Opioid control of behaviour in sheep: Effects of morphine and naloxone on food intake, activity and the affective state

Applied Animal Behaviour Science 142 (2012) 18–29

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

Opioid control of behaviour in sheep: Effects of morphine and naloxone on food intake, activity and the affective state Else Verbeek a,∗ , Drewe Ferguson a , Patrick Quinquet de Monjour a,b , Caroline Lee a a b

CSIRO, Animal Welfare, Locked Bag 1, Armidale, NSW 2350, Australia ParisAgroTech, 16 rue Claude Bernard, 75005 Paris, France

a r t i c l e

i n f o

Article history: Accepted 7 September 2012 Available online 29 September 2012 Keywords: Affective states Arousal Ear postures Opioid Sheep Welfare

a b s t r a c t The affective states of animals are important determinants of welfare, yet they are poorly understood. Here we investigate opioid involvement in the regulation of behaviours that may be indicative of the arousal and valence components of affective states in sheep. Ewes treated with sterile water (C), a low or high dose of the opioid agonist morphine (M1, M2) or opioid antagonist naloxone (N1, N2; n = 8 per treatment) were exposed to a range of different situations, including availability of concentrate feed, grazing in a paddock, novel object test and isolation box test; behavioural indicators, activity and ear postures were assessed. Morphine treated ewes crossed more zones (78.1 ± 5.4 for M1, P = 0.025 and 99.3 ± 5.4 for M2, P = 0.01) compared to C ewes (37.3 ± 5.4) and vocalized more (49.9 ± 6.5 for M1, P < 0.001 and 43.2 ± 6.5 for M2, P = 0.005) compared to C ewes (9.7 ± 6.5) during the novel object test. Morphine treated ewes also attempted to escape more often (3.7 ± 0.6 for M1, P = 0.01 and 4.3 ± 0.6 for M2, P < 0.001) compared to C ewes (0.7 ± 0.6) and showed a higher duration of the backward ear posture (14.9 ± 2.0 s for M1, P = 0.02) compared to C ewes (6.1 ± 2.0 s) during the novel object test. While grazing, morphine treated ewes (M1, P = 0.025 and M2, P < 0.001) also walked more compared to compared to C ewes. Opioid treatment did not affect agitation during the isolation test. Concentrate feed intake (square-root g) was slightly reduced in the N2 ewes (3.6 ± 0.9, P = 0.02) compared to the C ewes (6.6 ± 0.9) at 1.5 h after injection, while morphine had no effect on intake. In conclusion, morphine had a major impact on activity suggesting that the opioid system may be involved in regulating the arousal component of affective states in sheep. Morphine may have reduced the negative experience in the novel object test as indicated by the higher duration of the backward ear posture; however, further investigation is needed to determine the impact of opioid administration on emotional valence. © 2012 Elsevier B.V. All rights reserved.

1. Introduction It has been accepted for some time that (mammalian non-human) animals can experience basic emotions such as fear and distress (Broom, 1991; Cockram, 2004; Fraser, 1984). More recently, there is emerging evidence that

∗ Corresponding author. Tel.: +61 267761347. E-mail addresses: [email protected], [email protected] (E. Verbeek). 0168-1591/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.applanim.2012.09.001

animals are able to experience a range of other higher order emotions (Burgdorf and Panksepp, 2006; Panksepp, 2005), also called affective states. The affective state of the animal is likely to have a large impact on its welfare (Fraser, 2009; Paul et al., 2005), yet animal affective states are difficult to assess due to their subjectivity and hence are poorly understood. A theoretical framework of animal emotion proposed by Mendl et al. (2010) describes affective states being represented in a two-dimensional space, with one axis representing emotional valence, ranging from pleasant (positive) to unpleasant (negative), and the other axis

E. Verbeek et al. / Applied Animal Behaviour Science 142 (2012) 18–29

representing arousal, ranging from high to low. Affective states are often accompanied by behavioural, physiological and cognitive reactions (Paul et al., 2005; Reefmann et al., 2009b), and such reactions can potentially be used as indicators of specific affective states. For example, locomotor activity and the number of vocalizations may be indicative of arousal (Forkman et al., 2007). Few measures of valence are available, although recent evidence suggests that ear postures are potential indicators of the valence of affective states in sheep (Boissy et al., 2011; Reefmann et al., 2009a, 2009b). In addition, the amount of food consumed, or approach/avoidance behaviour of a particular object can be used to assess the palatability or valence of a resource or object (e.g., see Doyle et al., 1993; Romeyer and Bouissou, 1992; Woods and Leibowitz, 1985). Pharmacological approaches could be useful in obtaining a better understanding of animal affective states and their neurophysiological control (e.g., see Donald et al., 2011; Doyle et al., 2011; Hymel and Sufka, 2012). While pharmacological approaches have been widely used to assess the efficacy of human anti-depressants and anxiolytics in rodents (Prut and Belzung, 2003), their application in animal welfare science is relatively new. The opioid system may be involved in the regulation of animal affective states. Opioid receptor subtypes (␮, ␦, and ␬) are widely distributed throughout the sheep brain (Dunlap et al., 1986) and bind endogenous ligands (e.g., endorphins, dynorphins, etc.) as well as exogenous ligands (e.g., morphine, heroin, etc.). Opioid receptors are involved in the regulation of pleasurable (hedonic) experiences and have been extensively studied in the context of food intake in rodents, monkeys and humans (Berridge, 2003, ˜ and Berridge, 2000). Opioid ˜ 2008; Pecina 2009; Pecina, agonists of the ␮-receptor such as morphine enhance behavioural expressions of pleasure following the consumption of a pleasantly sweet sucrose solution and reduce aversive reactions after consuming unpalatable bitter food (Berridge, 2004; Cagniard and Murphy, 2009; Doyle et al., 1993; Rideout and Parker, 1996). Furthermore, ␮-opioid agonist administration increases food intake in rats (Woods and Leibowitz, 1985) and sheep (Obese et al., 2007). In contrast, ␮-opioid antagonist administration (naloxone) decreases food intake in sheep (Alavi et al., 1991, 1993). Therefore, opioid agonists may play a role in enhancing the rewarding properties of food, while opioid antagonists have the opposite effect. Opioids may also be involved in the regulation of other affective behaviours, such as social behaviours and behavioural responses to stress and fear (Kalin et al., 1988; Keverne et al., 1989; Sanders et al., 2005; Sasaki et al., 2002; Van den Berg et al., 1999; Vanderschuren et al., 1996; Yamada and Nabeshima, 1995) and maternal behaviour in sheep (Caba et al., 1995; Keverne and Kendrick, 1991). A number of behavioural tests have been designed to induce specific behavioural reactions in sheep, and these may be useful in studying the impact of opioid administration on behaviour and affective states in sheep. A novel object/arena test is commonly used to induce behavioural reactions indicative of fear (Desire et al., 2004; Romeyer and Bouissou, 1992) while social separation tests have been designed to induce isolation distress behaviours

