Physiology & Behavior 143 (2015) 151–157

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Evidence for a link between tail biting and central monoamine metabolism in pigs (Sus scrofa domestica) Anna Valros a,⁎, Pälvi Palander a, Mari Heinonen a, Camilla Munsterhjelm a,b, Emma Brunberg b,c, Linda Keeling b, Petteri Piepponen d a

Department of Production Animal Medicine, Faculty of Veterinary Medicine, P.O. Box 57, 00014 University of Helsinki, Finland Department of Animal Environment and Health, Swedish University of Agricultural Science, P.O. Box 7068, 750 07 Uppsala, Sweden Norwegian Institute for Agricultural and Environmental Research/Bioforsk, Section for Organic Food and Farming, Gunnars veg 6, NO-6630 Tingvoll, Norway d Division of Pharmacology and Pharmacotherapy, Faculty of Pharmacy, P.O. Box 56, 00014 University of Helsinki, Finland b c

H I G H L I G H T S • • • •

There appears to be a link between brain neurotransmission and tail biting in pigs. Tail biters tended to have an increase in their prefrontal cortex 5-HT metabolism. Tail biting victims had altered limbic and striatum dopamine and 5-HT metabolism. The results support the theory that tail biting is a stress-related behaviour.

a r t i c l e

i n f o

Article history: Received 25 November 2014 Received in revised form 24 February 2015 Accepted 25 February 2015 Available online 26 February 2015 Keywords: Tail biting Pig Neurotransmission Serotonin Dopamine

a b s t r a c t Tail biting in pigs is a major welfare problem within the swine industry. Even though there is plenty of information on housing and management-related risk factors, the biological bases of this behavioral problem are poorly understood. The aim of this study was to investigate a possible link between tail biting, based on behavioral recordings of pigs during an ongoing outbreak, and certain neurotransmitters in different brain regions of these pigs. We used a total of 33 pigs at a farm with a long-standing problem of tail biting. Three equally big behavioral phenotypic groups, balanced for gender and age were selected, the data thus consisting of 11 trios of pigs. Two of the pigs in each trio originated from the same pen: one tail biter (TB) and one tail biting victim (V). A control (C) pig was selected from a pen without significant tail biting in the same farm room. We found an effect of tail biting behavioral phenotype on the metabolism of serotonin and dopamine, with a tendency for a higher 5-HIAA level in the prefrontal cortex (PFC) of TB compared to the other groups, while V pigs showed changes in both serotonin and dopamine metabolism in the striatum (ST) and limbic cortex (LC). Trp:BCAA and Trp:LNAA correlated positively with serotonin and 5-HIAA in the PFC, but only in TB pigs. Furthermore, in both ST and LC, several of the neurotransmitters and their metabolites correlated positively with the frequency of bites received by the pig. This is the first study indicating a link between brain neurotransmission and tail biting behavior in pigs with TB pigs showing a tendency for increased PFC serotonin metabolism and V pigs showing several changes in central dopamine and serotonin metabolism in their ST and LC, possibly due to the acute stress caused by being bitten. © 2015 Elsevier Inc. All rights reserved.

1. Introduction Tail biting is a common behavioral problem in pigs. Under suboptimal housing and management conditions, pigs are prone to start biting each other's tails, causing wounds, amputation of the tail, and infections [1,2]. Tail biting is thus a serious animal welfare problem, indicating an underlying welfare problem, and causing additional stress to the victim ⁎ Corresponding author. E-mail address: anna.valros@helsinki.fi (A. Valros).

http://dx.doi.org/10.1016/j.physbeh.2015.02.049 0031-9384/© 2015 Elsevier Inc. All rights reserved.

[3] as well as production losses due to increased need for medication, reduced growth and increased prevalence of carcass condemnation of affected pigs [4,2,5]. The external risk factors for tail biting are reasonably well-known, including factors that cause stress, such as lack of manipulable materials to fulfill their need for exploration; crowding in the pen; competition by the feeder; poor environmental conditions, such as air quality; and feeding-related problems [6]. However, even in clearly suboptimal situations, such as barren housing conditions, not all pigs end up biting tails [7,8], indicating that there are individual differences between pigs,

