Meat quality in relation to swine well-being after transport and during lairage at the slaughterhouse

Accepted Manuscript Meat quality in relation to swine well-being after transport and during lairage at the slaughterhouse

L. Rey-Salgueiro, E. Martinez-Carballo, P. Fajardo, M.J. Chapela, M. Espiñeira, J. Simal-Gandara PII: DOI: Reference:

S0309-1740(17)31510-3 doi:10.1016/j.meatsci.2018.04.005 MESC 7517

To appear in:

Meat Science

Received date: Revised date: Accepted date:

2 December 2017 3 April 2018 6 April 2018

Please cite this article as: L. Rey-Salgueiro, E. Martinez-Carballo, P. Fajardo, M.J. Chapela, M. Espiñeira, J. Simal-Gandara , Meat quality in relation to swine well-being after transport and during lairage at the slaughterhouse. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Mesc(2017), doi:10.1016/j.meatsci.2018.04.005

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ACCEPTED MANUSCRIPT Meat quality in relation to swine well-being after transport and during lairage at the slaughterhouse

Running title: Corticosteroids and meat quality

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L. Rey-Salgueiro,a E. Martinez-Carballo,a P. Fajardo,b M.J. Chapela,b M. Espiñeirab and

Nutrition and Bromatology Group, Department of Analytical and Food Chemistry;

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a

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J. Simal-Gandaraa,*

Faculty of Food Science and Technology, University of Vigo – Ourense Campus, E-32004

ANFACO-CECOPESCA, Carretera Colegio Universitario, 16, Vigo, Spain.

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b

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Ourense, Spain.

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Corresponding author: E-mail address: [email protected] (J. Simal-Gandara).

ACCEPTED MANUSCRIPT Abstract Cortisol and corticosterone in saliva were evaluated as pig stress biomarkers, using pig genotype (Duroc, L62 or Pietrain) and lairage time in the slaughterhouse (0, 2.0, 4.0 or 6.0 h) as controlled variables. Although some pigs were found to be carriers of stress susceptibility, all were healthy heterozygous individuals. Pre-slaughter transport increased

slaughterhouse

raised

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cortisol levels in saliva above 3.0 µg/L (medium stress), and 4.0 h of lairage in the them above 6.0 µg/L, whereas corticosterone concentrations

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exceeded 4 µg/L, which are suggestive of high stress. The highest cortisol levels were

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detected in the Duroc genotype. Other factors such as food deprivation, background noise, the presence of a large number of animals waiting to be slaughtered, mixing with

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unfamiliar animals or recent mixing of genders may also influence stress. Corticosterone proved a reliable indicator of high stress only. Meat quality from the pig breeds studied

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was not affected by lairage in the slaughterhouse for up to 6.0 h.

Keywords: Pig stress; Corticosteroids; Pig genotypes; Transport; Slaughterhouse lairage

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time; meat quality.

ACCEPTED MANUSCRIPT 1. Introduction Pig stress depends on the particular breed. The origin of porcine susceptibility to stress is a mutation in RYR1 gene: RYR1 c.1843C>T (Fujii et al., 1991). High levels of stress increase

susceptibility

to

disease,

decrease

life

expectancy,

impair

growth

and

reproduction, cause body damage and behavioural abnormalities, and decrease meat

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quality (Guzik et al., 2006; Bonizzi and Roncada, 2007; Chaloupková et al., 2007). Crowding, loading and unloading, adverse weather conditions, feed and water deprivation,

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lairage, length of travel, mixing with animals from other groups, restraint and fatigue are

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known pig stressors (Adenkola, Ayo, Sackey & Adelaiye, 2011; dalla Costa et al., 2007; Gajana, Nkukwana, Marume & Muchenje, 2013; Ritter et al., 2007; Terlouw, 2005) to

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which swine respond with an increase in adrenocortical activity and corticosteroids levels (Cook et al., 1996; De Jong et al., 1998; Escribano et al., 2012; Martín et al., 2013).

