

i n f o

Article history: Accepted 1 July 2013 Available online 23 July 2013 Keywords: Oxytocin Prenatal stress Offspring Social Behaviour Heart rate

a b s t r a c t Developmental changes in response to prenatal stressors (PNS) can result in anxiety and abnormal social development in the offspring. Oxytocin (OT) reduces anxiety, whereas OT deficiencies are associated with social behaviour deficits. Hence, we hypothesized that OT could reverse some of the PNS effects. Female offspring from three socially stressed (PNS 35–56 days of gestation) and three control sows were tested at 18 days of age. In each litter, two piglets received 24 IU of OT intranasally and two piglets received saline as a control treatment. After 45 min, each piglet was isolated for 15 min in a separate room. The PNS piglets displayed fewer alert behaviours during social isolation than control piglets (saline-control: 93.6 ± 0.2, OT-control: 82.5 ± 0.2, saline-PNS: 52.0 ± 0.2, and OT-PNS piglets: 67.8 ± 0.2). All other behavioural differences were the result of the administration of OT to PNS piglets, which reestablished a few behavioural changes such as the time spent standing in PNS piglets, with OT-PNS piglets standing more than saline-PNS piglets (77.8 ± 6.7 s vs. 57.1 ± 6.7 s respectively), and as much as saline- and OT-controls (76.7 ± 6.7 s and 70.0 ± 6.7 s respectively). Furthermore, saline-PNS piglets spent more time sitting than control piglets, and OT reversed this effect in OT-PNS piglets, with OT-PNS piglets sitting no more than controls. Prenatal stress and the administration of OT interacted in their effect on the heart rate, with OT-control and PNS piglets (saline or OT) having lower mean heart rate than saline-control piglets (saline-control: 168 ± 4, OT-control: 159 ± 4, saline-PNS: 146 ± 5, and OT-PNS piglets: 149 ± 5). Neither PNS nor OT administration influenced the RMSSD of the heart rate. In conclusion, these preliminary results suggest that social stress in mid-gestation results in some behavioural differences and alters the development of the autonomic nervous system in the sow’s offspring, in this case female offspring. Exogenous OT administration reversed some behavioural changes, suggesting a common physiological basis. © 2013 Elsevier B.V. All rights reserved.

1. Introduction ∗ Corresponding author. Present address: Animal Welfare Science Centre, School of Land and Environment, University of Melbourne, VIC 3010, Australia. Tel.: +61 3 9035 7542; fax: +61 3 9035 7849. E-mail addresses: [email protected], [email protected] (J.-L. Rault). 0168-1591/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.applanim.2013.07.001

Developmental changes in response to prenatal stressors have been proposed to constitute an adaptive strategy aimed to enhance offspring’s survival traits (Sheriff et al., 2009). Prenatal stressors of diverse natures, whether it be nutritional (Brown et al., 2000), psychological (Clarke and