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(Hernandez et al., 2010; Parrott et al., 1988; Price and Thos, 1980). These tests, in particular the arena/novel object test, have been extensively used and validated in sheep, and give reasonably repeatable and consistent results (Forkman et al., 2007). The advantage of using such tests as a first step to assess the impact of opioid administration on behaviour, is that typical behavioural reactions induced by these tests are well defined (e.g., changes in locomotor activity, vocalization, urination, defecation, escape attempts, etc.). In addition, the consumption of palatable concentrate food may induce a mildly positive affective state and may also be modified by opioid administration. A group grazing situation may be useful to assess more general effects of the opioids on locomotor activity while sheep are not being stimulated (most likely to be a neutral or mildly positive situation). We hypothesize that administration of morphine, an opioid receptor agonist, and naloxone, an opioid receptor antagonist, will alter behavioural reaction across a range of different situations in sheep; morphine is expected to increase and naloxone to reduce food intake; morphine is expected to reduce and naloxone to enhance behavioural distress during arena/novel object and isolation tests and it is expected that the effect of opioid administration on locomotor activity when animals are grazing will be minimal (i.e., animals will not show any signs of sedation or apathy). In addition, we aim to assess ear postures across some of the different situations described. The behavioural changes induced by opioid administration may be indicative of shifts in the arousal and/or valence components of affective states. We expect that assessing the effects of opioid administration on behavioural reactions during different situations will contribute to a better understanding of opioid regulation of animal behaviour and affective states. 2. Methods 2.1. Ethical note This study was approved by the Armidale Animal Ethics Committee (Animal Research Authority #11-05). All animals were closely monitored during and after the experiments. No long-term effects on animal health and welfare were observed. 2.2. Animals and housing Forty merino ewe lambs (7–9 months of age) with a mean Body Weight (BW) of 27.5 ± 0.4 kg and a body condition score (BCS, scored on a scale of 1–5: Russel et al., 1969) of 2.6 ± 0.02 were used in this experiment. The ewes were born on the same experimental farm and reared in the same group after weaning. In the initial phase of the experiment, all ewes were grazed on pasture and supplemented with concentrate sheep pellets (11.9 MJ/kg DM containing wheat, lucerne, pollard, bran and salt with 22% crude protein) and oaten chaff (8.9 MJ/kg DM with 11% crude protein). Nutritional supplementation was slowly increased over a 3-week period. Following the dietary habituation period, ewes were housed indoors in individual

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pens (1 m × 1 m) and were given another 11 days to habituate to the indoor conditions. Sheep were fed twice a day during the indoor housing period (total 700 g pellets and 300 g of chaff per sheep), with the first feed between 8:30 and 10:00 h and the second between 15:00 and 16:30 h. Ten wide-angle video cameras (SCC-B2315P, Samsung, Seoul, South Korea) were placed above the pens and each camera recorded data continuously from four sheep (16 Channel Digital video recorder, PACOM, Port Melbourne, Victoria, Australia). After completion of the food intake test (see below) sheep were returned to pasture and grazed as one group. They continued to receive the pellets at 500 g per sheep/day while undergoing further testing (see below). 2.3. Treatments and opioid drugs An opioid agonist (morphine sulphate, Hospira, Lake Forest, IL, USA) and an opioid antagonist (naloxone hydrochloride, Tocris, Bristol, UK) were used in the experiment. The drugs were dissolved in sterile water to a concentration of 10 mg/mL and were administered intravenously (jugular vein). Ewes were randomly divided into five different treatments, balanced for body weight and BCS: low dose morphine (M1), high dose morphine (M2), low dose naloxone (N1), high dose naloxone (N2) and a sterile water control (C), with eight ewes per treatment. Drug dosages used during the feed intake test (see below) were 0.25 mg/kg for M1, 0.75 mg/kg for M2, 0.5 mg/kg for N1 and 1.5 mg/kg for N2. Because the effects of the opioid drugs on food intake were minor, the doses were increased to 0.75 mg/kg for M1, 1.0 mg/kg for M2, 1.5 mg/kg for N1 and 2.0 mg/kg for N2 for the novel object, isolation and activity tests (see below). In sheep, the half-life of morphine is approximately 119 min in blood plasma, and about 320 min in the interstitial fluid in the cortex, the clearance rate is about 34.3 mL min−1 kg−1 (Bengtsson et al., 2009). The half-life of naloxone is approximately 43 min in blood plasma and the clearance rate is about 90 mL min−1 kg−1 (Alavi et al., 1994). 2.4. Food intake test The food intake test was repeated twice, once without opioid administration (baseline test) and 4 days later with opioid administration. All uneaten food was removed at 16:30 h on the day before the experiment. All sheep were injected by 0 h (corresponding to 8:21 h for the baseline test and 8:36 h for the opioid test) and were left undisturbed for 45 min in order to record ear postures, see below. After 45 min, they received 1000 g of concentrate pellets and the weight of any food remains (g) was recorded at 1, 1.5, 2, 3, 4, 8, 12 and 24 h after opioid administration. Fresh concentrate pellets were provided at each measuring point (500 g for short measuring periods of less than 1 h and 1000 g for longer periods). No oaten chaff was provided during the feed intake test.