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Pig stress can be monitored via saliva (De Jong et al., 1998; Merlot et al., 2011; Escribano, Fuentes-Rubio & Cerón, 2012; Martín, Ovejero, Mateos & Villarroel, 2013). Cortisol and corticosterone are the main glucocorticoids secreted in pig saliva, usually in a

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ratios of about 4 (Bottoms et al., 1972). Cortisol in saliva is a good proxy for free cortisol

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in plasma (Kirschbaum and Hellhammer, 1989; Parrott and Misson, 1989). Rey-Salgueiro et al. (2015) were the firstto report on corticosterone in saliva; other authors have only monitored cortisol levels, using specific methods (Seshoka et al., 2013; Jama et al., 2016).

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Thus, Rey-Salgueiro et al. (2015) measured non-stress on-farm baseline corticosterone levels in pigs 1–3 days before transport to the slaughterhouse. In this work, we examined changes in salivary corticosteroids from pigs suffering mild to acute stress, and their relationship to genotype and meat quality, using genotype (Duroc, L62 or Pietrain), location (farm vs slaughterhouse) and lairage time before slaughter (0.0–6.0 h) as controlled variables. 2. Materials and methods The animal care and experimental procedures used in this study complied with EU Directive 2010/63 and Spain’s Royal Decree 53/2013, concerning the protection of animals used for scientific research. All procedures involving animals were developed by staff at

ACCEPTED MANUSCRIPT Frigolouro, a company complying with the requirements of welfare and protection of animals set in Spain’s Royal Decree 54/1995, which was further repealed by Spain’s Royal decree 37/2014.

2.1. Animals and experimental design

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The animals used included Pietrain x (Landrace x Large White), Duroc x (Landrace x Large White) and L62 (a crossed of several genotypes) x (Landrace x Large White) female

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and male pigs (barrows for the Duroc genotype and intact male pigs for the others) aged 15

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weeks and weighing 88 ± 8 kg.

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2.1.1. On-farm baseline stress

The pigs studied were from 12 different farms (four per genotype) in the province of

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Ourense (Galicia, NW Spain), where they were housed in groups of 10–12 animals per pen (the stocking density, 1.0 m2 /pig, equivalent to 88 kg/m2 , was identical on all farms). Three pens per farm were used. Thus, 30–36 pigs per farm, and 120–144 animals per genotype in

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total, were studied. Saliva samples were collected by suspending a twisted 100% cotton

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rope into each pen for 30 min (Rey-Salgueiro et al., 2015). This time is long enough for 75% or more of the animals to encounter the rope, chew it and deposit saliva (Detmer, 2011). The ropes were suspended in the home pens at 8:00, 10:00, 12:00 and 14:00 (i.e., at

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times 0, 2, 4 and 6 h, respectively). The initial ropes were withdrawn after 30 min and new ropes suspended at the previous sampling times. A total of 48 saliva samples were collected (12 farms × 4 replicates for each of 3 genotypes × 4 times). After the exposure period, saliva samples were collected from the ropes by wringing the wet for centrifugation and storage at –80 ˚C until analysis. The analytical results can be found elsewhere (ReySalgueiro et al., 2015).

2.1.2. Stress levels at the slaughterhouse The pigs were transported to a commercial slaughterhouse in Porriño (Pontevedra, Galicia, NW Spain), 80–120 km from the experimental farms. Transportation was done during

ACCEPTED MANUSCRIPT Spring 2014, between 07:00 h and 08:00 h, under commercially accepted conditions (0.47 m2 /pig, equivalent to 186 kg/m2 ) as regards mixing, handling, transportation, driving and new housing, among others. The animals were unloaded on arrival at the slaughterhouse and placed in mixed gender groups in a lairage area (stocking density 0.65 m2 /pig, equivalent to 135 kg/m2 ) with free access to water. The average gender proportion of male

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and female pigs was 50:50 for Duroc and Pietrain, and 40:60 for L62. No pigs from different farms were mixed. Saliva samples were collected as described in the previous

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section immediately after unloading at the slaughterhouse (approximately at 8:00; 0 h), and

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also after 2.0 h (10:00), 4.0 h (12:00) and 6.0 h of lairage (14:00). Two ropes were suspended in each pen (50 pigs per pen) and the amount of saliva collected with both was

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combined. A total of 48 saliva samples were analysed: 12 farms (4 per for each of the three

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genotypes) × 4 times.