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making some more prone to bite than others. Based on behavioral patterns and suggested motivational background it has been suggested [9] that there might, in addition, be several different types of tail biting behavior. Some forms of tail biting, such as the sudden-forceful type, where tail biting occurs in situation of competition, such as at the feeder, have been proposed to be linked to aggressive behavior, while other, more common types do not show a link to aggression [9]. Being a tail biter does not appear to be a stable individual trait [8], but rather a temporary behavioral change, due to challenges in the environment. These changes might, in turn, be related to changes in neurotransmission, as have been indicated in relation to behavioral problems in other captive animals, eg. feather pecking in chicken [10,11] and stereotypic behavior in bank voles and horses [12,13]. The serotonergic system is involved in the regulation of mood, eating behavior, aggressive behavior and mental disorders in humans and other species (for a review, see [14]). In pigs, the serotonergic system has been linked to aggressive behavior [15,16]. Furthermore, genetically stress-susceptible pigs have been shown to have lower levels of serotonin in several brain regions than stress-tolerant pigs [17], while increased feeding levels of tryptophan, which is the precursor of serotonin, inhibit physiological stress activity [18] and decreases aggression and stress reactions in pigs [15,19,20]. Increased level of orally administered tryptophan has also been shown to reduce tail and ear biting in pigs [21]. Ursinus et al. [8] were the first to show a link between tail biting behavior and the serotonergic system, by showing a decrease of blood serotonin in both tail biting and victim pigs. Stressful situations change serotonin activity in the brain [22,23]. The dopaminergic system is of great importance for both the reward system and for handling environmental challenges and emotional responses, such as fear and anxiety (for reviews, see [24,25]). In pigs, the dopaminergic system has been related to exploratory propensity [26] and the frustration level during a delay test [27]. Dopamine responds to stressful situations [22,23], for example, it has been shown that stress caused by restraining pigs increases dopamine turnover in certain brain areas [23]. Also noradrenaline activity in the brain has been linked to aggressive behavior and dominance in pigs [15]. As for the other monoamines, stress causes changes also in central noradrenaline levels and noradrenaline metabolism [23,28]. The aim of this study was to investigate a possible link between tail biting, based on behavioral recordings of pigs during an ongoing outbreak, and certain neurotransmitters in different brain regions of these pigs. The motivational background of tail biting appears to be complex [9], and we do not know, for example, if the behavior involves learning. Thus, the brain regions were selected to represent areas important for the organization of stress-related processes on different cognitive levels (for reviews, see [14,29]). Chronic and/or acute stress is assumed to be the main underlying factor behind the development of tail biting. As tail biting is known to induce stress in the victims [3,30], we hypothesized that both pigs performing tail biting and victims of tail biting should show changes in central monoamine levels and metabolism. 2. Material and methods The study protocol was approved by the Ethical Committee at the University of Helsinki (ECUH). 2.1. Animals and housing The pigs used in this study originated from a farm with a long-term tail biting problem. The farm housed altogether 900 fattening pigs in six all-in–all-out rooms from approximately 25 kg weight until slaughter. The pigs were a mix between Finnish Yorkshire, Landrace, Duroc and Hampshire and all pigs had undocked tails, as tail docking is not allowed in Finland. The pens in the different rooms differed in structure, size and

feeding systems, with 7–26 pigs per pen. All pens had partly slatted floor and a small amount of peat was used as enrichment. No bedding was provided. The different housing systems are described in more detail in Brunberg et al. [7] and Palander et al. [31]. Two different diets were offered to the pigs according to a phase feeding regimen. A grower diet was fed for 4 weeks after arrival, at 10 to 14 weeks of age, and finisher diet for the remaining time until slaughter. Both feeds consisted of barley mixed with a commercial concentrate. Details of the feeds and their composition are reported in Palander et al. [31].

2.2. Selection of pigs The pigs used for the study had been on the farm for 6–75 days before the study commenced. Their weight varied between 23 and 83 kg. A total of 33 pigs from 22 pens were selected for the trial based on behavioral observations in relation to the ongoing outbreak of tail biting. Potential tail biting pens were first selected using visible signs of tail injuries or information from the caretaker. Similar control pens from the same room of the farm, with no signs of tail damage, were selected as potential control pens. The tail biting status of these pens was further controlled by 2 ∗ 30 min behavioral observations for two days (− 3 and − 2 in relation to the day of euthanasia (day 0)), ending up with 11 tail biting pens, and 11 control pens, in which no significant tail biting had been observed. Based on all-occurrence sampling of performed and received tail bites, pigs were selected and allocated to one of three behavioral phenotypic groups: one tail biter (TB) and one victim of tail biting (V) from each tail biting pen, as well as a nonbiting, non-bitten control pig (C) from the control pen. Furthermore, individual pig observations were performed also on days − 2 and − 1 within these pens, by observing pigs for a total of 8 ∗ 15 min for performed and received tail bites. Frequency of performed and received tail bites is based on all the above-described observations. Tail biting frequency includes mild (no reaction from the receiver) to severe (receiver reacting severely, by eg. running away, screaming or biting back) bites (ethogram described in Brunberg et al. [7]). As a result of this selection procedure we ended up with a total of 11 trios of pigs, matched for gender, approximate age (based on arrival date on the farm), room within the farm, pen size and structure, and group size within pen, within each trio. Three of the trios were castrated males and the remaining eight were females. Further information about the pigs is shown in Table 1. The selection of the individuals for the study aimed to be as ‘pure’ as possible regarding the phenotypical classification. However, as can be seen from the behavioral data shown in Table 4, a few of the tail biters also received some bites and a few of the victims bit tails of other pigs, while tail biting was very uncommon in the control pens. This pattern is also seen in the tail damage scores of these pigs (Table 4).