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Schneider, 1993), physical (Lemaire et al., 2000), or physiological (Haussmann et al., 2000) can have long-lasting physiological and behavioural consequences. However, the same strategies that have evolved as adaptive in natural environments may be maladaptive in captive environments. Captive animals are usually kept at high densities or in larger groups relative to their wild counterparts. Sows in semi-natural environments form groups of two to five familiar and related individuals (Stolba and Wood-Gush, 1989). However, pigs are often kept in larger and less stable groups in commercial settings (Gonyou, 2001). Unnatural social group dynamics, such as maintaining animals at high densities, in uniform sex and age groups, or mixing them with unfamiliar peers can all contribute to social stress, while disturbing the use of evolutive social strategies such as hierarchy or social support (Estevez et al., 2007; Rault, 2012). Spatial and social constraints are intrinsic to animal holding facilities, and could affect both the dams and their offspring. The implications of prenatal stress (PNS) on the offspring are highly relevant to the welfare of animals kept in captive environments (Braastad, 1998; Sachser et al., 2011). Prenatal stress can alter the offspring’s behaviour, leading to anxiety-related behaviours (Fride and Weinstock, 1988; Thompson, 1957; Welberg and Seckl, 2001), cognitive changes (Kofman, 2002), and abnormal social development (Clarke and Schneider, 1993; Jarvis et al., 2006), which may not be suited to that particular captive environment. The hypothalamic–pituitary–adrenal (HPA) axis is suggested as a mediator of PNS effects (Weinstock, 2008), but catecholamines and opioids have also been proposed (Lieberman, 1963; Merlot et al., 2008). The specific neurophysiological substrates involved remain to be clarified in our effort to understand the socio-behavioural modifications induced in the offspring by early stressful experiences (Kofman, 2002). Oxytocin (OT) is a mammalian neuropeptide with a major involvement in various social processes (Carter, 1998; Ross and Young, 2009). In particular, OT reduces anxiety in rodents (Blume et al., 2008), whereas OT deficiency results in impaired social behaviour (Amico, 2008). Therefore, OT can bestow effects opposite to the anxiety and social deficits seen in PNS offspring. Indeed, a few studies in rodents suggest that OT can reverse PNS effects. Lee et al. (2007) reported that submitting rat dams to an intense series of unpredictable stressors altered the offspring’s social interactions, both in the quantity (76% less interaction time) and the quality of interactions (avoid nonaggressive contact interaction), whereas OT administration in the central amygdala reversed those social behaviour deficiencies (Lee et al., 2007). Severe food restriction of rat dams is associated with increases in corticosterone and blood pressure in adult offspring, but chronic administration of OT for the first two weeks of life alleviated those effects (Olausson et al., 2003; Sohlstrom et al., 2000). Lee et al. (2007) also found that the OT system was affected by PNS, with less OT mRNA in the paraventricular nucleus, one of the major brain regions producing OT, and an increase in OT receptor binding in the central amygdala, a region known to regulate fear and anxiety (Neumann, 2008).

While stressful events may be commonly encountered by captive animals during gestation, the consequences on the offspring remain poorly understood. If PNS modifies the OT system, this could have far-reaching consequences on the individual’s socio-behavioural development (Carter, 2003). In the present study, we hypothesized that OT could reverse some of the effects of PNS. Since the majority of the literature suggests that PNS results in greater anxiety, we predicted that PNS piglets would show a greater behavioural and physiological arousal to social isolation than control piglets, and that OT administration could reduce those effects. 2. Materials and methods All animals were cared for by the Purdue Swine Unit Animal Care Staff according to the research protocol approved by the Purdue Animal Care and Use Committee and in accordance with the Federation of Animal Science Societies’ Animal Care Guidelines (FASS, 2010). 2.1. Prenatal stress procedure Six multiparous Landrace × Yorkshire sows were artificially inseminated with semen from Duroc sires and housed in 2.1 m × 0.6 m gestation stalls until day 35 ± 0.5 (mean ± SD) of gestation. Sows were then subjected to one of two treatments. On day 35, three sows were introduced individually into one of three 2.3 m × 2 m pens with two unfamiliar sows of similar body weights. The hierarchy formation between mature sows usually results in substantial social stress and agonistic interactions (Arey, 1999), and a similar paradigm has been used by other researchers to study PNS in pigs (Jarvis et al., 2006). The timing of mixing was chosen based on recent legislatory or voluntary changes for the pig industry in many countries (e.g. European Union, United States), to keep sows in individual stalls for the first 4–6 weeks of gestation and in group-housed pens thereafter for the remaining of gestation, hence mixing sows around that day 35 of gestation. This social mixing was repeated every week for 3 weeks by moving each experimental sow to another pen with two other unfamiliar sows in order to minimize population variability. At the end of the 3 weeks, day 56 of gestation, these three sows were moved back into gestation stalls and represented the socially stressed sows giving birth to prenatally stressed offspring. These sows were scored for skin lesions 4 days after each mixing, using a 6-point scale adapted from Arey (1999) based on number and severity (scratch, cut, wound), and their body weight collected at the end of the 3 weeks mixing treatment. The three control sows were kept in the same gestation stalls with the same neighbours throughout the gestation period. At day 110, all the sows were moved into farrowing crates and farrowed on day 114 ± 0.2 (mean ± SD). Sows were not induced for parturition, nor did they receive OT during pregnancy or parturition. 2.2. Experimental subjects A total of 24 female suckling piglets of 17.8 ± 0.8 days of age were tested, comprising four piglets from each of the six