2.2. Determination of corticosteroids in pig saliva

The methodology used to extract, purify, identify and quantify stress corticosteroids in pig

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saliva is described in detail elsewhere (Rey-Salgueiro et al., 2015). Briefly, saliva samples

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(1.0 mL) were passed through a C18 cartridge that was previously activated with 3.0 mL of methanol and 1.5 mL of water. The cartridge was then washed with 0.25 mL of water, followed by 0.5 mL of water:acetone (4:1) and 0.25 mL of hexane. In order to remove co-

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extractives and interfering substances, a cartridge of silica that was previously activated with 2.0 mL of hexane and 1.0 mL of ethyl acetate was set in connection with the C18 cartridge. Then, hormones and precursors were eluted from the C18-silica cartridges by passing 6.0 mL of ethyl acetate:ethyl ether (1:1). The eluate was evaporated to dryness, dissolved in 50 µL of methanol and centrifuged at 850 g for 10 s for analysis of the supernatant by liquid chromatography–tandem mass spectrometry (LC–MS/MS). Method performance was assessed by evaluating various figures of merit including recovery, repeatability, reproducibility, linearity and limits of detection and quantification (Rey-Salgueiro et al., 2015). The regression analysis was validated by verifying linearity with Mandel’s fitting test (P = 99%; Mandel, 1964). Acceptable linearity was obtained for

ACCEPTED MANUSCRIPT all target compounds (r > 0.9920). Recoveries were calculated from the ratio of the slope of the calibration curve for spiked samples to that for the calibration line for hormone standards and ranged from 74% to 90% except for aldosterone (60%). The limits of detection (LOD) and quantification (LOQ) were calculated in an unfortified saliva sample, following the signal-to-noise criterion (S/N = 3 for LOD and S/N = 10 for LOQ)

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(MacDougall et al., 1980). LODs ranged from 0.020 to 0.10 μg/L and LOQs from 0.050 to

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0.31 μg/L.

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2.3. Pork technological and sensory quality

Samples used were from the same farms and transport conditions than samples used to

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measure corticosteroids. It was just one subset of samples showing cortisol levels at slaughtering ≤ 6.0 µg/L to show that parameters of both technological and sensory pork

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quality were not affected. After 6.0 h of lairage, 5 pigs from each farm (60 pigs in total) were slaughtered and their meat quality parameters evaluated in samples taken from the Longissimus muscle. At the meat processing plants, pH measurements were made 45 min

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post-mortem (pH45min ). All remaining analyses were done at the laboratory. pH was measured 24 (pH24h ) and 48 h post-mortem (pH48h ) by using a portable pH meter: model

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FG4: FiveGo™ DO from Mettler Toledo International, Inc., (Columbus, OH, USA). Meat colour was measured according to the CIE L*a*b* system, using a CR310 Minolta

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Chroma Meter with D65 lighting and a 10° standard observer. Drip loss was determined gravimetrically (Honikel, 1998). After 24 h of refrigeration (48 h post-mortem), exudate on the meat surface was carefully removed and pieces (50 ± 5 g) were re-weighed. Meat texture was determined by using Warner–Bratzler shear force (Akit et al. 2014; Honikel, 1998). Samples were cut with a metal template into rectangular strips 2 cm in diameter parallel to the muscle fibres. Shear force blade (V-shaped) was fitted to a texture meter from Metrotec (Lezo, Spain) and the crosshead speed set at 1 mm/s. The mean of the peak shear force was used as a proxy for tenderness.

2.4. RYR1 genotype

ACCEPTED MANUSCRIPT RYR1 genotype was studied in the same meat samples used for technological and sensory pork quality evaluation (20 pigs per genotype), which were subjected to the RT-PCR assay (Burgos et al., 2005; Lapusan et al., 2011). This assay uses two internal primers and two fluorescent probes (TTGGAGCGCACGGC-TAMRA labelled with the dye VIR and TTGGAGCACACGGC –TAMRA labelled with FAM) that are designed to bind

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specifically to each of the two possible alleles of the RYR1 gene. DNA was extracted from muscle tissue according to Lago et al. (2011). The concentration and quality of extracted

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DNA were determined by measuring the absorbances at 260 and 280 nm on a Nano Drop

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ND-1000 spectrophotometer from Thermo Fisher Scientific (Waltham, MA, USA) to calculate the 260/280 ratio. DNA extracts were labelled and stored at –20 ºC until

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subjected to the Real-Time PCR reaction. The reaction were carried out according to Burgos et al. (2005), using a Viia7 thermocycler from Applied Biosystems (Foster City,

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CA, USA) and a final volume per reaction of 10 µL containing 3–10 ng DNA/µl. The

2.5. Statistical analysis

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optimum concentrations of primers and probes were 900 and 200 nM, respectively.

