Livestock Science 122 (2009) Contents lists available at ScienceDirect. Livestock Science. journal homepage:

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1 Livestock Science 122 (2009) Contents lists available at ScienceDirect Livestock Science journal homepage: Effect of rearing and slaughter conditions on behaviour, physiology and meat quality of Large White and Duroc-sired pigs Claudia Terlouw, Alban Berne, Thierry Astruc INRA de Theix, Meat Research Station, St-Genès-Champanelle, France article info abstract Article history: Received 22 November 2007 Revised 27 August 2008 Accepted 30 August 2008 Keywords: Duroc Large White Stress Individual differences Meat quality Slaughter The present study investigated the effect of outdoor rearing and genetic background on behaviour and meat quality traits in heavyweight pigs. Large White and Duroc-sired pigs were reared in groups of 3 castrated males and 3 females, conventionally or in fields, from April to October, in a study with two replicates. Compared to indoor pigs, outdoor pigs were more active and showed a larger range of behaviour suggesting improved welfare from a behavioural point of view. During 5 h of isolation, outdoor pigs were less active and had lower heart rates, and in another test, they reacted less to a non-familiar object (traffic cone). Pigs were slaughtered at 150 kg live weight, half of each treatment group after mixing, short transport, and overnight lairage and half immediately following short transport. Outdoor pigs were less aggressive during pre-slaughter mixing. Ante- and post-mortem glycogen content of the Longissimus lumborum (LL), Semimembranonus (SM) and Semispinalis capitis (SC) muscles depended on slaughter conditions, sire breed, rearing conditions, gender and year of experimentation, sometimes influencing ultimate ph. Effects of rearing on muscle glycogen content and postmortem ph were stronger during the second year of rearing. Effect of slaughter conditions on glycogen content and ultimate ph depended on fighting levels during pre-slaughter mixing. Outdoor rearing increased muscle redness. Drip and cooking loss were higher in Large White than Duroc-sired pigs. Drip and cooking loss were correlated with early post-mortem and ultimate ph. Thawing loss was correlated with early post-mortem temperature and ultimate ph. Finally, reactivity to isolation had a predictive value as pigs more active during isolation had less skin damage due to fighting during pre-slaughter mixing. In conclusion, despite large effects of year of experimentation, outdoor rearing, sire breed, and slaughter conditions influenced behaviour and muscle characteristics Elsevier B.V. All rights reserved. 1. Introduction Outdoor pig production may be an interesting alternative to conventional systems. To the consumer, outdoor pig production presents a better image, probably due to perceived better welfare or meat quality (Oude Ophius, 1994; Dransfield et al., 2005). In reality, the effect of rearing environment on technological meat quality varies (Guy and Edwards, 2002). Some studies report that early post-mortem and ultimate ph did not differ between indoor and outdoor reared pigs (Gentry Corresponding author. Present address: INRA, UR1213 Herbivores, F Saint-Genès Champanelle, France. address: claudia.terlouw@clermont.inra.fr (C. Terlouw). et al., 2002a; Lebret et al., 2002). Other studies found lower initial and ultimate ph values in outdoor pigs with negative consequences for water holding capacity (Sather et al., 1997; Enfält et al., 1997). In addition, we have limited knowledge on adaptability of pig breeds to outdoor rearing and choice of breed may also have consequences for meat quality (Guy and Edwards, 2002; Edwards, 2005). The effects of outdoor pig breeding on welfare are also complex. Outdoor rearing may improve animal welfare aspects because it allows better behavioural expression. Little is known of stress reactions to the slaughter procedure of outdoor compared to indoor pigs (Edwards, 2005). Prior experience may influence reactivity to slaughter procedures. Pigs used to handling were less reactive to transport than /$ see front matter 2008 Elsevier B.V. All rights reserved. doi: /j.livsci

2 200 C. Terlouw et al. / Livestock Science 122 (2009) their non-handled counterparts (Geers et al., 1995). Although pigs reared under standard intensive conditions were easier to load (Geverink et al., 1999), they were more reactive to transport and lairage at the abattoir compared to pigs reared in larger pens or in an enriched environment, with negative effects on drip loss (De Jong et al., 2000; Klont et al., 2001). Opposite results have also been reported (Lambooij et al., 2004). Outdoor rearing may further directly influence carcass and muscle composition, and consequently, meat quality. Genetic background may also influence stress reactivity at slaughter. To clarify some of these influences on pig production we have studied the interactive effects of i) genetic background and rearing environment on behaviour and stress reactivity during rearing and at slaughter and ii) genetic background, rearing environment, slaughter conditions and individual stress reactivity on technological meat quality traits. Effects of outdoor rearing increase with time spent outdoors (Oksbjerg et al., 2005; Carrapiso et al., 2007). In addition, the production of heavy weight pigs using outdoor rearing could have a market interest. Therefore, the rearing period was extended and pigs were slaughtered at 150 kg. 2. Materials and methods 2.1. Animals and housing The experiment was organised in two replicates, one per year (2002 and 2003). Each year, 10 Large White Landrace sows were inseminated with mixed semen from pure Duroc and Large White sires (PIC/Genus Plc Hampshire, UK). Each year, from the about 100 piglets, 48 median weight piglets were selected, based on fatherhood. The aim was to carry out identical replicates, but for unknown reasons, less piglets were born from Large White than Duroc sires for the second replicate. Consequently, the second year only 12 Large White sired pigs could be selected, and 36 Duroc-sired pigs were selected for completion. Pigs were born on the rearing site in an outdoor farrowing system. They were weaned at 