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Table 1 Ethogram used for behavioural observations. Behaviours of each piglet were recorded continuously for each category: posture, activity, and arousal. Behaviours within the posture and activity categories were mutually exclusive. Category

Behaviour

Definition

Posture (% of time)

Stand Lie Sit

Up on 4 legs Lie down on belly or side in contact with floor Sit on 1 or 2 back legs

Activity (% of time)

Locomotion Exploration Inactive Other

Any locomotor behaviour, walk or run Nose in contact with the floor or the walls, root or chew Immobile Any other behaviour not listed above

Alert Eliminate Squeal

Immobile with head and ears up Defecation or urination High-pitch vocalization; extended sound (0.5–2 s) of both high amplitude and high frequency with an open mouth Low-pitch vocalization; sound of low to medium amplitude with a closed mouth Rear on its hind legs and jump against the walls. All limbs lose contact with the floor

Arousal (number of events)

Grunt Escape attempts

litters. Only female subjects were used because one of our previous studies (Rault, 2011) found the strongest OT effect in females, which concurs with the literature suggesting that OT may be of particular importance in females (Carter, 2007; Taylor et al., 2006). 2.3. Social isolation test The experiment was carried out over two days. On the day prior to the test, two pairs of female piglets were chosen from each litter on the basis of similar weight (mean of the within pair weight difference ± SD: 0.2 ± 0.2 kg). On the day of testing, one piglet of each pair (n = 12, six from PNS sows and six from control sows) received intranasally 24 IU (equivalent to 48 ␮g) of OT (Bachem, Torrance, CA, USA) diluted in 0.5 mL of 0.9% saline, with a half-dose in each nostril. The other piglet of each pair (n = 12, six from PNS sows and six from control sows) received intranasally 0.5 mL of 0.9% saline as a control. Piglets were submitted to either one of the two treatments by matched pairs, i.e. by giving one piglet of the pair OT and the other piglet of the same litter saline. Therefore, we obtained six piglets for each of the four treatment groups: saline-control piglets, OT-control piglets, saline-PNS piglets and OT-PNS piglets, with treatment comparisons blocked by litter to account for genetic and environmental effects. Treatments were delivered using a Mucosal Atomizer Device (MAD 300, Wolfe Tory Medical Inc., Salt Lake City, UT, USA) connected to a 1 mL syringe, while the piglets were maintained in a headup position. Piglets were picked up briefly from their pen to administer their treatment and immediately put back in their pen. This procedure took from 30 to 45 s. If the piglet expelled the solution, a second administration (halfdose) was delivered in that nostril within those 45 s. This occurred once in three out of six PNS piglets and three out of six control piglets. We did not account for suckling, i.e. whether piglets were tested just before or after a nursing bout, other than no treatment was administered during a suckling bout or to a piglet that was massaging the sow’s udder. Forty-five minutes post-administration (a time expected to be sufficient for OT to reach the brain based on an extrapolation from Born et al., 2002), each

piglet was picked up, fitted with a telemetric heart rate belt, carried to a different room 20 m away, and placed in an isolation box for 15 min. The piglet had no olfactory or visual contact with other pigs. The isolation box (floor area = 0.95 m × 0.95 m, height = 0.84 m) was built of interlocking plastic panels that slid into four corner pieces. The four plastic corner pieces were connected to steel pipes for stability. Lines were drawn on the floor using chalk to form a visual 3 × 3 grid (floor area of each grid: 0.32 m × 0.32 m) to measure locomotor activity. After placing the piglet into the isolation box, the experimenter immediately left the room and returned 15 min later. After the 15 min isolation test, the piglet was returned to its home pen. The isolation box was cleaned with bleach (2400 ppm) between each piglet. The ambient temperature of the isolation room was 24.5 ◦ C and always within a 1.5 ◦ C range of their home pen. 2.4. Sampling and measurements 2.4.1. Behaviour Behaviours were continuously recorded using a camcorder (DCR-TRV280, Sony, San Diego, CA, USA) fixed 2.1 m above ground level and recording at 30 frames per seconds. Videos were analysed using the “Observer” software (version 5.0, Noldus, The Netherlands) with a continuous recording method using the ethogram shown in Table 1. Videos were played at one-half the real-time playback speed in order to enhance the accuracy of analysis. Behavioural categories were posture, activity, and arousal. Locomotor activity was calculated by counting the number of grids crossed, based on the two rear legs relative to the lines drawn on the floor. All observations were conducted by a single individual who was blind to treatments. 2.4.2. Heart rate A telemetric heart rate monitor (Polar® RS800CX, Polar Electro Oy, Kempele, Finland) was fitted on the piglet prior to the isolation test when the piglet was picked up from the farrowing pen. The apparatus consisted of a belt containing two electrodes fitted around the thorax behind the forelegs, with both electrodes positioned on the left