Table 1 Characteristics of matched trios of behaviorally categorized tail biters, victims and controls from non-biting pens. Trio

Gendera

Weightb

Days on farmc

1 2 3 4 5 6 7 8 9 10 11

F F M F F M F F M F F

35.7 (2.1) 40.3 (1.5) 33.7 (7.1) 37.3 (2.5) 56.8 (5.3) 76.0 (6.3) 30.3 (3.8) 32.8 (1.0) 28.7 (4.9) 55.2 (8.5) 47.0 (5.9)

19 26 25 26 74 75 6 14 13 35 34

a b c

M = castrate, F = female. Weight given as mean and (sd) within the triplet. The pigs arrived at the farm at approximately 10–12 weeks of age.

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2.3. Data collection One to three days after the behavioral observations, sometime between 7 am and 1 pm, the pigs were sedated in their home pen. Pigs were sedated one by one, taking all pigs from one trio successively. The sedation order of the pigs within the trio was randomized between trios to avoid the effect of sedation order on the results. Sedation was done with an intramuscular injection of midazolam (0.5 mg/kg estimated liveweight, LW), butorphanol (0.2 mg/kg LW) and ketamine (10 mg/kg LW) and then carried to a separate room where they were euthanized with an intracardial injection of pentobarbital (20 mg/kg LW). The protocol is described in more detail by Munsterhjelm et al. [3]. As there is a risk for the sedation protocol to affect the neurotransmitters and their metabolism, we tested for possible differences in duration from sedation until death between different phenotypic groups. There was no between-group difference (Kruskal–Wallis, p = 0.6), and the duration was on average 18:46 min (sd: 8:27), which is probably short enough not to affect central neurotransmitter metabolism. Just before euthanasia, blood was taken from the jugular vein into heparinized tubes and centrifuged at 3000 rpm, 1400 g, for 10 min. The plasma was transported in dry ice and frozen at − 80 °C until analysis. Immediately following death, defined as no sign of corneal reflex, the pigs were decapitated, the skulls opened and their brains extracted and sectioned using an anatomical dissection and placed in liquid nitrogen. The brain sections used in this study included the left side of the prefrontal cortex (PFC), the hypothalamus (H), the striatum (ST) and the limbic cortex (LC). 2.4. Analyses of amino acids, neurotransmitters and their metabolites The amino acid concentration in plasma was analyzed using ultra performance liquid chromatography (UPLC) on an Acquity system (Waters Finland, Helsinki, Finland), equipped with an Acquity photodiode array optical detection system. The sample preparation and analyze protocols are described in detail by Palander et al. [31]. The pig brain samples were homogenized and pulverized in liquid nitrogen prior to analyses. Analyses of dopamine (DA) and its main metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), noradrenaline (NA) and its main metabolite 3-methoxy4-hydroxyphenylglycol (MOPEG) and serotonin (5-HT) and its main metabolite 5-hydroxyindoleacetic acid (5-HIAA) were performed. The brain samples were homogenized in 0.5 ml of homogenization solution consisting of six parts 0.2 M HCLO4 and one part antioxidant solution containing oxalic acid in combination with acetic acid and L-cysteine. The homogenates were centrifuged at 20,800 g for 35 min at 48 °C. The supernatant was removed to 0.5 ml Vivaspin filter concentrators (10,000 MWCO PES; Vivascience AG, Hannover, Germany) and centrifuged at 8600 g at 4 °C for 35 min. Filtrates containing monoamines were analyzed using high-pressure liquid chromatography with electrochemical detection. The analytes were separated on a Phenomenex Kinetex 2.6 μm, 4.6 × 50 mm C-18 column (Phenomenex, Torrance, CA, USA). The column was maintained at 45 °C with a column heater (Croco-Cil, Bordeaux, France). The mobile phase consisted of 0.1 M NaH2 PO4 buffer, 120 mg/l of octane sulfonic acid, methanol (5%), and 450 mg/l EDTA, the pH of mobile phase was set to 3 using H3PO4. The pump (ESA Model 582 Solvent Delivery Module; ESA, Chelmsford, MA) was equipped with two pulse dampers (SSI LP-21, Scientific Systems, State College, PA) and provided a flow rate of 1 ml/min. One hundred microliters of the filtrate was injected into chromatographic system with a Shimadzu SIL-20AC autoinjector (Shimadzu, Kyoto, Japan). Monoamines and their metabolites were detected using ESA CoulArray Electrode Array Detector with 12 channels. The applied potentials were +30 mV (channel 1), +60 mV (2), +90 mV (3), +120 mV (4), +150 mV (5), +180 mV (6), +210 mV (7), +240 mV (8), +270 (9), +300 (10), +330 mV (11) and + 360 mV (12).