4 weeks and kept indoors until fattening at 2 months of age. Each year, half of each genetic type was fattened indoors in four m pens with slatted floors in a heated (25 C) and ventilated animal room of m. The other half was fattened outdoors on 850 m 2 parcels on sandy soil, in which pigs could root but on which grew too little grass to contribute significantly to their nutrition. The near-absence of pasture for outdoor pigs is not unusual in certain regions or countries, where depending on season, rain may be sparse (e.g. France or Spain). Each parcel contained a 7.5 m 2 hut, a trough, a bowl drinker and a mud hole with water (pool). There were 6 pigs (3 females, 3 castrated males) of a single genetic type to a pen or parcel. This implies that in 2002, 2 indoor pens and 2 outdoor parcels with Duroc-sired pigs, and 2 indoor pens and 2 outdoor parcels with Large White pigs were reared. In 2003, 3 indoor pens and 3 outdoor parcels with Duroc-sired pigs, and 1 indoor pen and 1 outdoor parcel with Large White pigs were reared. Pigs were put into the fattening system in April and slaughtered in October. Outdoor pigs were fed ad libitum, indoor pigs were restricted by 10% in order to standardise slaughter weight at similar ages. Liquefied food was distributed at 6 and 18 h Behaviour and physiology Throughout the fattening period, undisturbed behaviour (Table 1) was observed every 14 days from 8h00 to 18h00. Every 20 min, for each pig, position, posture, and activity were recorded (time sampling technique), either by direct observation in the field (outdoor) or indirectly with a camera fitted above each pen (indoor). For position, indoor pens were divided in equal halves by an imaginary line distinguishing a dunging and feeding area. In the fields, a trough/drinker area and pool area were identified, both representing about 50% of the total area. Within the trough/drinker area, the hut and a hut area (2 m surrounding the hut) were also identified. Table 1 Definitions of postures and activities recorded during observations of undisturbed behaviour, the isolation and unfamiliar object tests, and preslaughter mixing. Posture a Standing Sitting Lying on the sternum, head lifted Lying on the sternum, head resting Lying on the side Resting on 3 or 4 hoofs Resting on thighs and fore hoofs Resting on belly and sternum, head not resting on the floor Resting on belly and sternum, head resting on the floor Resting on flanks Activities a Walking Moving at least 3 legs Sniffing and/or touching substrates b Rooting disk close to and/or touching surfaces or objects Vigilant None of the above overt activities and eyes open Inactive None of the above overt activities and eyes closed Undisturbed behaviour only Eating from trough Ingesting food delivered by animal caretaker Rooting Outdoor: rooting disk lifting soil Bathing Outdoor: at least 2 legs in the pool Aggressive interactions Pushing and/or biting another pig apparently with the aim to displace it Non-aggressive Touching and/or sniffing another pig with the interactions rooting disk or head apparently with no objective to displace it Unfamiliar object test only Close to object Pig in pen half containing the traffic cone Away from object Pig in pen half not containing the traffic cone Looking at object Head lifted, oriented towards the traffic cone Approach object Walking towards traffic cone Avoiding object Avoiding the object which is moving (dangling) Object in sight Any activity apart from the above while head oriented towards the traffic cone Pre-slaughter mixing only Fight Mutual pushing and biting between two pigs Head-knock c Intentional knock with the head on any part of the body Threat c Threatening head movement without obvious physical contact Push c Physical displacement of a pig by another pig Bite c Biting of any part of the body Escape c Moving away from a pig showing any of the activities above Inactive Any posture, no overt activity a For undisturbed behaviour, isolation and unfamiliar object tests, and preslaughter mixing. b Floor, wall, bars, trough, drinker, hut, soil. c Receiver and perpetrator were recorded.

3 C. Terlouw et al. / Livestock Science 122 (2009) At the age of 4 to 5 months, each pig was subjected to a 5 h isolation test to measure responses to a longer-term aversive situation. Each indoor pig was taken individually from its rearing pen and introduced in a 2 3 m pen in an unfamiliar pig house. Each outdoor pig was introduced into the 2 3 m space of an unfamiliar small lorry situated at 10 m from the rearing fields. Before introduction, each pig was transported in a trolley from its home pen to the isolation area for a similar duration (3 min) to standardise physical effort of indoor and outdoor pigs. To assess responses to relatively acute aversive events, at the age of 7 months, pigs were subjected to an unfamiliar object test in a 2 3 m test arena, different from the area used for the isolation test. Indoor pigs were tested in a pen adjacent to their animal room and outdoor pigs were tested in a test pen temporally erected in their rearing field for this purpose. Each pig was individually taken from its group and introduced into the test area where it was left undisturbed for 3 min. Subsequently an unfamiliar object (a 60 cm high white and orange traffic cone) was lowered from the ceiling (indoor pigs) or from a 4 m high perch (outdoor pigs) over 2 to 3 s and left dangling 5 cm above the floor or ground, for a period of 7 min. During both tests, indoor and outdoor pigs could hear, but not see other pigs. Tests were conducted in the morning at temperatures below 25 C. For both tests, pigs were fitted with a Polar heart rate monitor (Monitor, Anglet, France), which recorded heart rate averaged over 5 s intervals. Above each test area, a camera was fitted to record behaviour (Table 1). For the isolation tests, tapes have been analysed by recording every 10 min, during 1 min, posture, activity and where relevant, substrate (continuous recording). For the unfamiliar object tests, tapes were analysed using continuous recording, from the moment of lowering of the object. Behavioural analyses used The Observer 5.0 software (Noldus, Wageningen, Netherlands). At the age of 6 months, basal heart rate was measured in the morning