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The statistical analysis was performed with the software package Statgraphics Plus v. 5.1 (Manugistics, Rockville, MD, USA). Significant differences in the target parameters (cortisol and corticosterone concentrations) among pig groups (location, genotype and

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lairage time) were detected by one- and two-way ANOVA at the 95% confidence level. A Fisher’s least significant difference LSD test at the 95% confidence level was additionally used to seek interactions between variables. Any factor of confusion between farms and genotypes was overcome by following a threefold strategy, namely: (a) comparing on-farm baseline stress levels for each genotype with those of the same kind of animal at the slaughterhouse; (b) measuring corticosteroids in a combined saliva sample from about 50 animals obtained in a single experiment; and (c) running four independent experiments with animals of the same class to calculate a mean level and its standard deviation based on a replication of experiments under identical conditions. Significant differences at the 95% probability level would be found if the confidence interval for the mean did not overlap

ACCEPTED MANUSCRIPT between treatments. One-way ANOVA was also used to test for differences in meat quality parameters among the three genotypes since two-group comparisons were based on a Student’s t-test. Pearson correlation coefficients between the levels of the two stress hormones were also calculated. 3. Results

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Table 1 shows the measured cortisol and corticosterone concentrations in saliva samples collected at the slaughterhouse. As can be seen, the concentrations ranged from

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undetectable (0.050 µg/L for cortisol and 0.080 µg/L for corticosterone) to 17 µg/L for the

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former hormone to 22 µg/L for the latter. The mean value was 2.9 µg/L. Pearson correlations among the levels of both hormones (Table 1) were significant (n = 24 groups

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× 4 samples per group = 96, r = 0.542, P < 0.0001). Statistical differences among cortisol levels were detected in the following two situations:

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(a) After transport, between farms and at slaughterhouse time 0 (time of arrival), where cortisol rose above 3.0 µg/L (medium stress); and (b) between time 0 at the slaughterhouse and after 4.0–6.0 h of lairage before slaughter,

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with a marked increase in cortisol levels (above 6.0 µg/L) suggesting high stress.

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The interaction between location (farm vs. slaughterhouse) and lairage time had a statistically significant effect on cortisol levels (F-ratio = 4.6, P = 0.005; Figure 1). This suggests that a stress response was triggered. Stress increasing factors, as the ratio of post-

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stress cortisol to pre-stress levels; the stressor representing relocation and transport increased by a factor of 4–7 (Table 2). A lairage time of 2.0 h resulted in the lowest cortisol levels that were similar to baseline values (Figure 1). After 2.0 h, cortisol levels increased and peaked at 6.0 h, where they were 1.4–3.3 times greater than immediately after transport (Table 2). The interaction between location type (farm or slaughterhouse) and lairage time was also significant for corticosterone (F-ratio = 4.5, P = 0.035; Figure 2). As with cortisol, corticosterone levels peaked after 6.0 h in the slaughterhouse, with values up to 5.0 times higher than the baseline levels after transport. A residence time of 6.0 h in the slaughterhouse increased corticosterone levels in pig saliva by 4.0–19 μg/L. No differences

ACCEPTED MANUSCRIPT in corticosterone concentrations among genotypes were found, however. The average cortisol/corticosterone ratio for the Duroc genotype (Figure 3) evolved differently from those for L62 and Pietrain. Overall, the ratio decreased with increasing lairage time at the slaughterhouse. In Duroc, the ratio peaked in the central hours of lairage (2–4 h); in L62 and Pietrain, however, the ratios at 2–4 h were the lowest. This difference was a result of

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Duroc exhibiting the lowest corticosterone levels, and hence the highest average cortisol/corticosterone ratios (2.0 vs 1.5 for L62 and 1.3 for Pietrain. No previous studies

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have used corticosterone in pig saliva as a stress biomarker and only a few on other animal

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species such as rabbits have been reported where commercial transport had a significant effect on corticosterone levels in blood (Liste et al., 2008).