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Dopamine was detected on channel 3, NA, DOPAC, 5-HT and 5-HIAA on channel 4, HVA on channel 11 and MOPEG on channel 12. The chromatograms were processed and concentrations of monoamines calculated using CoulArray for windows software (ESA, Chelmsford, MA).

2.5. Data analyses The different brain tissue samples were weighed prior to analyses and levels of neurotransmitters and their metabolites are expressed as ng/g of brain tissue. Amino acids analyzed for the present study included tyrosine (Tyr) and phenylalanine (Phe), which are the precursors for dopamine and noradrenaline and tryptophan (Trp), which is the precursor for serotonin. In addition, the ratio of tryptophan to large neutral amino acids (LNAA) and to branched chain amino acids (BCAA) was calculated, as Trp and these amino acids share the same transporter through the blood brain barrier. Thus a high LNAA or BCAA to Trp ratio limits the transportation of Trp into the brain [33]. A full analysis of amino acids from pigs included in this study is presented in [31]. Behavioral signs of tail biting, ie. received and performed bites, are presented as frequency of observed bites per 3 h of observations, according to the calculation method described by Brunberg et al. [7].

2.6. Statistical analyses The PASW 18 software (SPSS Inc., Chicago, IL, USA) was used for all statistical analyses. All variables were tested for normality using Wilk– Shapiro tests and by visual investigation of distribution diagrams. HVA and HVA:DA in the LC were found not to be normally distributed, and these were normalized prior to testing using Log10-transformation. All numerical results are presented as original data. As the frequency of performed and received bites and time from sedation until death of the pigs were far from normally distributed, these variables were tested using non-parametric statistics. The effect of feeding phase (growing or finishing) and gender on the amino acids and neurotransmitters and their metabolites were tested with independent t-tests. Only 5-HT in the LC was found to differ between the genders, and gender was included in the respective model as a fixed factor. Each neurotransmitter, metabolite, ratios between neurotransmitters and their main metabolites, and amino acid, as well as Trp:LNAA and Trp:BCAA were tested with a separate univariate analysis of variance, with pig tail biting behavioral phenotype as the fixed effect, followed by pair-wise comparisons when appropriate. Differences in performed and received bites between the behavioral phenotypes were tested with the Kruskal–Wallis test. Correlations between neurotransmitters, metabolites and amino acids were tested using Pearson's correlations and correlations between the frequencies of received and performed bites and neurotransmitters, metabolites and amino acids were tested using Spearman rank correlations.

Table 2 Descriptive overall data (mean and (sd)) for the 11 trios of tail biters, victims and control pigs in control pens in plasma concentration of amino acids. Concentrations are given as μmol/l. Mean (sd) Tyrosine Phenylalanine Tryptophan Try:LNAAa Try:BCAAb a

67.1 (16.3) 78.4 (14.3) 54.1 (15.4) 0.31 (0.08) 0.08 (0.02)

Ratio of tryptophan to large neutral amino acids (LNAA). Ratio of tryptophan to branched chain amino acids (BCAA). b

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Table 3 Descriptive overall data (mean and (sd)) for the 11 trios of tail biters, victims and control pigs in control pens on concentrations of neurotransmitters and their metabolites, as well as the ratio between these, in the different brain areas. Concentrations are given as ng/g of brain tissue.

DA DOPAC DOPAC:DA HVA HVA:DA NA MOPEG MOPEG:NA 5-HT 5-HIAA-t 5-HIAA:5-HT

Received tail bites Brain region

Prefrontal cortex

Hypothalamus

Limbic cortex

Striatum

19.0 (8.7) 8.6 (3.3) 0.48 (0.12) 10.3 (8.3) 0.56 (0.30) 133 (24.5) 5.0 (2.7) 0.04 (0.2) 63.3 (27.8) 3.8 (1.3) 0.07 (0.03)

119 (32.2) 32.8 (10.8) 0.28 (0.08) 25.2 (14.0) 0.22 (0.12) 892 (313) 12.1 (3.5) 0.01 (0.004) 123 (42.7) 10.3 (3.4) 0.09 (.03)

300 (183) 56.9 (37.3) 0.20 (0.08) 84.6 (99.2) 0.33 (0.37) 76.9 (39.4) 9.3 (2.4) 0.18 (.18) 124 (44.1) 4.5 (5.0) 0.03 (.03)

3507 (935) 70.4 (29.2) 0.10 (0.28) 381 (244) 0.11 (0.07) N/A N/A N/A 150 (34.8) 8.1 (5.2) 0.05 (0.03)

Neurotransmitter/metabolite Spearman correlation Significance

Limbic cortex DOPAC: DA HVA HVA:DA 5-HIAA 5-HIAA:5-HT NA Striatum DOPAC HVA 5-HIAA 5-HIAA:5-HT

0.345 0.375 0.419 0.357 0.358 −0.310 0.409 0.406 0.457 0.402

0.05 0.03 0.02 0.05 0.05 0.09 0.02 0.02 0.008 0.02

between any of the neurotransmitters, metabolites and their respective precursors (p N 0.1 for all).