handling yards on the morning of the test. IceTags (3D, IceRobotics Ltd, Edinburgh, UK) were attached just below the knee on the left front leg and the opioid drugs were administrated at the same time. After administration of the drugs, sheep were immediately released into a paddock and were left undisturbed. The IceTags recorded the number of steps/min and the percentage of time spent standing. Sheep were observed closely during the activity test and no discomfort caused by the IceTags was observed during the period of data collection. 2.6. Novel object and isolation box tests The five treatment groups were subdivided into two groups for testing on two separate days as a maximum of 20 ewes could be tested per day. The tests took place 5 or 6 days after the activity test, respectively. The opioid drugs were administered 10 min before the start of testing. Half the sheep were pseudo-randomly assigned (balanced for treatment) to perform the isolation test first, directly followed by the novel object test, and the other half to perform the novel object first, directly followed by the isolation test. Both tests were completed within 10 min and sheep only received one injection. The 4 m × 4 m novel object arena was located on a concrete floor surrounded by a fence covered with opaque mesh. Prior to the start of the study, sheep were habituated to the arena on three separate occasions (without the novel object) for 10 min in groups of three. The novel object was an unfamiliar orange traffic cone that was placed in the centre of the arena. The arena was divided into four circular zones (indicated by white paint on the floor). All sheep were tested individually for 5 min, and latency to approach the object, number of zones crossed, vocalizations, defecations and urinations were recorded by two motionless persons located near the arena. A sheep was considered to have crossed a zone when the two front legs had crossed the white line. The tests were also video recorded with a handicam (Sony HDR-XR550, Sony corporation, Tokyo, Japan) and the number of jumps (all four legs off the ground), the number of rearings (two front legs off the ground) and the number of escape preparations (defined as a small and abrupt “hop” movement with the hindquarters placed under the body) were observed from the video recordings. The total number of escape attempts (the sum of the number of jumps, rearings, and escape preparations) was also calculated. Located next to the novel object arena was a wooden isolation box measuring 1.5 m × 0.5 m × 2 m. Sheep were isolated for 30 s and agitation was measured by an electronic meter (custom built on site), which generated a numerical value of the sheep’s agitation based on vibrations from movement and vocalizations in the box (Bickell et al., 2009). The number of vocalizations was also recorded separately. 2.7. Ear postures

2.5. Activity test The activity test took place 17 days after the feed intake test. Sheep were taken from the paddock to familiar sheep

Specific ear postures were analyzed from video recordings taken during the food intake and novel object tests at the following time points: (I) 15 min after drug

E. Verbeek et al. / Applied Animal Behaviour Science 142 (2012) 18–29

administration during the food intake test while no food was available (before feeding), (II) starting immediately when sheep placed their head in the feeding troughs after receiving food, about 30 min after the first observations (during feeding), (III) 30 s in the first min and 30 s in the third min after the start of the novel object test. All ear postures were continuously recorded until a total of 60 s was obtained in which both ears could be clearly seen. Because the ear postures changed very quickly during the novel object test (up to three posture changes/s), every ewe was observed by at least two different persons and the different observations were compared. In case of disagreement, the particular video data were observed again by all observers until a final posture was agreed upon. Only one person observed the ear postures before and during feeding, when the ear postures did not change as often. The following ear postures were recorded based on the postures identified by Reefmann et al. (2009a, 2009b): (I) Neutral ear posture (both ears perpendicular to the head–rump axis). (II) Forward ear posture (tips of both ears towards the front at an angle of more than 30◦ from the perpendicular). (III) Backward ear posture (tips of both ears towards the back at more than 30◦ from the perpendicular). (IV) Asymmetrical ear posture (one tip of the ear towards the front at an angle of more than 30◦ from the perpendicular and the other tip of the ear towards the back at more than 30◦ from the perpendicular). The total duration (s) of each posture was recorded.

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number of s standing was then averaged in 30 min blocks and analyzed by repeated measures analysis to test for differences between treatments with ewe as a random effect. Specific ear posture durations were analyzed by REML. For the novel object test, treatment, the testing date, order of testing and time (min 1 and min 3) were included as fixed effects and ewe as a random effect. The N posture was almost never observed during the novel object test, and was therefore not analyzed. For the feed intake test, treatment and situation (before or after feeding) were included as fixed effects and ewe as a random effect. All post hoc analyses were conducted using orthogonal contrasts (Bonferroni corrected). 3. Results 3.1. Food intake Baseline food intake levels were not different between the treatments (Fig. 1A). After opioid administration, there was a significant time × opioid treatment interaction for food intake (F(32,274) = 2.87, P < 0.001, Fig. 1B). The ANOVAs showed that there were significant opioid treatment effects at 1.5 h (F(4,34) = 4.52, P = 0.005) and 2 h after injection (F(4,34) = 3.07, P = 0.029); the N2 ewes ate less food at 1.5 h (F(1,34) = 9.48, P = 0.02) and tended to eat less at 2 h after injection (F(1,34) = 4.01, P = 0.1) compared to C ewes. At 24 h, intake in the M2 ewes tended to be reduced (F(1,34) = 6.53, P = 0.075). The total daily food intake tended to be affected by opioid treatment (F(4,34) = 2.35, P = 0.074), mostly due to the reduced intake in the morphine treated ewes.