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Based on allele distributions in the genotypes, all Duroc x (Landrace x Large White) pigs were homozygous (CC) in the RYR1 gene, whereas 48% of Pietrain and 15%

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of L62 pigs possessed the mutant allele (T). None of the animals studied was homozygous (TT), however.

Table 3 shows the values of the quality-related parameters studied, namely: meat

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pH (pH45min , pH24h and pH48h ), drip loss, texture and colour (L*a*b* at 24 and 48 h). No

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statistically significant differences (p <0.05) in any parameter between genotypes were found. Technologically, fresh meat is classified into three quality categories according to pH value, colour, texture and drip loss, namely: PSE (pale, soft, exudative), RFN (reddish-

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pink, firm, normal pork) and DFD (dark, firm, dry). Pork with pH45min < 6 is classified as potential PSE and pork with pH45min ≥ 6.0 as normal (Tomovic et al., 2014). pH45min exceeded 6 in all samples, so all meat was potentially RFN. Based on 24 h post-mortem pH, meat quality can be classified as normal (pH24h < 6.0) or DFD (pH24h ≥ 6.0). Only meat from one farm (L62 genotype) was DFD; it was dark meat with a decreased redness value (a* = 4.3 ± 0.6), pH24h ≥ 6, increased hardness (texture, 15.3 ± 2.3 N/cm2 ) and drier —and hence containing little exudate (drip loss = 1.3 ± 0.6%). 4. Discussion From farm to slaughter, pigs encounter many different stressors such as loading and unloading, transportation and mixing with other pig groups (Liste et al., 2008). Cortisol

ACCEPTED MANUSCRIPT and corticosterone were previously used as biomarkers of stress in animals (Cook, 2012), but only the former had been examined in non-invasively obtained samples such as saliva (De Jong et al., 1998). Consistent with previous results (Cook et al., 1996), our results confirm that stress during transport increases salivary cortisol levels in pigs, probably as a consequence of the strong effects of loading, transport and unloading. Not many studies of

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this type have been performed at the slaughterhouse level, however. Some authors have reported a marked increase in pre-transport salivary cortisol and

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a rapid subsequent decline to baseline levels after 30 min of lairage (Soler et al., 2013).

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Chaloupková et al. (2007) found decreased cortisol levels in saliva pigs 1.0 h after transport. Similarly, Jama et al. (2016) found a significant increase in salivary cortisol after

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2 h of transport, and the stressing effect to be greater in males than in females; after 20 h of lairage at the slaughterhouse, however, cortisol levels in males decreased to the levels of

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females after transport (Table 2). Warriss et al. (1992), and Pérez et al. (2002), found cortisol levels in plasma to be lowest after 2.0–3.0 h. Although a long enough lairage period could in theory allow pigs to recover from the stress of previous transport and

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associated handling, cortisol levels rose after 2.0 h and peaked after 6.0 h following

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transport (levels increased by a factor of 1.4–3.3). These values are similar to those for other stressors associated to pig handling on farms such as nose-snare, isolation or weighing (Table 2). An increasing trend was previously observed in plasma cortisol levels

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after 9.0 h of rest before slaughter (Pérez cet al., 2002) that was ascribed to fasting. Food deprivation may be responsible for the significant increase in salivary cortisol after 4.0 h of lairage; however, other factors relating to specific conditions in the slaughterhouse may also have contributed to the effect. We hypothesize that additional factors such as background noise, a large number of animals waiting to be slaughtered, mixing with unfamiliar animals or recent mixing of genders could also account for the significant increase observed. Tume and Shaw (1992) found cortisol concentrations in cattle slaughtered in commercial abattoirs to be higher than in animals slaughtered at research abattoirs owing to the inherent noise and movements of animals and people in the yards during routine management, and also to handling in the race when driving steers to the

ACCEPTED MANUSCRIPT stunning box. These are obvious indicators of the animals having difficulties, and the situation is aversive (Pérez et al., 2002; EFSA, 2004). Based on the results, pigs should be allowed to rest for at least 2.0 h or even 4.0 h before slaughtering —the corresponding cortisol levels ranged from 3.0 to 6.0 μg/L. In fact, a residence time of 6.0 h resulted in increased levels of salivary cortisol (6.0 to 17

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μg/L). Based on these values, 6.0 μg/L can be set as the threshold for high stress in the pigs. Likewise, 6.0 h of lairage before slaughter led to increased levels of corticosterone

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(4.0–19.0 μg/L), so 4.0 μg/L can be set as the threshold concentration for high pig stress.