3. Results

3.4. Effect of tail biting behavioral phenotype on amino acids and neurotransmitters

3.1. Descriptive data Overall levels of plasma amino acids are presented in Table 2. Levels of brain neurotransmitters and their metabolites, as well as the ratio between neurotransmitters and metabolites, are presented in Table 3. The number of performed and received tail bites in the different phenotypic groups is given in Table 4. 3.2. Correlations neurotransmitters

Table 5 Correlations between number of received tail bites and neurotransmitters in the brain. Only significant correlations and tendencies are given (n = 33).

between

performed

and

received

bites

and

None of the neurotransmitters nor their metabolites measured in PFC or H correlated with the number of performed or received tail bites (p N 0.1 for all). In LC a positive correlation between received bites and DOPAC:DA, HVA, HVA:DA, 5-HIAA, and 5-HIAA:5-HT was found. In addition, NA tended to correlate negatively with the frequency of received bites. In the ST, frequency of received bites correlated positively with DOPAC, HVA, 5-HIAA and 5-HIAA:5-HT (Table 5). 3.3. Correlations between amino acids and neurotransmitters Trp:BCAA and Trp:LNAA both correlated positively with 5-HIAA in the PFC (rp = 0.359, p = 0.04 and rp = 0.352, p = 0.04 respectively). When looking in more detail at this correlation, it was found that these were significant in TB pigs only (rp = 0.569, p = 0.07 and rp = 0.719, p = 0.01, respectively). In addition, in TB pigs Trp:BCAA and Trp:LNAA correlated positively also with the level of 5-HT (rp = 0.758, p = 0.007 and rp = 0.823 p = 0.002, respectively), and 5-HT tended to correlate positively with Trp (rp = 0.528, p b 0.1). 5HIAA:5-HT tended to correlate negatively with Trp:BCAA (rp = − 0.568, p = 0.07). No correlations occurred in any of the other brain regions

None of the plasma amino acids differed between the groups of pigs with differing tail biting behavioral phenotype (p N 0.1 for all). In PFC, 5-HIAA tended to differ between the groups of pigs with differing tail biting behavioral phenotype (F = 3.1. p = 0.06). Pairwise tests revealed that this difference was caused by a higher 5-HIAA level in TB pigs than in both V and C pigs (Fig. 1). Several tendencies for differences in the neurotransmitters and their metabolites between pigs of different tail biting behavioral phenotypes were found in both LC and ST. 5-HIAA and 5-HIAA:5-HT tended to differ between tail biting behavioral phenotypic groups in LC (F = 2.6, p = 0.09 and F = 2.9, p = 0.07, respectively), being highest in V pigs (Fig. 2a and b). In addition, HVA:DA tended to differ between behavioral phenotypes (F = 2.6. p = 0.09) in LC, being highest in V pigs (Fig. 2c). Similar differences were found in ST: 5-HIAA and 5-HIAA:5-HT differed between tail biting behavioral phenotypic groups (F = 4.8, p = 0.02 and F = 4.6, p = 0.02, respectively), being highest in V pigs (Fig. 3a and b). In ST HVA was higher in V pigs than in the other pigs (F = 3.9, p = 0.03) and also HVA:DA differed between behavioral phenotypes (F = 5.5, p = 0.004), being highest in V pigs (Fig. 3c and d). 4. Discussion We were able to show evidence for the expected link between neurotransmission in the pig brain and tail biting. Changes in neurotransmission were seen both in pigs performing tail biting and pigs that had been victims of tail biting. As this study was conducted during an 7

p = 0.04 p = 0.04

6

Tail biters

Victims

Control pigs

N

11

11

11

Received tail bites Performed tail bites Pigs with tail damage (n) - Mild damagea - Moderate damageb - Severe damagec

1 (0–8) 51 (30–98)

13 (3–38) 0 (0–3)

0 (0–1) 0 (0–1)

1 2 1

1 1 9

2 0 0

a b c

Wounds not deeper than subcutis. Wounds deeper than subcutis or moderate infection. Part of tail missing, from [31].

Significance

p b 0.001 p b 0.001

5 5-HIAA ng/g

Table 4 The number of received and performed tail bites in tail biters, victims and control pigs in control pens during a total of 3 h of observation; given as median (min–max), as well as number of pigs with tail damage in the same pig groups.