by fitting on 4 pigs at the time the heart rate monitoring equipment while they were left undisturbed in their rearing environment. The experimenter waited for pigs to lie down for 15 min to use heart rate measurements obtained over the last 5 min of this period Slaughter Pigs were slaughtered at the age of about 8 months in a small local abattoir after a fasting period of 24 h. They were stunned on the floor with manually operated tongs (Morphée, Lelong & Cie, Savigny Le Temple, France, 220 V). For each experimental year, slaughter took place on two different days, at one-week interval, balanced as best as possible for rearing conditions, genetic background and live weight. Thus, the first year, each slaughter day, pigs of two pens (1 of each breed) and pigs of two parcels (1 of each breed) were slaughtered. The second year, one slaughter day contained two pens and the other, two parcels of the same breed, as Large White pigs were partly lacking. Half of each rearing group was slaughtered following mixing (1.5 h) in an unfamiliar pen on the rearing site followed by 45 min of transport and overnight lairage in 5 5 m stocking pens in the abattoir. Mixing groups contained 3 indoor and 3 outdoor individually marked pigs of 4 different rearing groups. This implies that each slaughter day, 12 unmixed, non-lairaged pigs, and 2 mixed groups each containing 6 pigs, were slaughtered. One of the mixing groups contained 1 pig from one pen and 1 pig from one parcel, and two pigs from the other pen and two from the other parcel. The other mixing group was constructed the other way around. Mixing groups were balanced for gender. Pigs of the mixing/lairaging treatment were mixed at 16h30 the day before slaughter, and loaded at 18h00. Behaviour during mixing was recorded during the first 60 min with the help of a video camera overlooking the mixing pens to subsequently quantify duration of aggressive interactions (Table 1), while indicating receiver and perpetrator, over 15-min periods (continuous observation. The other half of the rearing groups was not mixed, and loaded and transported (45 min) the next morning, that is, on the morning of slaughter, to be slaughtered immediately upon arrival Muscle sampling and measurements Muscle temperatures of the Longissiumus Lumborum (LL), Semispinalis Capitis (SC), Semimembranosus (SM) were measured at 25 and 45 min after bleeding, using a thermocouple (98004PK) connected to an electronic thermometer (Sefram 9810, St-Etienne, France). Three gram samples of the three muscles were taken 45 min after bleeding of which 2 g was ground immediately in 18 ml of iodoacetate 5 mm, to measure ph of the homogenate with a glass electrode (Mettler Toledo, Switzerland), connected to a portable ph meter (Schött-Geräte, Germany). The remaining part of the sample was immediately frozen in liquid nitrogen and stored at 80 C for later assaying of lactate and glycogen. Skin damage was evaluated in the rear, middle and shoulder area of each half carcass using the scale ranging from 0 (intact carcass) to 3 (many and/or profound lacerations) described by Barton- Gade et al. (1996). Carcasses were cut subsequently. Twentyfour hours after slaughter, a 1-g sample was taken of each muscle and stored as above for subsequent lactate and glycogen (i.e. residual glycogen) assays. Temperature, ph, colour (a, b ) and lightness (L ) of the freshly cut surfaces were directly recorded on the tissue of the three muscles. Colour was measured using a Minolta Chromameter (CR-300, Minolta Corp., Osaka Japan) equipped with a 0 viewing angle and using illuminant C. Measurements were repeated on days 4 and 8 after slaughter for the LL and SC muscle. To evaluate drip losses, a 2-cm-thick slice of the LL was cut on the level of the last rib 24 h after slaughter, weighed and suspended in a plastic bag and left at 4 C. The amount of water lost was measured 72 (drip loss d3) and 120 h later (drip loss d5) and expressed as a percentage of the initial sample weight (i.e. 24 h post-mortem). To evaluate thawing losses, 24 h postmortem, a 7-cm-thick sample was cut from the LL, weighed, vacuum packed, and frozen after ageing (4 days after slaughter). About 4 weeks later, samples were thawed overnight at +4 C, unpacked, and weighed without the water lost. Thawing losses were expressed as the percentage of the initial sample weight. Samples were subsequently cut to a standardised size (5 8 4 cm.), weighed and vacuum packed. They were then heated in a water bath at 70 C for 50 min and cooled in a +4 C water bath. They were then unpacked and weighed after removal of precipitated protein. Cooking losses were expressed as the percentage of the initial weight of the standardised-cut sample.

4 202 C. Terlouw et al. / Livestock Science 122 (2009) The day after slaughter, a 2-cm think slice was taken from the LL at 11 cm from the last rib and ground with a food processor (Moulinex 76 type 320, SEB, Ecully, France). Two 5-g samples were taken from the ground meat and kept in a stove at 105 C for 24 h. Dry matter was expressed as percentage of weight after drying relative to initial weight, averaged over the two samples. Backfat thickness was measured over the middle of the LL on the cut at the level of the last rib and over the middle of Trapezius muscle on the cut in the shoulder region between the third and forth vertebra in sternum Glycogen and lactic acid assays Two hundred mg of lyophilised muscle was ground and suspended in 10 ml of perchloric acid 0.5 M. After hydrolysis of the glycogen by amyloglucosidase (Bergmeyer, 1974), glucose content of the homogenate was determined. Lactic acid was determined on the supernatant after centrifugation (20 min at 2500 g) of the perchloric homogenate following Bergmeyer's (1974) procedure. Concentrations are expressed in µmol/gram of fresh tissue. Glycolytic potential, the sum of compounds likely to produce lactic acid post-mortem, was calculated using the formula proposed by Monin and Sellier (1985) and expressed as µmol lactate equivalents/g of fresh tissue Statistical analysis Average frequencies of posture, behaviour and place were calculated over the whole rearing period (10 observation days) over 10-hours per rearing system, for an overall analysis (Chi-square). The six