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side of the animal. Conductivity between the piglet’s skin and the electrodes’ contact point was enhanced by applying ultrasound transmission gel (Spectra 360, Parker Lab. Inc., Fairfield, NJ, USA). The recording watch-style device was fixed to the belt on the animal’s back. The device was used to record and store successive interbeat intervals, following a previously validated method (Marchant-Forde et al., 2004). Data were manually corrected for artefacts and 1 min sections containing more than 10% artefacts were excluded from the analysis and accounted as missing data during statistical analyses. Time domain analyses were performed to determine heart rate (HR) mean, HR maximum, and HR minimum for each minute of the test, all in beats per min (bpm). The root mean square of successive differences (RMSSD, ms) in the beat-to-beat heart rate time series for the first 8 min of the test was extracted using the Polar Pro Trainer software (Polar Electro Oy, Kempele, Finland). The RMSSD is highly correlated with methods that describe the amplitude of respiratory sinus arrhythmia, e.g. high frequency heart rate variability, and has been used as an index of vagal cardiac control reflecting the parasympathetic activity (Berntson et al., 2005; von Borell et al., 2007). 2.5. Statistical analysis All data met the criteria for normality and homogeneity of variance except the alert behaviour which had to be transformed using the square root transformation. Data were analysed using a mixed model (Proc Mixed, SAS Inst. Inc., Cary, NC, USA). For each behaviour, the model included as fixed effects the two main effects prenatal stress and oxytocin as well as their interaction and as random effect litter nested within prenatal stress. For heart rate data, the model included the same effects and accounted for repeated measures over time, except for the RMSSD measure which was analysed as a single measure over the first 8 min of the test as a standardized length. When significant differences (P < 0.05) were detected, appropriate Tukey–Kramer adjustments were used for pairwise comparisons between treatments. Data are presented as least square means ± SEM. 3. Results This project was part of a larger study looking at the effects of PNS on the productivity of sows and their litters (Mack et al., 2012). The social stress treatment reduced the body weight of the sows (group mixed sows: 200.4 ± 1.3 kg vs. control sows: 207.0 ± 1.3 kg, F(3,43) = 9.16, P < 0.001) and resulted in higher head lesions (group mixed sows: 4.2 ± 0.2 vs. control sows: 3.6 ± 0.1, F(3,28) = 4.59, P < 0.01), confirming that the social stress treatment imposed on the sows was stressful. However, the social stress treatment had no effect on sow body, rump, leg or foot lesions, sow cortisol or progesterone 24 h after each weekly mixing, sow farrowing rate, sow gestation length, litter size, litter sex ratio, piglet mortality, piglet birth weight, piglet weight until 140 days of age, number and symmetry of piglet teats, anogenital distance, or male testis weight (P > 0.1).