4

3

2

1

0 Tail biters

Victims

Control pigs

Fig. 1. Concentration of 5-HIAA (ng/g) in the prefrontal cortex of tail biters, victims and control pigs (n = 33), given as mean and standard deviation.

A. Valros et al. / Physiology & Behavior 143 (2015) 151–157

155

a

a

20

14

p = 0.03

p = 0.008

p = 0.02

18

12

16 14 12

5-HIAA ng/g

5-HIAA ng/g

10

8

10 8

6

6

4

4 2

2

0 Tail biters

0 Tail biters

Victims

Control pigs

b

0.12

0.08

Control pigs

p = 0.008

p = 0.03

p = 0.02 0.10

0.07

0.06

0.08 5-HIAA:5-HT

5-HIAA:5-HT

Victims

b

0.05

0.04

0.06

0.04

0.03

0.02

0.02

0.01

0.00 0.00

Tail biters Tail biters

Victims

Victims

Control pigs

Control pigs

c

c

1,000

1.20

p = 0.01

p = 0.06

p = 0.06

p = 0.02

900 1.00

800 700 600

HVA ng/g

HVA:DA

.80

.60

500 400

.40

300 200

.20

100 0

.00 Tail biters

Victims

Control pigs

Fig. 2. a. Concentration of 5-HIAA (ng/g) in the limbic cortex of tail biters, victims and control pigs (n = 33), given as mean and standard deviation. b. 5-HIAA to 5-HT ratio in the limbic cortex of tail biters, victims and control pigs (n = 33), given as mean and standard deviation. c. HVA to DA ratio in the limbic cortex of tail biters, victims and control pigs (n = 33), given as mean and standard deviation.

Tail biters

Victims

Control pigs

d 0.30

p = 0.02

p = 0.02

0.25

HVA:DA

0.20

actual outbreak of tail biting, we cannot make conclusions about cause and effect of the observed differences. However, this is, to our knowledge, the first study showing that there exists a link between central monoamines and tail biting, in support of the results by Ursinus et al. [8], showing a link between plasma serotonin and tail biting behavior. Thus, even though we can only speculate on the mechanisms behind these changes, our results warrant further investigation into the subject. While there was no difference between tail biters and the other phenotypic classes of pigs in level of serotonin, the level of the main metabolite of serotonin, 5-HIAA, was increased in the prefrontal cortex of tail biters. This increase in serotonin metabolism indicates that the tail biters had experienced stressful challenges [14,23]. However, we cannot conclude if this occurred prior to, or during the currently ongoing tail biting outbreak.

0.15

0.10

0.05

0.00 Tail biters

Victims

Control pigs

Fig. 3. a. Concentration of 5-HIAA (ng/g) in the striatum of tail biters, victims and control pigs (n = 33), given as mean and standard deviation. b. 5-HIAA to 5-HT ratio in the striatum of tail biters, victims and control pigs (n = 33), given as mean and standard deviation. c. Concentration of HVA (ng/g) in the striatum of tail biters, victims and control pigs (n = 33), given as mean and standard deviation. d. HVA to DA ratio in the striatum of tail biters, victims and control pigs (n = 33), given as mean and standard deviation.