skin damage scores obtained for each carcass served to calculate average skin damage score per pig. Analysis of variance using SAS software (version 9, SAS Inst., Inc; Cary, NC) was used to study treatment effects on heart rate and behaviour during the isolation and unfamiliar object tests (per hour and per minute, respectively) and on meat quality aspects. To study behavioural and physiological data obtained during rearing, the analysis of variance (mixed models) contained 4 inter-individual effects (2 experimental years, 2 genetic types, 2 rearing systems, and 2 sexes). Rearing group was included in the analysis of variance as random effect to control for rearing group effects. To study carcass and meat quality data, the analysis of variance contained, in addition, 2 slaughter conditions. Mixing group was added as random effect to analyse behaviour during mixing (analysis included only mixed pigs). Where relevant, t-tests or subset analyses were used to identify differences. Time effects (heart rate) were studied with analysis of variance for repeated measures. Relationships between variables were studied with analysis of co-variance and Pooled Pearson correlations. Standing, locomotion or touching behaviour were introduced as co-variables in the analysis of variance to study relationships between general activity level, specific activities or heart rate, and breed and rearing differences. Glycolytic potential, fat cover, ph, temperature and level of skin damage were introduced as co-variables to study relationships with meat quality parameters. Pooled Pearson correlations were calculated over i treatment groups and j observations using the formula (x ij x i )(y ij y i )/M( (x ij x i ) 2 (y ij y i ) 2, where x i and y i are means of the ith treatment group. As these correlations are based on treatment rather than overall means they allow to pool data over treatments while correcting for treatment effects (Crunch manual, 1991). If a variable was correlated with several other variables, multiple step-by-step forward regression was used to determine whether effects were complementary, or, if there were treatment effects to control for, they were simultaneously introduced as co-variables in the analysis of variance. Three, four and five-way interactions are not reported. 3. Results 3.1. Behaviour and physiology Undisturbed behaviour and heart rate Housing system influenced behaviour. Differences in posture did not reach statistical significance, despite the fact that outdoor pigs were more often standing both during season 2002 (42.3 vs 21.5% for indoor pigs) and 2003 (29.3 vs 17.5% for indoor pigs). Main activities defined as occupying more than 2.5% of time differed (Chi-square) at the p=0.07 level. For outdoor pigs, main activities were being vigilant (2.7%), eating from trough (3.8%), rooting (5.7%), walking (6.1%), sniffing/touching (6.6%), bathing (9.3%; 2.7 and 16.0% for the first and second replicate, respectively), and inactive (41.5%). Behaviour of pigs in the hut could not be recorded (14.4%). For indoor pigs, main activities were non-aggressive interactions with other pigs (2.6%), eating from trough (5.2%), walking (3.3%), sniffing/touching of pen fittings (13.9%), and inactive (68.4%). Aggressive and non-aggressive interactions showed also different patterns according to housing conditions (pb0.01), primarily because much less non-aggressive interactions were observed in outdoor (0.4%) than in indoor pigs. Aggressive interactions were low in both housing systems (b0.03%). Indoor pigs spent slightly more time in the feeding (55%) than in the dunging area (45%). Outdoor pigs spent 32% of time in the pool area, 26% in the hut, 24% in the trough/drinker area and 18% within 2 m of the hut. Basal heart rate was on average 93.4±1.4 beats per min and did not differ according to any of the treatment factors Isolation and unfamiliar object tests Effects are only reported if the first and second replicate showed similar tendencies. Considering the whole period of 5 h of isolation, compared to outdoor pigs, indoor pigs were less often lying on the side (31.6±4.2% vs 45.3±4.4%; pb0.01) and more often on the sternum with the head lifted (19.3±6.7 vs 5.2±2.8%; p=0.05). Indoor pigs were generally more active. They tended to perform more walking (2.46±0.42 vs 1.47± 0.33%; p=0.07), moresniffing/touching substrates (13.55±1.77 vs 9.24±1.59%; pb0.01) and more of these combined activities ( walking while sniffing/touching : 2.12±0.38 vs 0.78±0.21%; pb0.0001). Differences in resting behaviour and activities were apparent for each of the 5 h, but generally significant during the first 3 h only. Indoor pigs stood significantly more during the second hour (pb0.005). After correction for the higher standing levels of indoor pigs by introducing standing levels as a covariable (pb0.001) into the analysis, the effect of rearing conditions on sniffing and/or touching substrates, and walking while sniffing/touching remained significant (rearing effect: pb0.05 and p=0.02, respectively).

5 C. Terlouw et al. / Livestock Science 122 (2009) Fig. 1. Average heart rate of indoor (grey rhombuses) and outdoor pigs (black squares) during 5 h of isolation. Bars indicate standard errors of means. Heart rate was high during the first hour of isolation and declined significantly (pb0.0001) over time (Fig. 1). No consistent effects of rearing conditions or genetic background were found. Considering heart rate with respect to the initial heart rate response to the test situation (first 5, 10 or 15 min of isolation), by fitting initial responses as a co-variable in the analysis, showed significant correlations (e.g. second hour: pb0.01). This analysis showed lower heart rates for outdoor pigs the first, (p=0.03) second (p=0.05), third (p=0.08) and fourth hour (p=0.02) of isolation (c.f. Fig. 1). Analysis of co-variance found that heart rate was positively correlated with sniffing/touching substrates (pb0.001). Indoor pigs had more (pb0.01) often the unfamiliar object in sight while performing activities unrelated to the object (46.9±2.8%) and touched the object more (pb0.05) often (23.3±2.3%), than outdoors (object in sight: 32.5±3.1%; touch: 14.5±2.0%). Total amount of activities directed towards the object (looking