3.1. Behaviour Prenatally stressed piglets, from group-mixed sows, displayed fewer alert behaviours than control piglets (F(1,16) = 5.23, P = 0.04, Fig. 1A), and showed a trend to have a longer latency to the first escape attempt (PNS piglets: 320.8 ± 42.6 s vs. control piglets: 136.4 ± 44.9 s, F(1,4) = 6.68, P = 0.06). All other behavioural differences appeared as the result of the OT treatment to PNS piglets. Oxytocin reversed the differences in time spent standing in PNS piglets (PNS × OT interaction: F(1,16) = 4.41, P = 0.05, Fig. 1B), with OT-PNS piglets standing more than saline-PNS piglets (Tukey test: OT-PNS vs. saline-PNS P = 0.05), and as much as controls (Tukey test: OT-PNS vs. saline-control P > 0.1). Furthermore, saline-PNS piglets spent more time sitting than saline-control piglets (PNS × OT interaction: F(1,16) = 10.06, P = 0.006, Fig. 1C; Tukey test: saline-PNS vs. saline-control P = 0.02), and OT reversed this effect in OT-PNS piglets (Tukey test: OT-PNS vs. saline-PNS P = 0.02), with OTPNS piglets sitting no more than controls (Tukey test: OT-PNS vs. saline-control P > 0.1). Oxytocin administration also tended to re-establish alert behaviour in PNS piglets (PNS × OT interaction: F(1,16) = 3.03, P = 0.1, Fig. 1A). In contrast, OT administration increased differences between PNS and control piglets with regard to the number of eliminations, i.e. defecation and urination, with OT-PNS piglets eliminating more than OT-control piglets (PNS × OT interaction: F(1,16) = 5.29, P = 0.04; Tukey test: OT-PNS vs. OT-control P = 0.02, Fig. 1D). We did not notice any elimination due to handling between the home pen and the isolation pen. Vocalizations, whether grunts or squeals, did not differ between treatments, nor did the total number of grids crossed, locomotion, escape attempts, exploration, lying, or the time spent inactive (P > 0.1, Table 2). The readministration of a half-dose of OT for half of the piglets which expelled the solution did not affect any results (P > 0.1). 3.2. Heart rate Data were collected and analysed from 21 out of 24 piglets, with an 11% exclusion rate due to artefacts. Prenatally stressed piglets showed a trend to have a lower mean HR compared to control piglets (F(1,4) = 6.22, P = 0.07, Fig. 2), but no difference in maximum or minimum HR (F(1,4) = 4.07, P > 0.1 and F(1,4) = 1.29, P > 0.1 respectively). The administration of OT resulted in a lower maximum and minimum HR in all piglets (F(1,267) = 10.84, P = 0.001 and F(1,267) = 12.83, P < 0.001 respectively), but no main effect of OT on the mean HR (F(1,267) = 1.59, P > 0.1). However, the effects of PNS and OT administration were interactive on the mean HR (PNS × OT interaction: F(1,267) = 6.07, P = 0.01), with OT-control piglets and PNS piglets (saline or OT) having lower mean HR than saline-control piglets (Tukey tests: saline-control vs. OT-control or saline-PNS both P = 0.01, saline-control vs. OT-PNS P = 0.02, Fig. 2). Neither PNS nor OT administration influenced the RMSSD of the heart rate (P > 0.1, Table 2). Because the HR measures could be influenced by the level of activity of the piglets, we included behavioural

J.-L. Rault et al. / Applied Animal Behaviour Science 148 (2013) 99–107

Alert (number)

10

Saline a OT

200 a b

Heart rate (bpm)

A 12

b

8

1

6 4 2 0 Control

B 100 80

Stand (% of time)

a b

160

b

b

140 120 100

PNS b

ab a

60

PNS

Fig. 2. Effects of prenatal stress and oxytocin administration on the heart rate mean (bars), heart rate maximum (䊉), and heart rate minimum () performed during the 15 min isolation test. Data are presented as least square means ± SEM. For clarity purposes, post hoc differences are only shown for heart rate mean; bars with different letters indicate significant differences (P < 0.05, Control = control piglets, PNS = prenatally stressed piglets; n = 6 per treatment).