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The positive correlation between the level of tryptophan in the blood, as well as the Trp:LNAA ad Trp:BCAA ratios and brain serotonin and 5-HIAA levels was, interestingly enough, only evident in tail biters. This correlation might additionally support the theory of an activated serotonin metabolism in tail biting pigs, causing tryptophan to become a limiting factor for these pigs only. This is in line with the findings by Ursinus et al. [8], who showed a decrease in serotonin blood level in tail biters, which, again, could indicate a higher usage of tryptophan for the formation of central serotonin. Increasing tryptophan levels in the diet has been shown to reduce activity and aggressive behavior and to improve stress recovery in pigs, as well as to decrease tail and ear biting frequency [15,19–21]. It is thus possible that demands for tryptophan in the diet are altered in tail biting pigs due to increased demand for stress coping. Thus, the current results indicate a need for further studies of the effect of dietary tryptophan on tail biting. It is also possible that there is only an increased need for tryptophan in pigs with a propensity for tail biting. In humans, it has been shown that only mentally unhealthy individuals benefit from an increased level of tryptophan, while it has no effect on healthy individuals [33]. Tail biting pigs are often anecdotally reported to be smaller than average. Even though there are contradicting results from recent studies [30,32], it is possible that tail biting is somehow linked to dietary challenges in pigs. Tryptophan deficiency in the feed causes reduced feed intake in pigs [34] and increased tryptophan level in diets has been shown to enhance gut development in young pigs [35]. It has further been shown that pigs prefer a diet with a higher level of tryptophan when given a choice [36]. If tail biting pigs have a higher level of serotonin metabolism they might also have a higher demand for tryptophan in their diet, although we cannot exclude that a higher demand for tryptophan, such as in pigs with a high growth potential [37] means that there is less tryptophan available for serotonin synthesis. The serotonergic system is affected by both chronic and acute stresses. Situations causing acute stress result in increased serotonin metabolism [22,23]. It has also been shown that previously experienced stress has a long-term effect on serotonin activity. Social stress changed serotonin levels in the prefrontal cortex in macaques for at least 14 months after the exposure [38]. As the tail biting behavior in the pigs in the current study was observed 2–3 days prior to the euthanasia and sampling, and as it is well-known that different acute and chronic stressors can trigger tail biting [6], it is not possible to conclude if the changes in neurotransmission in the pigs in this study were due to current occurring stress, or to a more chronic challenge. The motivational background of tail biting is still not clear, and, for example, no consistent link between tail biting and aggression has been proven. Taylor et al. [9] suggested that there exist several types of tail biting: “two-stage” type tail biting usually results in rather mild wounds, and occurs in situations not indicative of aggression as a motivator. However, in the less commonly occurring, so called “suddenforceful” cases of tail biting, the pig may attack another pig without prior indication of tail manipulation. This typically happens in a situation of frustration and/or competition, for example at the feed trough. In these cases, it is possible that tail biting is related to aggressive behavior. As aggressiveness has been extensively connected to the serotonergic activity [15,20] this might be a common mechanism behind at least this form of tail biting. Victim pigs, ie. pigs that had been suffering from biting of their tail, showed several changes in serotonin and dopamine metabolism in the striatum and limbic cortex. This effect is likely due to the stress caused to these pigs by the biting per se. As this study was conducted during a period of acute tail biting, and as histological examination of a larger set of pigs (including all pigs from this study) showed that none of the victim pigs had signs of healed tail damage [32], it is probable that the stress caused to the victim pigs by being bitten was fairly acute. This might explain the fact that there was no correlation between brain serotonin or its metabolites and tryptophan in these pigs, but only in tail

biting pigs, which, in turn, might have suffered from a more chronic stress, causing a more chronically increased tryptophan utilization. The fact that we also found that there was a correlation between the amount of received bites recorded during the behavioral observations and several of the monoamine metabolites further supports this notion. However, as this correlation was found only in the overall data (i.e. when including all animals) it must be considered with caution. There is probably a confounding effect due to the victims being the ones receiving the majority of bites, and also the ones showing elevated monoamine levels. Tail bites were received also by tail biters and control pigs, but, at a much lower level. As we only have information from the victims during the ongoing tail biting outbreak, we cannot, however, exclude the possibility that the increases in monoamine metabolism we observed were not individual characteristics actually predisposing the pigs to become victims of tail biting. Early stressful experiences [24] and environmental factors [8,39] can affect the development of the dopaminergic and serotonergic systems, and thus the mental state of animals both on short- and long-term. Our study showed that tail biters differed from other phenotypes in their serotonin metabolism in the prefrontal cortex, but not in any of the other brain areas studied (hypothalamus, striatum and limbic cortex). On the other hand, victims of tail biting had increased levels of both dopamine and serotonin metabolism in the striatum and limbic cortex, but not in the prefrontal cortex. One possible explanation for this difference, in keeping with our previous discussion, is that victims were probably currently suffering from acute stress due to the biting per se, while the tail biters might have become tail biters due to a more chronic stress experience. Long term coping with stress is more probable to have included the need for cognitive processes to adapt to the challenges. The function of the prefrontal cortex, which governs higher-level cognitive processes, can be negatively altered by stress, causing changes in behavior [40]. The limbic system, on the other hand, is more primitive, and involved in processing of highly emotional situations [41], as well as in fear reactions [42]. We used pigs from pens where tail biting either occurred (tail biters and victims) or did not occur at a significant level (control pigs). Thus, it is difficult to conclude what the control pigs really represent. Did the pens remain free from significant tail biting due to differing environmental conditions, or due to the lack of animals prone to bite or become victims in these pens? The pens were all on the same farm, and within each trio, biting pens and control pens were from the same room, and as similar as possible. This should minimize the effect of differing environmental conditions. On the other hand, several anecdotal reports indicate that tail biting might occur more frequently in certain problem pens. Reasons for this might be that factors with a potential local variability, such as air quality, pen hygiene and ambient temperature [6] might differ between pens even within the same room. Thus, we can only speculate on the possible individual phenotype of the control animals, and again, the difference between control animals and tail biters and victims does not tell us anything about the cause and effect relationship between tail biting and neurotransmission. After selection of the control pens, some incidences of tail biting were recorded also in these pens, and two of the control pigs had mild tail damage at the end of the experiment. Thus, the pens, were not perfect controls, even though tail biting was very infrequent in them. However, even though the control pens were included in the analysis, differences in central monoamine metabolism were clearly apparent between the tail biter and victim pigs as well. These came from the same pen and were thus exposed to the same environmental conditions. 5. Conclusions This is the first study indicating a link between brain neurotransmission and tail biting behavior in pigs. We showed that during an ongoing tail biting outbreak, tail biters tended to have an increase in their