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Pearson correlations among the levels of the stress hormones in pig saliva were significant for cortisol and corticosterone (24 groups × 4 samples/ group = 96 samples, r =

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0.542, P < 0.0001). This result suggests that the two hormones respond very similarly to stress and hence that they could be used as stress indicators. As can be seen from Figure 3,

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the temporal variation of the average cortisol/corticosterone ratio differed between the Duroc genotype and the other two; this may have resulted from the corticosterone levels of Duroc being the lowest or from corticosterone conversion into aldosterone (Muller, 1988).

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In any case, cortisol proved a more sensitive marker than corticosterone and was not so

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variable. Therefore, cortisol could be used as the main stress biomarker in pig saliva and corticosterone as a confirmation biomarker. No significant interaction between cortisol levels immediately before slaughter and

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pork technological quality was found. However, the average stress levels for the pigs before slaughter fell below the threshold for cortisol in saliva (6 µg/L), which is indicative of mild stress. All meat samples were classified as RFN (reddish-pink, firm, normal pork); by exception, one sample was DFD (dark, firm and dry). Although to a lesser extent than PSE (pale, soft, and exudative) meat, DFD meat is less favoured by the processing industry and consumers owing to its high susceptibility to microbial contamination (Kauffman et al., 1993). Whereas all Duroc pigs were homozygous (CC), lower proportions of free T/C mutation were found in L62 (85%) and Pietrain pigs (52%). Although the desirable allele C at the RYR1 c.1843 C>T locus confers resistance to malignant hyperthermia, the mutant

ACCEPTED MANUSCRIPT allele is associated to superior productive performance and lean meat content of pigs, so it has been selected for breeding commercial pig populations for decades (MacLennan and Phillips, 1992). Wide genetic variation at RYR1 loci have been found among breeds, countries and farms. Duroc is the breed with the highest frequencies of favourable alleles to resist stress: 100% as reported by Carolino et al. (2007) and Roman (2006), 91%

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according to Ruan et al. (2013) and 75% according to Jasek (2006). Consistent with our results, the mutant allele is highly prevalent in Pietrain pigs.

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5. Conclusions

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All Duroc pigs were healthy and non-carriers (CC) of the RYR gene mutation predisposing to porcine stress. By contrast, 48% of Pietrain pigs and 15% of L62 pigs showed some

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mutation; all, however, were healthy heterozygous pigs (CT) —no homozygous recessive individuals (TT) were identified. For the first time, various corticosteroids in pig saliva

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were evaluated as stress biomarkers with provision for variables such as genotype (Duroc, L62 and Pietrain), and lairage time before slaughter (0–6.0 h). The highest cortisol levels were found in the Duroc genotype. Pre-slaughter transport increased salivary cortisol

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above 3.0 µg/L, which is suggestive of medium stress; also, cortisol levels after 2.0-4.0 h

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of lairage in the slaughterhouse rose above 6.0 µg/L, which is suggestive of high stress. Cortisol concentrations above 6.0 µg/L in saliva, equivalent to corticosterone levels above 4.0 µg/L, were thus indicative of high stress in the pigs. Although both cortisol and

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corticosterone were easily detected in saliva, cortisol determinations were more sensitive and their results less variable. Therefore, cortisol could be used as the main stress biomarker in pig saliva, and corticosterone as confirmation biomarker. In practice, transport inevitably causes mild stress; also, 4 h of lairage in the slaughterhouse results in mild stress (< 6 µg/L) that is acceptable because it does not affect meat quality. Lairage for more than 4 h leads to greater variability in stress levels among animals and to very high levels (17 µg/L) in some cases that are better avoided by slaughtering the animals before. Food deprivation after transport can play a role in increasing hormone levels; however, other factors such as background noise, a large number of animals waiting to be

ACCEPTED MANUSCRIPT slaughtered, mixing with unfamiliar animals or recent mixing of genders may also contribute.