40 20 0 Control

C 14 12 Sit (% of time)

180

Saline OT

Control Saline ab OT

103

PNS b

Saline OT

10 ab

8

a

a

6

4. Discussion

4 2 0 Control

Elimination (number)

D 4

measures of activity (total number of grid crossed, duration of locomotion and number of escape attempts) as covariates in the model and their interaction with treatments for the HR variables (mean, maximum and minimum HR and RMSSD). None of the behavioural measures of activity had significant effect on the HR variables (P > 0.1). Furthermore, to investigate if the HR response varied over the course of the 15 min isolation test, we included time, for each min of the test, as a main effect in the model as well as its interactions with the two main effects PNS and OT. Time or its interactions with PNS and OT were never significant on mean, maximum or minimum HR (P > 0.1).

PNS

Saline OT

b

3 ab ab

2 a

Prenatally stressed piglets from socially stressed sows displayed a lower level of vigilance, in our case recorded as alert behaviour, during social isolation. All other behavioural differences were the result of the administration of OT to PNS piglets, which reestablished a few behavioural changes such as the time spent standing for PNS piglets. Both PNS and the administration of OT resulted in lower heart rate. These preliminary results suggest that social stress during mid-gestation results in some behavioural differences and alters the development of the autonomic nervous system in the sow’s offspring, in this case female offspring.

1

0 Control

PNS

Fig. 1. Effects of prenatal stress and oxytocin administration on (A) the number of alert behaviour1 , (B) duration of standing, (C) duration of sitting, and (D) the number of eliminations performed during the 15 min isolation test. Data are presented least square means ± SEM. Bars with different letters indicate significant differences (P < 0.05; Control = control piglets, PNS = prenatally stressed piglets; n = 6 per treatment). 1 That behaviour had to be analysed using the square root transformation and is presented as transformed in the graph. The back-transformed least square means ± SEM were saline-control: 93.6 ± 0.2, OT-control: 82.5 ± 0.2, saline-PNS: 52.0 ± 0.2, and OT-PNS piglets: 67.8 ± 0.2.

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Table 2 Effects of prenatal stress and oxytocin administration on the behaviours: total number of grids crossed, escape attempts, the duration of locomotion, lie, exploration, and inactive behaviours, and the number of grunts and squeals during the 15 min isolation test and on the root mean square of successive differences (RMSSD) of the heart rate for the first 8 min of the isolation test (PNS = prenatally stressed piglets, Control = control piglets; n = 6 per treatment). Data are presented as least square means ± pooled SEM. Piglets

Saline

Oxytocin

SEM

Prenatal stress effect

Oxytocin effect

Prenatal stress × oxytocin effect

Total grids crossed (number)

PNS Control

125.0 185.7

159.8 196.7

±22.8

P = 0.13

P = 0.26

P = 0.55

Escape attempts (number)

PNS Control

9.3 20.8

12.7 29.8

±6.6

P = 0.14

P = 0.27

P = 0.61

Locomotion (%)

PNS Control

10.7 14.4

11.6 16.7

±2.8

P = 0.25

P = 0.48

P = 0.77

Lie (%)

PNS Control

31.8 19.8

17.4 24.0

±6.8

P = 0.70

P = 0.48

P = 0.19

Exploration (%)

PNS Control

27.0 35.6

34.7 34.4

±3.6

P = 0.27

P = 0.39

P = 0.24

Inactive (%)

PNS Control

13.5 7.7

10.2 11.1

±4.6

P = 0.13

P = 0.60

P = 0.48

Grunts (number)

PNS Control

502.2 675.2

609.5 658.2

±81.3

P = 0.19

P = 0.58

P = 0.46

Squeals (number)

PNS Control

191.0 186.2

138.0 151.0

±54.3

P = 0.94

P = 0.41

P = 0.87

PNS Control

12.0 11.9

11.7 13.0

±1.5

P = 0.82

P = 0.74

P = 0.69

Variables Behaviour

Heart rate RMSSD (ms)