2.8. Statistical analysis 3.2. Novel object test Data are presented as mean ± sem, unless stated otherwise. Statistical procedures were carried out in GenStat 13 (VSN International Lt, Hemel Hempstead, Hertfordshire, UK) and all data were checked for normality (Shapiro–Wilk test) and transformed where necessary (square root transformation: food intake test: all data; novel object test: urinations, escape attempts; activity test: number of steps; isolation test: number of vocalizations; ear postures: asymmetrical, forward and neutral postures during the feed intake test). Data from the food intake test were analyzed by repeated measures analysis to test for differences between opioid treatments with ewe as a random effect. The total food intake from the baseline test was included as a covariate. Additionally, individual time points were analyzed by ANOVA with the corresponding baseline value as a covariate. Data from the novel object test were analyzed by ANOVA for differences between treatments, testing days and order of specific tests with ewe as a random effect. Data from the isolation box test were analyzed by ANOVA for differences between treatments, testing days and order of specific tests with ewe as a random effect. Data from the first 6 h after opioid administration were used for the analysis of the activity test. The number of steps recorded in the activity test was analyzed by repeated measures for differences between treatments with ewe as a random effect. The IceTag data on percentage of time spent standing was first converted to number of s spent standing per min. The

The latency to approach the novel object was not affected by treatment (Table 1). The total number of zones crossed were increased in M1 (F(1,29) = 9.46, P = 0.025) and M2 ewes (F(1,29) = 21.48, P = 0.01) compared to C ewes, while observations for N1 and N2 ewes were not different from C ewes (Table 1). Test order also affected the number of zones crossed (F(1,24) = 4.39, P = 0.047) with ewes that performed the isolation test first crossing more zones during the novel object test (76 ± 11 zones crossed) compared to ewes that performed the novel object test first (55 ± 7 zones crossed). The test day did not affect the number of zones crossed. The number of vocalizations was higher in the M1 (F(1,29) = 19.29, P < 0.001) and M2 ewes (F(1,29) = 13.38, P = 0.005) than C ewes while there was no difference between N1, N2 and C ewes (Table 1). Test order and day did not affect the number of vocalizations. The number of defecations was lower in M1 (F(1,28) = 33.48, P < 0.001) and M2 ewes (F(1,28) = 25.76, P < 0.001) compared to C ewes (Table 1). The N1 and N2 ewes were not different from C ewes. Test day also affected the number of defecations (F(1,28) = 4.76, P = 0.038), with ewes tested on the first day defecating more (1.8 ± 0.4) than on the second day (1.2 ± 0.3). The urinations tended to be less in N2 ewes than in C ewes (F(1,28) = 6.15, P = 0.095, Table 1). Animals tested

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E. Verbeek et al. / Applied Animal Behaviour Science 142 (2012) 18–29

Fig. 1. The effects of opioid treatment on square root transformed mean ± sem food intake for (A) baseline food intake and (B) food intake after opioid treatment, T × T*** indicates a significant time × opioid treatment interaction (P < 0.001), T indicates a treatment difference at individual time points (T** P < 0.01 and T* P < 0.05). *Indicates a difference between treatment and control at individual time points (P < 0.05), + indicates a tendency for a difference between treatment and control at individual time points (P < 0.1). Footnote: M1, 0.25 mg/kg morphine; M2, 0.75 mg/kg morphine; N1, 1.0 mg/kg naloxone; N2, 1.5 mg/kg naloxone; C, control.

on the first day tended to urinate more (square-root transformed mean ± sem (back-transformed mean): 0.7 ± 0.2 (0.5)) compared to ewes tested on the second day (0.4 ± 0.2 (0.1); F(1,28) = 3.95, P = 0.057). Test order did not affect the number of defecations and urinations. The total number of escape attempts was higher in M1 (F(1,28) = 12.12, P = 0.01) and M2 ewes (F(1,28) = 16.98, P < 0.001) compared to C ewes (Table 1). M2 ewes also reared more often (F(1,28) = 16.66, P < 0.001) and prepared more often to escape (F(1,28) = 16.98, P < 0.001) than C ewes. M1 ewes tended to rear more often (F(1,28) = 7, P = 0.065) and prepared more often to escape (F(1,28) = 12.12, P = 0.01) than C ewes (Table 1). The number of jumps was not affected by opioid treatment (Table 1). There was no difference in escape behaviours between N1, N2 and C ewes. The number of rearings was higher on testing day 1 (square-root transformed mean ± sem (back-transformed mean): 1.6 ± 0.4 (2.4); F(1,28) = 5.34, P = 0.028) than on day 2 (0.7 ± 0.4 (0.5)), while the other escape behaviours were

not affected by day. Test order did not affect any of the escape behaviours. 3.3. Isolation box test The agitation score was not affected by opioid treatment (Table 2). However, there was an effect of opioid treatment on the number of vocalizations (F(4,34) = 2.81, P = 0.041, Table 2), with the M1 ewes (F(1,34) = 6.32, P = 0.085) tending to vocalize more than C ewes. There was no effect of test order or day on the score or the number of vocalizations. 3.4. Activity The number of steps were affected by a time × opioid treatment interaction between 1 and 360 min (F(44,385) = 2.75, P < 0.001), mostly because the M2 ewes walked a greater number of steps compared to C ewes (F(1,35) = 30.31, P < 0.001, Fig. 2A). M1 ewes did not walk more than C ewes over the 360 min period, but walked

Significance

ns P < 0.001 P < 0.001 P < 0.001 P = 0.019 ns P < 0.001 P < 0.001 P < 0.001

F

F(4,19) = 1.3 F(4,24) = 27.08 F(4,29) = 7.44 F(4,28) = 15.98 F(4,28) = 3.53 F(4,27) = 1.62 F(4,29) = 7.16 F(4,28) = 7.68 F(4,28) = 8.91

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more than C ewes between 0 and 180 min (F(1,35) = 5.2, P = 0.025). The naloxone treated ewes were not different from C ewes. The time spent standing between 1 and 360 min was affected by a time × opioid treatment interaction (F(44,376) = 3.6, P < 0.001, Fig. 2B), mostly because the M2 ewes spent the majority of the time standing while the other ewes lay down for some periods. The M1 ewes showed a significant reduction in standing time compared to C ewes at 180 min (F(1,34) = 15.9, P < 0.001) and 210 min (F(1,34) = 15.01, P < 0.001).