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prefrontal cortex serotonin metabolism as compared to victims of tail biting and control pigs from pens where tail biting hardly occurred. The mechanism behind this possible link can only be discussed and further studies are warranted on links between, among others, neurotransmission and tail biting, as well as on the importance of diet tryptophan in connection to tail biting. Furthermore, we found that victims of tail biting showed several changes in central dopamine and serotonin metabolism in their striatum and limbic cortex, possibly due to the acute stress caused by being bitten. The fact that tail biters and victims showed changes of neurotransmitter metabolism in different brain areas, might indicate changes on a different level of cognitive processing in these behavioral phenotypes. These results might help understand underlying individual differences in pigs that increase the risk of becoming a tail biter or a victim of tail biting. They also give further support for the theory that tail biting is, indeed, a stress-related behavior. As indicated by studies [6] on risk factors for tail biting, this increased stress, in turn, might be induced by a range of factors, both related to behavioral frustration due to e.g. a barren environment and to physiological challenges such as disease and feeding problems. Acknowledgments This study was a part of the NKJ (Nordic Joint Committee for Agricultural and Food Research)-funded project ‘Tail biting and tail docking in the pig: biological mechanisms, prevention, treatment and economic aspects’ (grant number NKJ 129) and national funding was provided by the Finnish Ministry of Agriculture and Forestry (grant number 45/ 502/2008). The authors wish to thank the farm for allowing the research team to use their facilities, and for all their help. A special acknowledgement goes to Anne Larsen for technical assistance during the data collection phase. References [1] M. Heinonen, T. Orro, T. Kokkonen, C. Munsterhjelm, O. Peltoniemi, A. Valros, Tail biting induces a strong acute phase response and tail-end inflammation in finishing pigs, Vet. J. 184 (2010) 303–307. [2] A. Valros, S. Ahlström, H. Rintala, T. Häkkinen, H. Saloniemi, The prevalence of tail damage in slaughter pigs in Finland and associations to carcass condemnations, Acta Agric. Scand. Sect. A Anim. Sci. 54 (2004) 213–219. [3] C. Munsterhjelm, E. Brunberg, M. Heinonen, L. Keeling, A. Valros, Stress measures in tail biters and bitten pigs in a matched case–control, Anim. Welf. 22 (2013) 331–338. [4] E.J. Hunter, T.A. Jones, H.J. Guise, R.H.C. Penny, S. Hoste, Tail biting in pigs, the prevalence at six UK abattoirs and the relationship of tail biting with docking, sex and other carcass damage, Pig J. 43 (1999) 18–32. [5] A. Sinisalo, J. Niemi, M. Heinonen, A. Valros, Tail biting and production performance in fattening pigs, Livest. Sci. 143 (2012) 142–150. [6] European Food Safety Authority, Scientific opinion concerning a multifactorial approach on the use of animal and non-animal-based measures to assess the welfare of pigs, EFSA J. 12 (2014) 3702 (101 pp.). [7] E. Brunberg, A. Wallenbeck, L.J. Keeling, Tail biting in fattening pigs: associations between frequency of tail biting and other abnormal behaviours, Appl. Anim. Behav. Sci. 133 (2011) 18–25. [8] W.W. Ursinus, C.G. Van Reenen, I. Reimert, J.E. Bolhuis, Tail biting in pigs: blood serotonin and fearfulness as pieces of the puzzle, PLoS ONE 9 (2014) e107040. [9] N.R. Taylor, D.C.J. Main, M. Mendl, S.A. Edwards, Tail-biting: a new perspective, Vet. J. 186 (2010) 137–147. [10] Y.M. van Hierden, S.M. Korte, E.W. Ruesink, C.G. van Reenen, B. Engel, G.A. KorteBouws, et al., Adrenocortical reactivity and central serotonin and dopamine turnover in young chicks from a high and low feather-pecking line of laying hens, Physiol. Behav. 75 (2002) 653–659. [11] M.S. Kops, E.N. de Haas, T.B. Rodenburg, E.D. Ellend, G.A.H. Korte-Bouws, B. Olivier, et al., Effects of feather pecking phenotype (severe feather peckers, victims and non-peckers) on serotonergic and dopaminergic activity in four brain areas of laying hens (Gallus gallus domesticus), Physiol. Behav. 120 (2013) 77–82. [12] B. Schoenecker, K.E. Heller, The involvement of dopamine (DA) and serotonin (5-HT) in stress-induced stereotypies in bank voles (Clethrionomys glareolus), Appl. Anim. Behav. Sci. 73 (2001) 311–319.

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