These results contradict our original prediction that PNS would make the offspring more aroused during social isolation. Prenatally stressed piglets were indeed less alert than control piglets, suggesting that PNS reduces stress in this test. Evidence supports that both the timing in the gestation period and the type of PNS exposure of the dam may dictate behavioural changes in the offspring (Kranendonk et al., 2006; Weinstock, 2008). Studies that found reductions in activity generally used stress exposure at an earlier stage of gestation (Kofman, 2002), which concurs with our results, whereas PNS in later gestation often results in higher level of activity (e.g. Otten et al., 2007). Yet, increased passive behaviours does not necessarily indicate lower stress. Studies in rodents (Batuev et al., 2000; Lehmann et al., 2000; Thompson, 1957; Vallee et al., 1997) found that PNS offspring showed decreased open-field locomotion but increased fear and anxiety, i.e. a shift to freezing fear response rather than active escape response. Aversive treatment of ewes by humans also results in lambs being more fearful of humans, with fewer vocalizations, less exploration, and a slower approach to the human (Coulon et al., 2011). Prenatal stress has been shown to result in reduced ability to cope with aversive or conflict-inducing situations (for review, see Weinstock, 1997). Prenatally stressed rats demonstrate less flexibility in their behavioural strategy in solving tasks and perseverate in their behaviour (Aleksandrov et al., 2001). In pigs, PNS piglets retreated more when attacked by conspecifics at weaning (Jarvis et al., 2006), and spent more time displaying submissive behaviours (Davis, 2010), with reduced non-aggressive encounters (Kranendonk et al.,

2006), suggesting that PNS alters the pig’s ability to cope with social confrontation. Our results demonstrate that PNS induced by maternal social experience also modifies the piglet’s reaction to social isolation. However, our results are the opposite of Otten et al. (2007), which reported more escape behaviour for piglets from sows treated with ACTH in late gestation. Again, this supports that changes may depend on the timing in gestation and/or the type of PNS exposure. It should be noted that the control sows were kept in gestation crates for their entire gestation, which can induce a chronic stress response (Barnett et al., 1991). Nonetheless, our results showed that the social stress treatment did impose additional stress on the group mixed sows which were housed in gestation crates as well apart from the social stress treatment between days 35 and 56 of gestation. Prenatal stress has been suggested to interfere with the normal development of the autonomic nervous system (Sontag, 1941), but empirical evidence has remained scarce. One earlier paper showed that prenatal hypoxia in rats starting in early gestation alters the development of brain regions that regulate the autonomic nervous system, reducing the maturation of the nucleus tractus solitaries in the medulla oblongata and increasing the maturation of the locus coeruleus in the pons (Peyronnet et al., 2002). Mid-gestation could be a time at which the sow’s foetus is particularly susceptible to maternal hormones (Jarvis et al., 2006; Lay et al., 2008), especially since the HPA axis is still highly reactive compared to the later hyposensitive effect of pregnancy hormones on the HPA axis (Hay et al., 2000; Ponirakis et al., 1998; Windle et al., 2006).

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Intranasal administration has been increasingly used this past decade to study the involvement of various hormones in behaviour and affective states (Bos et al., 2012). The anxiolytic properties of OT are well documented (e.g. Blume et al., 2008; Heinrichs et al., 2003; Kosfeld et al., 2005). We confirmed in a previous experiment that intranasal OT administration successfully lowered distress behaviours and heart rate in piglets (Rault, 2011), using similar methods and subjects and a larger sample size. In the present experiment, OT administration resulted in lower heart rate for control piglets, in agreement with studies in rodents (Grippo et al., 2009; Higa et al., 2002). High endogenous concentrations of OT have also been associated with lower heart rate in women (Light et al., 2005) and OT administration enhanced vagal regulation, hence parasympathetic tone, in rodents and humans (Grippo et al., 2009; Higa et al., 2002). We could not see any effect on the RMSSD in the present experiment, possibly due to the small sample size or the need for more stationary data which could not be obtained considering the distress induced by the isolation test. Oxytocin stimulates vagal regulation by acting on OT receptors in the brain stem, precisely in the dorsal motor nucleus of the vagus (Dreifuss et al., 1988; Higa et al., 2002). Intranasal OT may have acted centrally in our pigs as well, although the data collected here does not allow us to confirm or deny this effect. Nonetheless, there is evidence supporting that intranasal oxytocin application in various species acts directly on the central nervous system rather than through indirect pathways (MacDonald et al., 2011; MeyerLinderberg et al., 2011; Veening et al., 2010). However, OT receptors present on the surface of the heart could also explain autonomic effects through peripheral actions (Gutkowska et al., 1997). Oxytocin administration reversed some behavioural differences shown by PNS piglets. However, it increased rather than lowered arousal as we originally predicted. Intranasal OT reestablished behavioural indicators of vigilance, causing OT-administered PNS piglets to stand more and conversely sit less, more like control piglets. This suggests that OT and PNS effects are somehow linked in their effects on physiological pathways, but result in opposite effects on behaviour. This reversal of PNS behavioural effects could result from a direct effect of OT, possibly through the central amygdala, as seen for PNS rats (Lee et al., 2007). Alternatively, it could be a secondary effect as the OT system is known to interact with other physiological pathways such as the HPA axis (Engelmann et al., 2004), opioid (Kovacs et al., 1998), dopaminergic (Insel, 2003), and serotonergic systems (Emiliano et al., 2007). Indeed, PNS has been shown to alter various components of the HPA axis (Weinstock, 2008), serotonergic (Peters, 1988), and dopaminergic systems (Schneider et al., 1998). Collecting additional physiological measures, such as cortisol concentrations, might help clarify the mechanisms involved. Nonetheless, the OT effects observed in this study offer an interesting parallel with the observations from Jarvis et al. (2006), who noticed changes in later maternal behaviour with PNS piglets becoming more aggressive mothers. These abnormal material behaviours could be related to an effect of PNS on the oxytocinergic system, considering the role