13.3 99.3 43.2 0.4 0.3 0.9 10.0 2.8 4.3 7.8 51.4 18.3 2.7 1.0 0.7 2.3 0.5 1.6

ns, no significant treatment differences. a M1, 0.75 mg/kg morphine; M2, 1.0 mg/kg morphine; N1, 1.5 mg/kg naloxone; N2, 2.0 mg/kg naloxone; C, control. * P < 0.05 for differences between opioid treated and control ewes. ** P < 0.01 for differences between opioid treated and control ewes. *** P < 0.001 for differences between opioid treated and control ewes. + P < 0.1 for differences between opioid treated and control ewes.

1.4 78.1 49.9 0.0 0.4 1.0 8.5 1.8 3.7 7.0 37.3 9.7 2.8 0.8 0.2 0.3 0.3 0.7 8.4 34.4 17.7 1.8 0.2 0.2 1.3 0.2 0.9

N1

Latency to approach (s) Zones crossed Vocalizations Defecations Urinations Jumps Jump preparations Rearings Total escape attempts

± ± ± ± ± ± ± ± ±

5.3 5.4 6.5 0.3 0.2 (0.9) 0.3 (0.4) 1.7 0.4 (0.3) 0.6 (2.5)

N2

± ± ± ± ± ± ± ± ±

3.4 5.4 6.5 0.3 0.2 (0.0)+ 0.3 (0.1) 1.7 0.4 (0.0) 0.6 (0.8)

C

± ± ± ± ± ± ± ± ±

1.8 5.4 6.5 0.3 0.2 (0.7) 0.3 (0) 1.7 0.4 (0.1) 0.6 (0.4)

M1

± ± ± ± ± ± ± ± ±

0.8 5.4* 6.5** 0.3*** 0.2 (0.1) 0.3 (1.1) 1.7** 0.4 (3.3)+ 0.6 (13.4)**

M2

± ± ± ± ± ± ± ± ±

4.9 5.4*** 6.5* 0.3*** 0.2 (0.1) 0.3 (0.8) 1.7*** 0.4 (8.1)*** 0.6 (18.4)***

3.5. Ear postures

Variable

Table 1 The effects of opioid treatmenta on mean ± sem variables in the novel object test. Data on urinations, jumps, rearings and total escape attempts are square-root transformed mean ± sem (back-transformed mean).

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During the novel object test, the duration of the backward ear posture was higher in the M1 (F(1,34.6) = 9.79, P = 0.02) and tended to be higher in the M2 ewes (F(1,35.9) = 5.69, P = 0.1) compared to C ewes, while the naloxone treated ewes were not different from C ewes (Table 3). There was also a treatment effect on the duration of the forward ear posture (F(4,31) = 3.22, P = 0.025), mostly due to the M1 ewes (F(1,35.1) = 5.63, P = 0.1) and M2 (F(1,35.8) = 5.29, P = 0.1) ewes tending to have a lower duration compared to C ewes, while there was no difference between naloxone treated and C ewes. Test order, day and minute of observation did not affect the ear postures. During the feed intake test, treatment tended to affect the backward ear posture (F(4,34.1) = 2.59, P = 0.054), mostly due to the higher duration of the backward ear posture in the M1 and M2 ewes (Table 3). The duration of the backward ear posture also tended to be higher after feeding than before feeding (F(1,33.7) = 2.93, P = 0.096). There was no time × treatment interaction for the backward ear posture. The forward ear posture tended to be affected by a time × treatment interaction (F(4,32.9) = 2.25, P = 0.085), mostly due to the C, N2 and M1 ewes having a higher duration after feeding than before feeding and the M2 and N1 ewes having a higher duration before feeding than after feeding. 4. Discussion In this study, we assessed the effects of morphine, an agonist of the opioid receptor, and naloxone, an antagonist of the opioid receptor, on behaviour during a range of different situations. The effects of morphine and naloxone on behaviour are summarized in Table 4. Morphine induced high levels of locomotor activity and increased vocalizations, suggesting that morphine had an impact on the arousal component of the framework of animal emotions by Mendl et al. (2010). Furthermore, morphine induced a higher duration of the backward ear posture during the novel object test. Because the backward ear posture has mostly been observed in neutral and positive valence situations (Reefmann et al., 2009a, 2009b), the higher duration during a situation designed to induce fear suggests that the morphine treated ewes may have perceived the novel object test as less negative than control and naloxone treated ewes. Morphine did not enhance food intake or alter the ear postures after animals received food, suggesting that morphine did not increase the palatability of food.

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Table 2 The effects of opioid treatmenta on mean ± sem agitation score and number of vocalizations during the isolation box test. Data on vocalizations are square-root transformed mean ± sem (back-transformed mean). Variable

N1

N2

C

M1

M2

F

Significance

Agitation score Vocalizations

59 ± 10 0.4 ± 0.2 (0.1)

49 ± 9 0.7 ± 0.377 (0.5)

53 ± 11 0.4 ± 0.3 (0.2)

75 ± 10 1.5 ± 0.4 (2.1)+

59 ± 10 1.1 ± 0.3 (1.3)

F(4,31) = 1.08 F(4,34) = 2.81

ns P = 0.041

ns, no significant treatment differences. a M1, 0.75 mg/kg morphine; M2, 1.0 mg/kg morphine; N1, 1.5 mg/kg naloxone; N2, 2.0 mg/kg naloxone; C, control. + P < 0.1 for differences between opioid treated and control ewes. Table 3 Mean ± sem duration of ear postures (s) during the novel object test and food intake test, with A = asymmetrical, B = backward, F = forward, and N = neutral ear postures. Data on the asymmetrical, forward and neutral postures during the feed intake test are square-root transformed mean ± sem (back-transformed mean). Test