Acknowledgements: This work was funded by the Centro para el Desarrollo Tecnológico Industrial (CDTI), and co-funded by the FEDER–INNTERCONECTA research program,

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in the framework of Project INNOSABOR (Ref. ITC-20133017) The authors are grateful to Ruth Lois, Miguel Sarria and Borja Casales for help with the planning of the

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experimental set-up at farms and the slaughterhouse.

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ultimate pork quality. Meat Science 34:283–300.

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ACCEPTED MANUSCRIPT Table 1. Cortisol and corticosterone levels among genotypes, times and locations. Corticosterone 3 1.1 ± 0.23 a 3.9 ± 4.1 a 1.8 ± 0.32 a 0.53 ± 0.92 a 3.8 ± 0.30 a 2.4 ± 4.0 a 0.88 ± 0.20 a 5.9 ± 6.8 a 1.8 ± 0.23 a 0.61 ± 1.3 a 0.74 ± 1.1 a 2.5 ± 3.1 a 0.66 ± 0.40 a 6.1 ± 9.0 a 0.65 ± 0.35a 6.7 ± 7.5 a 0.69 ± 0.45 a 2.6 ± 5.4 a 1.7 ± 2.3 a 3.8 ± 5.0 a 0.74 ± 1.1 a 3.3 ± 1.3 a 1.1 ± 0.50 a 5.3 ± 2.7 b

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Time (h)1

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Location 2 Cortisol3 F 1.2 ± 0.20 0 S 5.2 ± 4.7 a F 1.4 ± 0.81 a 2 S 1.7 ± 0.10 a Duroc F 1.4 ± 0.22 a 4 S 5.6 ± 6.7 a F 1.1 ± 0.21 a 6 S 7.7 ± 4.7 b F 0.51 ± 0.05 a 0 S 2.1 ± 1.0 a F 0.59 ± 0.30 a 2 S 1.8 ± 0.91 a L62 F 0.45 ± 0.50 a 4 S 4.5 ± 3.2 a F 0.60 ± 0.14 a 6 S 6.9 ± 4.6 b F 0.74 ± 0.30 a 0 S 5.0 ± 0.93 b F 0.51 ± 0.23 a 2 S 2.7 ± 1.43 a Pietrain F 0.43 ± 0.08 a 4 S 4.1 ± 2.0 a F 0.95 ± 0.50 a 6 S 6.8 ± 3.2 b 1 Sampling every 2 h from 8:00 to 14:30. 2 F farm, S slaughterhouse. 3 Mean ± s.d. (standard deviation) in µg/L; n = 12. (4 farms × 3 pens each) a,b Significant differences at P ˂ 0.05 for cortisol and corticosterone. Genotype

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Landrace × Large White

Vena cava blood sampling Pen relocation Transport Nose-snare Weighing

≈ 1.7

≈ 5.1 ≈ 4.2

3.6 5.3 1.9

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≈ 1.4 ≈ 0.8

Nose-snare

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Table 2. Salivary cortisol levels in pigs found in previous studies (in bold, the most important stressors with stress increasing factors ≥ 4.0). Salivary Stress cortisol (µg/L) Genotype Stressors increasing Reference PrePostfactor stress stress Landrace-York Nose-snare 2.1 7.2 3.4 Cook et al. (1996) Pen relocation ≈ 1.51 ≈ 6.01 4.0 ≈ 2.52 ≈ 4.52 1.8 Great Yorkshire x (Great Isolation ≈ 2.01 ≈ 5.01 2.5 Yorkshire x Dutch Landrace) De Jong et al. (1998) ≈ 4.02 ≈ 8.02 2.0 1 1 Nose-snare ≈ 2.0 ≈ 4.5 2.3 ≈ 4.02 ≈ 8.02 2.0 Great Yorkshire x(Great 4.7 Geverink et al. Transport ≈ 1.5 ≈ 7.0 Yorkshire x Dutch Landrace) (1998) 5.2 Schönreiter and German landrace Transport 1.3 6.8 Zanella (2000)