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of OT in regulating maternal behaviour (Ross and Young, 2009). 5. Conclusions These preliminary findings suggest that social stress during mid-gestation alters the behavioural coping strategy and the development of the autonomic nervous system in the pig’s offspring. These behavioural modifications induced by PNS could be reversed to some extent by exogenous OT administration, suggesting a common physiological basis. Acknowledgements and disclaimer The authors thank Mathieu Lardiere for his help in conducting this experiment and analysing the behavioural observations. This research was supported by the United States Department of Agriculture – Agricultural Research Services. This funding agency had no further role in the study design, collection, analysis and interpretation of data, the writing of the report or in the decision to submit the paper for publication. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the United States Department of Agriculture (USDA). USDA prohibits discrimination in all its programmes and activities on the basis of race, colour, national origin, age, disability, and where applicable, sex, marital status, familial status, parental status, religion, sexual orientation, genetic information, political beliefs, reprisal, or because all or part of an individual’s income is derived from any public assistance programme. (Not all prohibited bases apply to all programmes.) Persons with disabilities who require alternative means for communication of programme information (Braille, large print, audiotape, etc.) should contact USDA’s TARGET Centre at (202) 720-2600 (voice and TDD). To file a complaint of discrimination, write to USDA, Director, Office of Civil Rights, 1400 Independence Avenue, S.W., Washington, D.C. 20250-9410, or call (800) 795-3272 (voice) or (202) 720-6382 (TDD). USDA is an equal opportunity provider and employer. References Aleksandrov, A.A., Polyakova, O.N., Batuev, A.S., 2001. The effects of prenatal stress on learning in rats in a Morris maze. Neurosci. Behav. Physiol. 31, 71–74. Amico, J., 2008. Conditional oxytocin receptor knockout mice: targeting the forebrain to understand behavior. Endocrinology 149, 3254–3255. Arey, D.S., 1999. Time course for the formation and disruption of social organisation in group-housed sows. Appl. Anim. Behav. Sci. 62, 199–207. Barnett, J.L., Hemsworth, P.H., Cronin, G.M., Newman, E.A., McCallum, T.H., 1991. Effects of design of individual cage-stalls on the behavioural and physiological responses related to the welfare of pregnant pigs. Appl. Anim. Behav. Sci. 32, 23–33. Batuev, A.S., Polyakova, O.N., Aleksandrov, A.A., 2000. The effect of prenatal “social stress” on anxiety. Zhurnal Vysshei Nervnoi Deyatelnosti Imeni I P Pavlova 50, 281–286. Berntson, G.G., Lozano, D.L., Chen, Y.-J., 2005. Filter properties of root mean square successive difference (RMSSD) for heart rate. Psychophysiology 42 (2), 246–252.

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