Factor

Ear postures A

B

F

N

N1 N2 C M1 M2

3.6 ± 0.8 2.8 ± 0.9 5.6 ± 0.8 3.4 ± 0.8 3.6 ± 0.8 F(4,32.9) = 1.52 ns

10.1 ± 2.0 8.3 ± 2.1 6.1 ± 2.0 14.9 ± 2.0* 12.9 ± 2.1+ F(4,31.1) = 3.06 P = 0.031

16.4 ± 1.9 18.8 ± 1.9 18.4 ± 1.9 11.8 ± 1.9+ 11.8 ± 2.0+ F(4,31) = 3.22 P = 0.025

N1 N2 C M1 M2

0.9 ± 0.3 (0.9) 1.1 ± 0.3 (1.1) 1.2 ± 0.3 (1.4) 1.0 ± 0.3 (1.0) 0.6 ± 0.3 (0.4) F(4,34.9) = 0.78 ns

44.9 ± 3.5 46.7 ± 3.5 39.6 ± 3.5 51.8 ± 3.9 54 ± 3.6 F(4,34.1) = 2.59 P = 0.054

2.7 ± 0.5 (7.5) 2.5 ± 0.5 (6.2) 2.6 ± 0.5 (6.7) 1.5 ± 0.5 (2.3) 1.6 ± 0.5 (2.4) F(4,33) = 1.49 ns

0.8 ± 0.3 (0.7) 0.3 ± 0.3 (0.1) 1.2 ± 0.3 (1.4) 0.6 ± 0.3 (0.4) 0.3 ± 0.3 (0.1) F(4,33.9) = 2.04 ns

Before food After food

1 ± 0.2 (1.0) 0.9 ± 0.2 (0.8) F(1,34.8) = 0.2 ns

45.2 ± 2.1 49.6 ± 2.1 F(1,33.7) = 2.93 P = 0.096

2.1 ± 0.3 (4.3) 2.3 ± 0.3 (5.2) F(1,32.8) = 0.14 ns

0.8 ± 0.2 (0.6) 0.5 ± 0.1 (0.3) F(1,33.3) = 2.31 ns

F(4,34.9) = 0.78 ns

F(4,33.8) = 0.93 ns

F(4,32.9) = 2.25 P = 0.084

F(4,33.3) = 1.13 ns

Novel object testa

F Significance Food intake testb

F Significance

F Significance Treatment × time interaction F Significance

ns indicates no significant treatment differences. a Novel object test: durations are the mean of the two 30 s blocks (min 1 and min 3). Opioid doses: M1, 0.75 mg/kg morphine; M2, 1.0 mg/kg morphine; N1, 1.5 mg/kg naloxone; N2, 2.0 mg/kg naloxone; C, control. b Food intake test: durations are the mean of the 60 s blocks. Opioid doses: M1, 0.25 mg/kg morphine; M2, 0.75 mg/kg morphine; N1, 1.0 mg/kg naloxone; N2, 1.5 mg/kg naloxone; C, control. * Significantly different from control (P < 0.05). + Tended to be significantly different from control (P < 0.1). Table 4 Summary of the effects of morphine and naloxone on behavioural indicators of affective states across the different situations. Situation Food intake test Food intake Ear postures Novel object test Locomotor activity Vocalizations Escape behaviour Defecations Urinations Ear postures Isolation test Agitation score Vocalizations Activity test Locomotor activity

Morphine

Naloxone

No effect early in experiment, tended to be reduced at 24 h Backward ear posture tended to be increased

Reduction at 1.5 h, tended to be reduced at 2 h No effect

Increased Increased Increased Decreased No effect Increased backward ear posture

No effect No effect No effect No effect Tended to be reduced No effect

No effect Tended to be increased

No effect No effect

Increased

No effect

E. Verbeek et al. / Applied Animal Behaviour Science 142 (2012) 18–29

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Fig. 2. The effects of opioid treatment on (A) square root transformed mean ± sem number of steps and (B) mean ± sem time spent standing (min) during the activity test, T × T*** indicates a significant time × opioid treatment interaction (P < 0.001). Footnote: M1, 0.75 mg/kg morphine; M2, 1.0 mg/kg morphine; N1, 1.5 mg/kg naloxone; N2, 2.0 mg/kg naloxone; C, sterile water control.

Naloxone had little effect on behaviour across the different situations, except for a small reduction in food intake suggesting that it may have slightly reduced hunger or the palatability of the food. 4.1. Food intake A reduction in absolute intake at 1.5 h after naloxone injection was found, and there tended to be a reduction at 2 h after naloxone injection. However, the total amount of food eaten in those measurement periods was relatively small. A study by Alavi et al. (1991) showed that sheep treated with 0.3 and 1 mg/kg naloxone showed major reductions in cumulative intake between 2 and 12 h after injection. Baile et al. (1981) also found small reductions in intake in sheep treated with 0.125 mg/kg naloxone. Our results are therefore in agreement with other studies, although the reductions observed in the current study were much smaller compared to previous reports (Alavi et al., 1991; Baile et al., 1981). Morphine administration did not affect food intake in the current study, which is in contrast to studies showing that morphine enhances intake in rats (Doyle et al., 1993; Woods and Leibowitz, 1985). In sheep, SD33 (a ␮-opioid receptor agonist) increases intake of concentrate feed, although this effect was only seen after 24 h (Obese et al., 2007). It has been suggested that

morphine mainly enhances the intake of palatable food as opposed to non-preferred foods (Berridge, 1991; Zhang and Kelley, 2000) although others have rejected this hypothesis (Olszewski et al., 2011). From experience we know that sheep consider the concentrate feed highly palatable and therefore the palatability of the diet is unlikely to explain these results. At 24 h, there tended to be a reduction in food intake in the morphine treated ewes leading to a tendency of reduced total 24 h food intake in the M2 ewes. The reduction in total 24 h intake in the M2 ewes is in agreement with a study in which rats were implanted with slow release morphine pellets leading to a dramatic decrease in intake after 1 day (Ferenczi et al., 2010). Some studies have also reported disorganized feeding behaviour and altered rhythmicity of feeding after opioid administration in rats, even shortly after administration (Chen et al., 2006; Ferenczi et al., 2010). Similar changes in feeding behaviour could have occurred in the morphine treated sheep in our study (although this was not measured) and could potentially have caused the reduction in intake. 4.2. Novel object test, isolation test and activity test All animals approached and sniffed the object during the novel object test, suggesting that the object was not perceived as particularly fearful. However, the