at, touching, sniffing, avoiding) was higher (p=0.03) for indoor (44.7±3.3%) than outdoor pigs (28.1± 1.7%). Duroc-sired tended to have lower (p=0.08) heart rates (128.4±3.0) than Large White-sired pigs (137.8±6.2) before lowering of the unfamiliar object (Fig. 2) Pre-slaughter mixing Outdoor reared pigs spent more (pb0.01) time inactive (93.7±1.2 vs 84.4±2.1%) and were less time engaged in Fig. 2. Heart rate during the unfamiliar object test for Large White (black circles) and Duroc (grey circles) sired pigs. Bars indicate standard errors of means. The arrow indicates the moment of lowering of the unfamiliar object. aggressive interactions: they spent less (pb0.05) time in head knocks (0.14±0.11%) than indoor pigs (0.86±0.23%) and (pb0.01) in fighting (1.5± 0.6 vs 8.3± 2.0%). Treatment factors did not affect posture (pn0.25). Aggressive interactions took mostly place during the first 15 min of mixing; for example, fighting (time effect: pb0.001) represented 12.7± 3.1; 4.6±1.7; 2.2±0.6; 0.83±0.3% of time over the four 15- minute-periods, respectively Carcass and meat quality The growing curve differed (pb0.0001) between the first and second replicate with respect to rearing conditions. The first year, the fourth month of fattening, outdoor pigs had a higher (pb0.01) average daily gain (1.01±0.05) than indoor pigs (0.78±0.06 kg/day). The second year no differences were observed. Consequently, the first year, carcass weight of outdoor pigs (134.9±2.4 kg) was higher (pb0.05) than of indoor pigs (123.0±4.2 kg) and than those of the second year (indoors: 123.4±2.1; outdoors: 122.9±2.3 kg). Castrated males had more (pb0.001) subcutaneous fat over the LL (2.2±0.1 mm) than females (1.6±0.1 mm). Effect of rearing conditions on subcutaneous fat depended on replicate (interaction: pb0.01). The first year, outdoor pigs had more (pb0.01) subcutaneous fat on the LL (2.4±0.2 mm) and Trapezius (3.3±0.3 mm) than indoor pigs (LL: 1.8±0.1; Trapezius: 2.5±0.2 mm). The second year, outdoor pigs had less (pb0.02) fat on the Trapezius (1.9±0.1) than indoors (2.0±0.1), while fat cover of the LL showed no difference (p=0.17: 1.6±0.1 and 1.9±0.1 for outdoor and indoor pigs, respectively). The LL contained more (pb0.02) dry matter the first (73.2±0.1%) than the second year (72.6±0.2%). The LL of indoor pigs contained more (pb0.001) dry matter (73.3± 0.2%) than outdoor pigs (72.5±0.2%). Indoor pigs of the mixing/lairage procedure had higher separate and overall skin damage scores (1.28±0.18) than outdoor pigs (0.64 ±0.13) of the same slaughter procedure (pb0.01), while levels were low for indoor (0.10±0.04) and outdoor pigs (0.04 ±0.02) of the no mixing/no lairage group. Tables 2 6 summarise main and 2-way interactive treatment effects on post-mortem muscle traits. For most of the meat quality parameters, treatment effect depended on replicate and muscle. For example, compared to the second

6 Table 2 Average values per treatment and p-values of significant main and 2-way interactive treatment effects for post-mortem traits of the Longissimus lumborum. Longissimus lumborum Time post-mortem Glycolytic potential Temperature ph Glycogen Lactate 25 min 45 min 24 h 45 min 24 h 45 min 24 h 45 min 24 h Rearing conditions Indoors 115.7± ± ± ± ± ± ± ± ± ±2.4 Outdoors ± ± ± ± ± ± ± ± ± ±1.8 Sire breed LW ± ± ± ± ± ± ± ± ± ±2.6 D 111.5± ± ± ± ± ± ± ± ± ±1.8 Gender Castrated males 112.5± ± ± ± ± ± ± ± ± ±1.9 Females 123.7± ± ± ± ± ± ± ± ± ±1.5 Slaughter conditionsmixing/lairage 110.4± ± ± ± ± ± ± ± ± ±2.2 No mixing/no 125.5± ± ± ± ± ± ± ± ± ±2.0 lairage Replicate Year ± ± ± ± ± ± ± ± ± ±1.5 Year ± ± ± ± ± ± ± ± ± ±2.2 p-values of significant effects Rearing conditions 0.05 Sire breed Gender Slaughter conditions Replicate b C. Terlouw et al. / Livestock Science 122 (2009) Interactions with replicate (p-value) Other interactions (p-value) rearing (0.002) Rearing slaughter (0.04) sire breed (0.01) rearing (0.001) sire breed (0.01) Bold and italic characters indicate larger values where significant and near-significant treatment differences were found, respectively. rearing (0.001) Sire breed gender (0.02) Replicate rearing (0.002) Rearing slaughter (0.01) Sire breed gender (0.005) sire breed (0.01) gender (0.0007)

7 Table 3 Average values per treatment and p-values of significant main and 2-way interactive treatment effects for post-mortem traits of the semimembranosus. Semimembranosus Glycolytic potential Glycolytic Temperature ph Glycogen Lactate Time post-mortem potential 25 min 45 min 24 h 45 min 24 h 45 min 24 h 45 min 24 h Rearing conditions Indoors 108.6± ± ± ± ± ± ± ± ± ±2.5 Outdoors 120.4± ± ± ± ± ± ± ± ± ±2.0 Sire breed LW 122.6± ± ± ± ± ± ± ± ± ±2.3 D 110.4± ± ± ± ± ± ± ± ± ±2.1 Gender Castrated males 111.4± ± ± ± ± ± ± ± ± ±2.1 Females 117.6± ± ± ± ± ± ± ± ± ±1.3 Slaughter conditions Mixing/lairage 109.0± ± ± ± ± ± ± ± ± ±2.7 No mixing/no lairage 119.4± ± ± ± ± ± ± ± ± ±1.7 Replicate Year ± ± ± ± ± ± ± ± ± ±1.6 Year ± ± ± ± ± ± ± ± ± ±2.2 p-values of significant effects Rearing conditions Sire breed Gender Slaughter conditions C. Terlouw et al. / Livestock Science 122 (2009) Replicate b b b Interactions with replicate rearing (0.01) rearing (0.01) rearing (0.01) Replicate rearing (0.02) slaughter (0.003) Replicate rearing (0.01) Other interactions (p-value) Rearing gender (0.01) Bold and italic characters indicate larger values where significant and near-significant treatment differences were found, respectively. 205

8 Table 4 Average values per treatment and p-values of significant main and 2-way interactive treatment effects for post-mortem traits of the Semispinalis capitis. Semispinalis capitis Time post-mortem Glycolytic potential Temperature ph Glycogen Lactate 25 min 45 min 24 h 45 min 24 h 45 min 24 h 45 min 24 h Rearing conditions Indoors 59.4± ± ± ± ± ± ± ± ± ±1.9 Outdoors 76.6± ± ± ± ± ± ± ± ± ±1.3 Sire breed LW 69.5± ± ± ± ± ± ± ± ± ±1.7 D 67.3± ± ± ± ± ± ± ± ± ±1.5 Gender Castrated males 64.9± ± ± ± ± ± ± ± ± ±1.8 Females 71.2± ± ± ± ± ± ± ± ± ±1.7 Slaughter conditions Mixing/lairage 59.8± ± ± ± ± ± ± ± ± ±1.4 No mixing/no lairage 76.8± ± ± ± ± ± ± ± ± ±1.8 Replicate Year ± ± ± ± ± ± ± ± ±1.3 Year ± ± ± ± ± ± ± ± ± ±1.5 p-values of significant effects Rearing conditions Sire breed 0.07 Gender Slaughter conditions b Replicate 0.02 b b b Interactions with replicate rearing (0.03) rearing (0.04) slaughter (0.01) rearing (0.0001) rearing (0.01) rearing (0.04) Other interactions (p-value) Slaughter gender (0.01) Slaughter gender (0.004) Slaughter gender (0.04) Rearing slaughter (0.001) Rearing sire breed (0.02) sire breed slaughter (0.05) slaughter gender (0.002) Sire breed gender (0.02) Bold and italic characters indicate larger values where significant and near-significant treatment differences were found, respectively. 206 C. Terlouw et al. / Livestock Science 122 (2009)