≈ 3.2

Merlot et al. (2011)

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≈ 1.5 ≈ 3.3 2.2 7.0 32.5 4.6 Escribano et al. 2.4 5.0 2.1 (2012) ≈ 2.0 ≈5.0 2.5 Martín et al. (2013) 3.915.23.9-8.6 Transport (♀-♂) 4.5 38.6 (♂) Duroc boars × Dutch LandraceJama et al. (2016) Large White gilts 20.0 h lairage (♀- 3.916.34.2-3.7 ♂) 4.5 16.5 (no diff.) Transport 0.5 2.1 4.2 0.7 5.0 7.1 L62 1.2 5.2 4.3 Pietrain This work 6.0 h lairage 2.1 6.9 3.3 Duroc 5.0 6.8 1.4 5.2 7.7 1.5 1 Poor environment: standard farrowing pens followed by standard rearing and fattening pens 2 Enriched environment: larger farrowing pens followed by larger rearing and fattening pens, straw supplied Duroc × (Landrace × Large White) No data

ACCEPTED MANUSCRIPT Table 3. Meat quality parameters (mean ± s.d.; 20 pigs for each genotype) after slaughtering after 6 h of lairage. No significant differences in any parameter among genotypes were found. Pietrain

L62

pH(slaughtering)

6.4 ± 0.3

6.4 ± 0.2

6.6 ± 0.2

pH24 h

5.7 ± 0.1

5.8 ± 0.2

5.8 ± 0.2

pH48 h

5.8 ± 0.2

5.7 ± 0.2

5.8 ± 0.2

L*24h

48.8 ± 2.4

48.1 ± 2.6

48.1 ± 2.2

a*24h

7.9 ± 2.8

7.1 ± 1.6

6.0 ± 1.9

b*24h

13.5 ± 1.0

13.0 ± 1.3

L*48h

50.4 ± 2.5

49.1 ± 2.8

a*48h

9.5 ± 3.8

8.5 ± 1.9

b*48h

15.2 ± 1.3

14.5 ± 1.5

14.3 ± 1.1

Texture (N/cm2 )

12.3 ± 2.7

12.1 ± 3.4

13.8 ± 2.6

Drip loss (%)

3.8 ± 2.5

Cortisol(slaughtering)

5.6 ± 3.9

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12.2 ± 1.1

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Duroc

48.6 ± 1.8 8.5 ± 1.8

4.3 ± 2.3

2.9 ± 2.5

5.4 ± 2.3

4.8 ± 3.8

ACCEPTED MANUSCRIPT Figure captions

Fig. 1. Significant differences (p < 0.05) in salivary cortisol levels for the interaction location (farm, F, or slaughterhouse, S) vs. time (0, 2, 4 or 6 h) found when the confidence interval for the mean did not overlap between treatments.

Fig. 2. Significant differences (p < 0.05) in the salivary corticosterone levels for the

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interaction location (farm, F, or slaughterhouse, S) vs. time (0, 2, 4 or 6 h) found when the

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confidence interval for the mean did not overlap between treatments.

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6 h) and pig genotype (Duroc, L62 and Pietrain).

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Fig. 3. Variation of the salivary cortisol/corticosterone ratio with lairage time (0, 2, 4 and

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Fig. 1. Significant differences (p < 0.05) in salivary cortisol levels for the interaction location (farm, F, or slaughterhouse, S) vs. time (0, 2, 4 or 6 h) found when the confidence

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interval for the mean did not overlap between treatments.

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Fig. 2. Significant differences (p < 0.05) in the salivary corticosterone levels for the

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interaction location (farm, F, or slaughterhouse, S) vs. time (0, 2, 4 or 6 h) found when the

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confidence interval for the mean did not overlap between treatments.

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2.5

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2.0 1.5

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1.0 0.5 0.0 2h

4h

6h

0h

2h

4h

6h

0h

2h

4h

6h

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Cortisol / Corticosterone ratios

3.0

Duroc

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L62

Fig. 3. Variation of the salivary cortisol/corticosterone ratio with lairage time (0, 2, 4 and

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6 h) and pig genotype (Duroc, L62 and Pietrain).