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combination of the relatively unfamiliar arena and social isolation would also have contributed to the fearfulness of the test (Forkman et al., 2007). The number of defecations was lower in the morphine treated animals, which may indicate reduced fear (Romeyer and Bouissou, 1992). However, the reduction in defecations could also have been caused by the reduced rumen motility due to morphine administration (Kania, 1994). The morphine treated animals had higher levels of locomotor activity, as measured by the total number of zones crossed. Generally, increased activity in arena or novel object tests is considered to be an indication of increased fear or distress in sheep (Forkman et al., 2007; Romeyer and Bouissou, 1992), and the increased activity would be contrary to the prediction that morphine would reduce behavioural distress. However, increased locomotor activity was also observed while sheep were grazing as a group in a familiar paddock. It is unlikely that morphine would have induced distress, resulting in increased activity, in the absence of a fearful stimulus (Hanks et al., 1995; O’Neill et al., 2000), although it cannot be completely ruled out. Other studies in rats and mice also found that morphine enhanced locomotor activity in a neutral situation (Deroche et al., 1993; Joyce and Iversen, 1979) and this effect was further enhanced during social isolation (Deroche et al., 1994, 1995; Francès et al., 2000). Our activity measures did not allow us to directly compare activity during grazing and the novel object test, but activity could potentially have been higher during the novel object test because it is likely to have induced a higher level of distress compared to the grazing situation (Francès et al., 2000). Therefore, morphine may increase general arousal resulting in increased activity independent of the situation. Activity in morphine treated animals could potentially be further increased when exposed to distress or fear, although whether this also occurs in sheep needs further investigation. It has been suggested that the locomotor effect induced by morphine is not a simple reflexive motor reaction, but is related to morphine’s potential as a positive reinforcer (Wise and Bozarth, 1987). Morphine activates dopamine neurons by disinhibition of ␮-receptor containing GABAergic interneurons, leading to increased dopamine release in the nucleus accumbens (Johnson and North, 1992; Nowycky et al., 1978; Trulson and Arasteh, 1985). Dopamine plays a major role in motivational and goal directed behaviour (Bromberg-Martin et al., 2010; Schultz, 1997) and may play a role in reward expectation (Schultz, 2002). Morphine may amplify the existing dopamine responses to natural rewards and rewardrelated environments, and more hypothetically, may even induce illusionary rewards (Schultz, 2002). Therefore, the increased locomotor activity after morphine administration is most likely mediated by dopaminergic pathways, although contradicting data has also been reported in mice (Murphy et al., 2001). We hypothesize that the increased activity in the morphine treated ewes is an expression of increased goal directed or approach behaviour. Therefore, the increased activity and escape attempts during the novel object test in the morphine treated ewes may reflect increased goal directed behaviour stimulating the ewes to look more actively for an escape from their negative

situation, while the increased activity in the paddock could be explained by the animals searching for rewards (e.g., social, nutritional or other types of rewards). The isolation distress agitation score measured by the isolation box test was not different between treatments, suggesting that the opioid drugs did not have an effect on isolation distress behaviours. This is contradictory to the increased activity found in the novel object test that also has an isolation distress component. However, sheep in the isolation test were confined and do not have much room for movement, which could have affected the results. There is some evidence that opioid drugs do affect separation distress in sheep; plasma cortisol concentrations increased during social isolation in all sheep, but cortisol was lower and increased for a shorter duration in sheep treated with morphine compared to control and naloxone treated sheep (Parrott and Thornton, 1989). Naloxone administration increased plasma cortisol concentrations even when sheep were not socially isolated (group housed) (Parrott and Thornton, 1989). However, naloxone failed to alter the behavioural and cortisol responses to social isolation in cattle (Rushen et al., 1999). Therefore, the effect of opioid drugs on separation distress behaviours is still unclear. Cortisol was not measured in the current study but may have provided further insight into stress responses in opioid treated animals during social isolation. Naloxone had no impact on any behavioural indicators measured during the novel object and isolation tests. This is consistent with other studies reporting that naloxone mostly reduces positive behavioural reactions derived from positively valenced experiences, such as the consumption of palatable food (Giraudo et al., 1993; Glass et al., 1996), while it is still unclear whether naloxone alone can augment negative states, such as fear and distress (Davis, 1979; Glover and Davis, 2008; Rushen et al., 1995). However, another explanation for the lack of effect of naloxone is that it may not have had a high biological activity or may not have bound to the opioid receptors in the sheep brain. There are also other indicators of arousal available (e.g., heart rate or plasma cortisol concentrations) that were not measured in this study but that could potentially provide more insight into the effects of naloxone administration on arousal. 4.3. Vocalizations Morphine increased the number of vocalizations in the novel object test and tended to increase vocalizations in the isolation test. Vocalizations are used to communicate “needs” and possibly emotions between individuals and may also function as a warning to conspecifics during fear eliciting situations (Manteuffel et al., 2004). A higher proportion of sheep vocalized when placed in a novel arena without conspecifics compared to a novel arena in which conspecifics were present (Ligout et al., 2011), suggesting that vocalizations expressed during social isolation may signal the need for social reunion. If morphine indeed enhances goal directed and motivational behaviours (Schultz, 2002; Wise and Bozarth, 1987), it is likely that the increased vocalizations were representative of an increased motivation for social reunion.