9 C. Terlouw et al. / Livestock Science 122 (2009) Table 5 Average values±standard errors of means for the Longissimus lumborum of Large White and Duroc-sired pigs according to gender (top: glycogen 24 h post-mortem) and experimental year (bottom: ph 45 min and 24 h postmortem). Sire breed Gender Glycogen 24 h Large White Castrated males 11.4±1.3 a Females 15.8 ±1.7 b Duroc Castrated males 9.6 ±0.8 a Females 9.4 ±0.8 a Sire breed Year ph 45 min ph 24 h Large White ±0.04 a 5.51±0.05 ab ±0.05 b 5.48±0.03 a Duroc ±0.03 a 5.52 ±0.03 ab ±0.03 a 5.59 ±0.03 b Within columns, values with different subscripts (a, b) differ significantly (ttest: pb0.05). year, the first year, glycolytic potential was higher for the LL and lower for the SC muscle. Similarly, the first year, ph at 45 min post-mortem was higher for the LL and lower for the SM muscle, compared to the second year. Finally, replicate showed many interactions with rearing conditions, and to a much lesser extent with slaughter conditions, sire breed or gender. The most consistent across replicate and muscle effect was the effect of slaughter conditions. Pigs that were mixed and lairaged had lower glycolytic potential and residual glycogen levels and higher ph 24 h post-mortem (ultimate ph) in the 3 muscles. In the case of the SC, ultimate ph was influenced by a slaughter conditions rearing conditions gender interaction (p=0.02) as mixing and lairaging had a greater effect in indoor castrated males than indoor females (Fig. 3). Large White-sired pigs had higher SM and LL glycolytic potential and SM residual glycogen than Duroc-sired pigs. LL residual glycogen was only higher in female Large Whitesired pigs (Tables 2 5). These effects did not result in overall differences in ultimate ph, but the second year, Large Whitesired pigs had lower (pb0.01) ph 45 min post-mortem and ultimate ph (pb0.02) than Duroc-sired pigs (Table 5). Rearing conditions influenced also muscle glycogen content, and sometimes, ultimate ph. Compared to indoor pigs, in outdoor pigs, the SM and, the second year, the LL and SC had higher glycolytic potential. Indoor reared pigs had less LL and SM residual glycogen and higher ultimate ph, especially the second replicate (Tables 2, 3, and 6). SM and SC muscles of outdoor pigs had higher glycogen levels (45 min postmortem) the second replicate (Tables 3, 4 and 6). LL and SC muscles of castrated males had lower glycolytic potential, and higher ultimate ph. Despite a sire breed gender interaction for the SC ultimate ph, the t-test found no significant differences in pair-wise comparisons. Castrated males had sometimes higher muscle temperature (LL, SC) but the opposite tendency (SM) could also be observed (Tables 2 4). Variations in LL ultimate ph were mostly explained by variations in muscle glycogen content at the moment of slaughter, as glycolytic potential introduced as a co-variable (pb0.01) in the analysis of variance removed the effect of rearing conditions (p=0.18) and most of the effect of slaughter conditions (p=0.08). Slaughter weight was not correlated with any of the meat quality traits. LL fat cover used as co-variable, was negatively correlated (pb0.05) with LL and SM glycolytic potential, and removed the effect of rearing system (p=0.13) and gender (p=0.44) on LL glycolytic potential. Slaughter, and to a lesser extent rearing conditions influenced colour (Table 7). Meat produced by the mixing/ lairage group was overall darker (lower L -values), redder (higher a -values) and more yellow (higher b -values). Outdoor reared pigs produced redder (SM, LL) and more yellow meat (LL). There were no significant treatment interactions for L, a and b values. Effects persisted over time: the LL of the no mixing/no lairage and outdoors group showed still higher (pb0.05) b values 4 and 8 days postmortem, and a values were higher (p=0.01) for outdoor pigs 4 days post-mortem. Introduction of ultimate ph as covariable into the analyses of L, a and b values of the 3 muscles found significant correlations (pb0.05) but did not remove the treatment effects. LL fat cover used as co-variable was positively (pb0.05) correlated with LL and SM redness, but did not remove treatment effects. Drip loss after 72 h suspension and cooking loss were higher for LW than D sired pigs (Table 8). Drip loss after 72 h but not after 96 h suspension was correlated (pb0.001) with LL ultimate ph (r= 0.54) and ph 45 min post-mortem (r= 0.42). When simultaneously introduced as co-variables in the analysis variance of drip loss (72 h) both ultimate ph (pb0.001) and Table 6 Average values±standard errors of means of various meat quality traits for the two experimental years and rearing conditions, for the Longissimus lumborum, Semimembranosus and Semispinalis capitis muscles. Muscle Year Rearing conditions Glycolytic potential Glycogen 45 min Glycogen 24 h ph 24 h Longissimus lumborum 2002 Indoors ±5.4 a 11.8 ± 1.1 a 5.52 ±0.03 a Outdoors ±4.8 ab 12.2 ±0.9 a 5.51±0.04 a no interaction 2003 Indoors 98.6±4.3 c 7.1±0.3 b 5.63 ±0.04 b Outdoors 119.0±4.4 b 12.8 ±0.9 a 5.49±0.02 a Semimembranosus 2002 Indoors 42.7 ±2.1 ab 11.8±1.4 a 5.60 ±0.02 ab Outdoors 41.8±2.5 bc 14.5 ±1.9 a 5.61±0.06 ab no interaction 2003 Indoors 36.0 ±2.4 c 7.5 ± 1.4 b 5.66±0.05 a Outdoors 48.7±2.4 a 14.8 ±0.8 a 5.54 ±0.01 b Semispinalis capitis 2002 Indoors 62.2±4.2 a 14.6±1.9 a 2.0 ±0.5 a Outdoors 62.5±3.6 a 16.1 ± 1.9 a 3.8 ±1.0 a 2003 Indoors 57.1±5.0 a 17.2±2.3 a 3.7 ±0.9 a No interaction Outdoors 90.2±6.3 b 36.3 ±3.0 b 21.7±2.3 b Within columns and muscles, values with different subscripts (a, b, c) differ significantly (t-test: pb0.05). Data are not presented if the treatment interaction was not significant.