E. Verbeek et al. / Applied Animal Behaviour Science 142 (2012) 18–29

However, a number of other studies have found that morphine reduced, rather than enhanced distress vocalizations during social separation. In infant rhesus monkeys and rats, ␮-opioid receptor agonists significantly reduced vocalizations when separated from the mother (Carden et al., 1991; Kalin et al., 1988; Kehoe and Blass, 1986). Furthermore, when the infants were reunited with their mothers, morphine reduced clinging behaviours and vocalizations (Kalin et al., 1995). The discrepancy between our results and the results reported in the literature could be due to differences between the species (i.e., sheep versus rats and monkeys) and the particular separation distress situation (i.e., separation from conspecifics versus separation of infant and mother) and dosages and routes of administration of the opioid drugs. Alternatively, the reduced vocalizations in morphine treated animals could potentially be a result of reduced activity at sedative doses of morphine rather than the distress itself (Winslow and Insel, 1991). Naloxone had no effect on vocalizations in the current study. 4.4. Ear postures Facial expressions are used by several species to express and communicate their emotions. However, facial expressions may be of limited use in sheep because sheep do not possess a wide range of facial expressions due to the limited network of superficial facial muscles (Boissy et al., 2011). Because ear postures, rather than facial expressions, may be reflective of the affective state of sheep (Boissy et al., 2011; Reefmann et al., 2009a, 2009b), we hypothesized that ear postures may be modulated by opioid administration. The morphine treated ewes had a higher duration of the backward ear posture during the novel object test compared to control and naloxone treated ewes. A high proportion of the backward ear posture has previously been observed during feeding, while socially separated sheep had a higher proportion of the forward ear posture (Reefmann et al., 2009a), which is similar to what we observed in the control and naloxone treated ewes. Furthermore, sheep being voluntarily groomed (positive valence) had a higher proportion of the backward posture compared to socially separated sheep (Reefmann et al., 2009b). Considering that the proportion of the backward posture appears to be increasing with increasing positive affect, the high proportion of the backward posture during the novel object test in the morphine treated ewes could indicate that they perceived the situation as less negative compared to the control and naloxone treated ewes. However, Boissy et al. (2011) offer a different interpretation because they observed the backward posture mostly during uncontrollable and unfamiliar negative valence situations and interpreted the backward posture as a sign of fear. All ewes showed a high duration of the backward ear posture during the food intake test, and the backward posture tended to be further increased in the morphine treated ewes. The backward ear posture also tended to be higher after feeding than before feeding in all ewes, although there was no time × opioid treatment interaction. It therefore appears that morphine tended to induce a higher duration of the backward posture independent of whether food was available or not. This suggests that

27

morphine did not enhance the palatability of food, which is consistent with the unaltered food intake. The reason for the slightly increased duration of the backward ear posture during the food intake test in the morphine treated ewes is unknown and would need further investigation. Naloxone did not affect ear postures but suppressed food intake at 1.5 and 2 h after injection. Possibly, ear postures in the naloxone treated ewes could have been altered at the time of suppressed food intake, when ear postures were not recorded. 5. Conclusions Overall, we have shown that the morphine doses used in this study were sufficient to induce behavioural changes, affecting arousal in particular. However, the impact of morphine on the valence component of affective states still needs further investigation. Naloxone had little effect on behaviour, except for a small reduction in food intake. Therefore, the results of this study have provided some evidence that opioids may be involved in regulating affective states in sheep. It would be valuable to investigate the impact of opioid administration during alternative situations of positive and/or negative valence and to use alternative measures of arousal and valence in future studies in order to determine the specific involvement of opioids in the regulation of affective states. Role of the funding source This study was entirely funded by CSIRO, and no external funding for this project was received. Acknowledgements Thanks to Dr. Carlos E. Hernandez Verduzco and Dr. Ian Colditz for their useful comments on the manuscript. Thanks to Dr. Alison Small for assisting with data collection and opioid administration. We also thank Dr. Nadine Reefman for advice on ear posture analysis. Finally, thanks to all students and technicians of CSIRO for assisting with data collection and animal care. References Alavi, F.K., Mccann, J.P., Mauromoustakis, A., Sangiah, S., 1993. Feedingbehavior and its responsiveness to naloxone differ in lean and obese sheep. Physiol. Behav. 53, 317–323. Alavi, F.K., Mccann, J.P., Sangiah, S., Clarke, C.R., 1994. Effect of dietary obesity on naloxone disposition in sheep. Can. J. Physiol. Pharmacol. 72, 471–475. Alavi, F.K., McCann, J.P., Sangiah, S., Mauromoustakos, A., 1991. Effects of naloxone on ad libitum intake and plasma insulin, glucose, and free fatty acids in maintenance-fed sheep. Domest. Anim. Endocrinol. 8, 109–115. Baile, C.A., Keim, D.A., Della-Fera, M.A., McLaughlin, C.L., 1981. Opiate antagonists and agonists and feeding in sheep. Physiol. Behav. 26, 1019–1023. Bengtsson, J., Ederoth, P., Ley, D., Hansson, S., Amer-Wåhlin, I., HellströmWestas, L., Marsál, K., Nordström, C.H., Hammarlund-Udenaes, M., 2009. The influence of age on the distribution of morphine and morphine-3-glucuronide across the blood–brain barrier in sheep. Br. J. Pharmacol. 157, 1085–1096. Berridge, K.C., 1991. Modulation of taste affect by hunger, caloric satiety, and sensory-specific satiety in the rat. Appetite 16, 103–120. Berridge, K.C., 2003. Pleasures of the brain. Brain Cogn. 52, 106–128.

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