10 208 C. Terlouw et al. / Livestock Science 122 (2009) Fig. 3. Ultimate ph of the Semispinalis capitis according to rearing and slaughter conditions, for castrated males (white and grey bars) and females (white hatched and grey hatched bars). Bars with different subscript (a, b, c) differ significantly. ph 45 min post-mortem (p=0.01) were significant (pb0.01) and removed the effect of sire breed (p=0.31). Thawing loss was positively correlated with temperature 45 min post-mortem (r=0.24;pb0.05) and negatively with ultimate ph (r= 0.51; pb0.0001). Both were simultaneously significant co-variables in the analysis of variance of thawing loss (pb0.001). Introduction of these co-variables introduced an effect of slaughter conditions (p=0.05). Cooking loss was negatively correlated with LL ultimate ph (r= 0.39; pb0.001); ultimate ph as co-variable in the analysis of variance of cooking loss partly removed the effect of sire breed (p=0.08). There were no significant treatment interactions on water holding capacity related parameters Behaviour and meat quality Total level of skin damage was positively correlated with time spent fighting (r=0.70; pb0.001) and negatively with time spent inactive (r= 0.49; pb0.05) and threatening other pigs (r= 0.46; pb0.05) during pre-slaughter mixing. In mixed/lairaged outdoor pigs, levels of skin damage were correlated (pb0.05) with LL (r= 0.65) and SM glycolytic potential (r= 0.56) and with (pb0.01) LL (r=0.62) and SM (r=0.82) ultimate ph. Mixed/lairaged outdoor pigs with less skin damage stood (r= 0.60; p=0.01), walked (r= 0.70; pb0.01; Fig. 4a) and touched/sniffed substrates (r= 0.49; pb0.05) longer during the social isolation test. Consequently, mixed/lairaged outdoor pigs showing more of these activities during isolation had higher LL glycogen content 45 min postmortem,(e.g. LLglycogenvs walking: r=0.70; pb0.01; Fig. 4b). Correlations obtained in mixed/lairaged indoor pigs are in accordance with those obtained for outdoor pigs. Level of skin damage was correlated with SC temperature 45 min post-mortem (r= 0.60; pb0.05), SM ultimate ph (r=0.49; p=0.06), andll drip loss (72 h) (r= 0.63; pb0.05). In these pigs, levels of sniffing and touching the floor during isolation were correlated with ultimate ph (LL, r= 0.55; pb0.05; SM, r= 0.45; p=0.07; SC,r= 0.57; pb0.05). Multiple regression on ultimate ph retained for the LL and SM sniffing and touching the floor Table 7 Average values±standard errors of means, and main effects of sire breed, rearing and slaughter conditions, gender and replicate on colour parameters, 24 h postmortem. Muscle Longissimus lumborum Semimembranosus Semispinalis capitis L a b L a b L a b Rearing conditions In 51.9± ± ± ± ± ± ± ± ±0.3 Out 53.0 ± ± ± ± ± ± ± ± ±0.3 Sire breed LW 52.9± ± ± ± ± ± ± ± ±0.4 D 52.2 ± ± ± ± ± ± ± ± ±0.3 Gender Castrated males 52.6± ± ± ± ± ± ± ± ±0.3 Females 52.3 ± ± ± ± ± ± ± ± ±0.4 Slaughter conditions Mixing/lairage 50.8± ± ± ± ± ± ± ± ±0.4 No mixing/no lairage 54.1± ± ± ± ± ± ± ± ±0.2 Replicate ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.2 p-values of significant effects Rearing conditions Sire breed Gender Slaughter Replicate 0.08 b There were no significant treatment interactions.

11 C. Terlouw et al. / Livestock Science 122 (2009) Table 8 Average values±standard errors of means, main and 2-way interactive effects of sire breed, rearing and slaughter conditions, gender and replicate on water losses. Drip loss 72 h post-mortem (% of initial weight) Drip loss between 72 and 96 h post-mortem (% of weight 24 h post-mortem) Thawing loss (% of weight before freezing) Breed of sire Large White sired 5.24± ± ± ±0.7 Duroc-sired 4.16± ± ± ±0.5 Rearing conditions indoor 4.01± ± ± ±0.5 outdoor 5.03± ± ± ±0.6 Slaughter conditions Mixing/lairage 4.57± ± ± ±0.6 No mixing/no lairage 4.47± ± ± ±0.5 Gender Castrated male 4.17± ± ± ±0.6 Female 4.87± ± ± ±0.5 Replicate ± ± ± ± ± ± ± ±0.5 p-values of significant main effects There were no significant treatment interactions. Breed of sire Rearing conditions Slaughter conditions Gender 0.05 Replicate Cooking loss (% of weight after thawing and before cooking) during isolation (pb0.05) and for the SC, total levels of skin damage at slaughter (pb0.05) in the model. The effect of slaughter conditions on LL and SC ultimate ph, LL glycolytic potential and on LL and SM residual glycogen depended on skin damage levels: introduction of skin damage levels as co-variable (pb0.02) in the analysis of variance including all pigs removed the effect of slaughter conditions (pn0.17). It did not remove the effect of rearing conditions (LL and SM residual glycogen) or sire breed (LL glycolytic potential and SM residual glycogen). Skin damage was negatively correlated (pb0.01) with drip loss (72 h), but this was due to its correlation with ultimate ph (pb0.01): introduction of ultimate ph as a co-variable in the analysis of variance (pb0.001) removed the effect of skin damage (p=0.10) on drip loss. No correlations were found between time spent fighting and behaviour during isolation or meat quality for either outdoor or indoor pigs after the mixing/lairage procedure. For pigs slaughtered following the no mixing/no lairage procedure, levels of standing during the isolation test were correlated (pb0.05) with LL (r=0.47) and SC(r=0.35) glycolytic portential. Behaviour during the unfamiliar object test showed no consistent correlations with behaviour during the isolation test or during pre-slaughter mixing, or with meat quality parameters. Heart rate after lowering the unfamiliar object was correlated with heart rate during the first, second, third fourth and fifth hour of the isolation test (e.g. first hour of isolation vs the 7 min in presence of the unfamiliar object: r=0.55; pb0.0001) and to a lesser extent to basal heart rate (r=0.28; p=0.07). Andresen (2003). Pigs appeared to prefer the field half containing the pool, but this was due to their bathing activity. When not bathing, time spent in the pool area was 23%, that is, similar to time spent in the trough/drinker area. Bathing was strongly related to weather conditions. For example, the second year of experimentation has known exceptionally high temperatures (first half of august temperatures N40 C) with very sunny weather. That year, bathing activity amounted to 16.0%, compared to 2.7% for the first replicate, suggesting that the presence of a pool may be particularly beneficial at higher temperatures as also reported by Stolba and Wood-Gush 4. Discussion As earlier demonstrated for pigs reared in enriched environments (Mendl et al., 1997; Johnson et al., 2001; Guy et al., 2002; Gentry et al., 2002b), outdoor pigs were more active and showed a larger range of activities than indoor pigs. The absence of feeding or grazing behaviour, reaching between 20 and 40% of observations in outdoor pigs under certain conditions (Stern and Andresen, 2003), is explained by the absence of grass or other feeding substrates in the present experiment. Amount of rooting in the outdoor pigs is similar to levels observed in ad libitum fed outdoor pigs by Stern and Fig. 4. Correlation between time spent walking during isolation (x-axis) and skin damage score (top; r= 0.70; pb0.01) and LL glycolytic potential (bottom; r=0.70; pb0.01) at slaughter for Large White (black circles) and Duroc-(grey circles) sired pigs.

12 210 C. Terlouw et al. / Livestock Science 122 (2009) (1989). Olsen (2001) and Olsen et al. (2001) found increased wallowing at higher temperatures, although it is not clear how time of day effects were accounted for the statistical analysis. They did show that pigs housed at the warmer south compared to the cooler north side spent more time making bubbles in the wallow water (Olsen et al., 2001). In the present study, non-aggressive interactions were observed less often in outdoor pigs, possibly because these took place in the hut, where animals could not be observed, or because outdoor pigs preferred other activities. Earlier work showed that the provision of a substrate such as straw increases interactions with the substrate (e.g. rooting) and decreases activities directed to other pigs (Day et al., 2002). One of the five freedoms of the Brambell report (1965) on animal welfare is the liberty to perform species-specific behaviour. The latter notion is difficult to define or to use in practical terms, as behaviour is in any case environment dependent. The increased behavioural richness and use of the whole surface of the field do suggest, however, better welfare of outdoor pigs from a behavioural point of view. In addition, pigs excrete mostly in areas where they do not forage or root (Stern and Andresen, 2003), a motivation they can only express if they have sufficient space. Heart rate increases due to physical effort or emotional stress. The similar heart rate response to introduction into the isolation pen, suggests that the physical and psychological disturbance related to the transport and handling was comparable in indoor and outdoor pigs. Outdoor pigs had a lower initial response to introduction into the test arena of the non-familiar object test which required walking a few meters for both in- and outdoor pigs, and thus little physical effort. This may indicate a lower emotional response of outdoor pigs, or alternatively, better cardiac resistance to this level of physical effort. Increased activity in a presumably aversive situation is often an indication of emotional stress (Boissy and Bouissou, 1995). Compared to outdoor pigs, indoor pigs were behaviourally more active during the first 3 h of the isolation test, until they settled down at levels similar to outdoor pigs. Their heart rate remained also more elevated compared to outdoor pigs. The positive correlation between sniffing/touching activities and heart rate may simply have a physiological background as respiratory rate and effort influence heart rate (Task Force, 1996). Indoor pigs kept the unfamiliar object more in sight and touched it more often than outdoor pigs. An earlier study found that indoor pigs explored more an unfamiliar object than outdoor pigs (Olsson et al., 1999). In the present study, the lower responsiveness and faster reduction in emotional and behavioural arousal in outdoor pigs may be related to lower arousal levels on a daily level, due to a better-adapted rearing environment. Alternatively, indoor pigs may be more reactive to environmental stimulation, as they were slightly food restricted (Brown et al., 1999; Campbell and Sheffield, 1953; Teghtsoonian and Campbell, 1960). Compared to Duroc, Large White-sired pigs had a slightly higher heart rate at the start of the unfamiliar object test, which may indicate that they were more aroused. Overall, the results indicate that rearing conditions and to a lesser extent genetic background influence behavioural and physiological reactivity to moderate stress-inducing situations, in accordance with earlier studies (Olsson et al., 1999; De Jong et al., 2000; Guy et al., 2002; Terlouw and Rybarczyk, 2008). In the present study, slaughter and rearing conditions, gender and genetic background influenced muscle glycogen levels sometimes resulting in changes in ultimate ph. Outdoor and Large White-sired pigs, as well as unmixed/nonlairaged pigs, had higher glycogen levels before and after slaughter. It is well known that slaughter conditions may have major effects on meat quality traits (Fernandez et al., 2002; Rosenvold and Andersen, 2003; Terlouw and Rybarczyk, 2008). The coherence of the effects across the two replicates of the present study is in line with this. Detailed analysis shows that some of the sire breed effects depended on gender with higher levels for female Large White sired pigs, and that the effects of rearing conditions were stronger the second year. Another study found differences in effect of outdoor rearing between replicates, but here, indoor rather than outdoor pigs had higher residual glycogen the second year of experimentation (Heyer et al., 2006). This could be related to the fact that in the latter study, indoor and outdoor pigs had different feeding regimes. The decrease in dry matter in outdoor compared to indoor pigs was also found in the Iberian breed (Cava et al., 2000; Carrapiso et al., 2007), and believed to be related to the effect of increased intramuscular fat content on water diffusivity (Ruiz-Cabrera et al., 2004). The increased redness in outdoor pigs in the present study is consistent with several studies (Bee et al., 2004; Gentry et al., 2002c, 2004; Carrapiso et al., 2007). In contrast, Heyer et al. (2006) found no differences in redness between indoor and outdoor pigs, while Millet et al. (2005) found that giving pigs access to a court yard reduced rather than increased LL redness. Increased exercise often increases proportions of oxidative muscle fibres (Lefaucheur et al., 2002) and consequently redness, but Gentry et al. (2002b) found that forced exercise of indoor pigs did not influence fibre type composition. They suggested that other aspects of outdoor rearing explain the effects on muscle redness; increased proportions of oxidative fibres seem to be due to spontaneous rather than forced exercise (Petersen et al., 1998), and pigs may ingest iron or plants (Gentry et al., 2004). In accordance with present results, Heyer et al. (2006) found increased yellowness in the LL of outdoor pigs, but only the second experimental year. The decreased redness in mixed, lairage pigs, compared to their counterparts, is also coherent with another study showing that increased pre-slaughter effort and/or emotional stress reduced a values (Terlouw and Rybarczyk, 2008). However, the effect of slaughter stress on redness seems to depend on the muscle and other factors (Rosenvold and Andersen, 2003; Fernandez et al., 2002). Variations in colour measurements were related to ultimate ph for all studied muscles, which is coherent with earlier research (Renerre, 1990; Lindahl et al., 2006). However, differences between treatments were only partly explained by variations in ultimate ph. This suggests that rearing and slaughter conditions influenced other muscle characteristics, possibly anti-oxidative capacity, NADH availability or lipid content, which in turn influenced colour (Renerre, 1990; Rosenvold and Andersen, 2003; Lindahl et al., 2006). This is consistent with the correlation between LL fat cover and LL and SM redness observed in the present study. Duroc-sired pigs had better water holding capacity than Large White sired pigs. This is in accordance with an earlier study showing that Duroc-sired pigs had better water holding