# A multi-suckling system combined with an enriched housing environment during the growing period promotes resilience to various challenges in pigs | Scientific Reports – Nature.com

Established principles of laboratory animal use and care and the Dutch law on animal experiments were followed. They comply with the European Directive 2010/63/EU on the protection of animals used for scientific purposes. The Animal Care and Use Committee of Wageningen University approved the experiment (AVD1040020186245). All methods applied in the study were performed in accordance with the ARRIVE guidelines and regulations. Due to the difference in housing system according to the treatment group, the investigators were aware of the treatment of the animals when collecting samples. However, lab analyses of tear staining measurements on pictures, behavioural observations during the transport challenge, respiratory rate and metabolic chamber parameters during the heat stress challenge, wound healing areas on pictures and scores related to organs during dissections were done blindly. The sample size estimation was based on the rise in cortisol levels using results obtained in pigs subjected to transport stress36 (α = 5%, power = 80%, SD = 2.42, δ = 2.8).

### Animals

A total of 144 Tempo × Topigs-20 pigs (n = 71 females; n = 73 males) were used during the experiment, spread over three batches (n = 48 pigs per batch). Piglets were offspring from 24 multiparous sows. During lactation, half of the sows and their piglets were housed in a conventional farrowing pen (CONV) (mean ± SD; sow parity = 4.2 ± 1.8) and the other half in an alternative group housing system (AHS) (parity = 4.0 ± 1.7) at the Swine Innovation Centre (Sterksel, The Netherlands). The piglets were not castrated, nor were their tails docked or teeth clipped. Average birth weight was similar for piglets from both systems: 1.46 ± 0.28 kg for the CONV and 1.44 ± 0.27 kg for the AHS.

### Housing systems

#### From birth to 9 weeks of age

Piglets were raised in two different housing systems (similar to van Nieuwamerongen et al.21). The AHS comprised of five farrowing pens of 3.2 × 2.2 m (mix of solid 2.2 × 2.2 m and slatted floor 1.0 × 2.2 m), adjacent to a communal area of 11.1 × 2.80 m (solid floor). Next to the communal area, were a dunging (2.8 × 3.3 m, slatted floor) and a feeding area (4.2 × 3.3 m, solid floor). Enrichment was provided in the form of four jute bags (110 × 80 cm) and during the first week a slice of straw was added to the farrowing pens (approximately 2.5 kg per pen). One week before the expected farrowing date, five sows per batch were put in this system. Two days before the expected day of farrowing, sows were moved to a farrowing pen and confined in a farrowing crate (1.9 × 0.6 m). Two days after farrowing, they were allowed to access the full system again. Newly born piglets were kept with their own litter in the farrowing pens for 1 week, after which they could access the entire system and mingle with the other litters. Piglets were provided with a heated piglet nest next to the farrowing pens (0.7 × 1.6 m), with a temperature of 33–35 °C (day 1 till day 7), 29–31 °C (day 7 till day 25) and 23–26 °C (day 25 till weaning). Piglets were fed in round bowls until 5 weeks of age and from a sensor-controlled automatic feeder (Rondomat, from 3 weeks of age). Besides this, the piglets could participate in feeding with the sows, which were fed in a large trough placed on the floor. Ingestion of solid feed was stimulated with the use of intermittent suckling from week 5 of age onwards. AHS piglets were weaned at an average of 62.6 ± 1.9 days and a body weight of 26.6 ± 4.9 kg. They received a starter diet from 35 days onwards.

In the CONV system, piglets were kept in farrowing pens of 2.8 × 1.8 m until weaning. Sows were confined in a crate (1.9 × 0.6 m). The floor consisted of metal slats within the crate. There was a solid floor of 1.2 × 0.3 m with heating lamp for the piglets and the remaining area consisted of plastic slats. Piglets received additional creep feed in the farrowing pens from 1 week after birth. CONV piglets were weaned at 27.4 ± 1.2 days of age and 8.7 ± 1.3 kg. After weaning, CONV piglets were housed with their litter mates in nursery pens of 3.18 × 1.0 m (0.40 m2 per piglet) for five additional weeks with a chain and a jute bag as enrichment. They received a commercial weaner diet for 10 days after weaning and a starter diet, similar to that provided to AHS piglets, from 35 days onwards.

Lights were on from 07:00 h till 19:00 h in both systems, giving the sows and piglets a 12 h light regime with 115 Lux. Besides that, the AHS had natural daylight through two windows. Transition between day and night light settings was done progressively in 10 min. Ambient temperature was 23 °C in both systems. Water was available ad libitum in both systems.

#### From 9 weeks of age onwards

After weaning of the AHS piglets at approximately 9 weeks of age, all piglets were moved to the Carus research facilities in Wageningen, the Netherlands. They were mixed in groups of six unfamiliar piglets originating from the same system. Litter, sex and weight were balanced between pens. Piglets were selected based on their sex (50:50% male and female), and weight at birth in order to choose piglets representative of the full litter. Per litter, 6 piglets were selected: 2 piglets with a birth weight between the minimum weight of the litter + 10% and the 1st weight quartile (light); 2 piglets with a birth weight between the 1st and 3rd quartile (medium); 2 piglets with a birth weight between the 3rd quartile and the maximum weight − 10% (heavy).

AHS pigs were housed in a 2.40 × 4.67 m pen, i.e. double the size of a conventional pen (1.87 m2 per pig), enriched with deep straw, peat and sawdust bedding, which was replenished regularly (2.5 kg of straw and 30 L of sawdust every day, 22.5 L peat every week). Besides that, AHS pigs were provided with a handful of hay, alfalfa or cardboard egg trays once a week and a chain, jute bag or rope (rotation every week). They were also provided with one extra toy (either a biting ball on a chain, a free chewing ball for dogs, a tyre dog toy, a porcichew® toy, a green MS Schippers Bite cylinder®, or a green MS Schippers Cross®) which was changed every 2 days to preserve the attractiveness of the toy. CONV pigs were housed in standard pens of 1.20 × 4.67 m with conventional space allowance (0.93 m2 per pig), with a partly solid and partly slatted floor without substrate. CONV piglets were provided with a ball and a chain with screws, which were not changed. AHS and CONV pens were placed alternately in the rooms. Pigs were all fed the same feed (a standard commercial diet for growing pigs) ad libitum from a single pig feeder and water was available ad libitum.

The light regime was similar to that before 9 weeks of age, giving the pigs 115 Lux in the pens during the day (from 7:00 h to 19:00 h; 5000 K ultraviolet A at an intensity of 42, 2700 K at 60) and 30 Lux during the night (5000 K ultraviolet A at an intensity of 3, 2700 K at 0). The transition between the day and night rhythm was done progressively for 10 min. No natural day light was available. Temperature was kept at 23 °C for the first 2 days, then at 22 °C for the 2 subsequent days and at 21 °C onwards.

### Challenges

Resilience of the pigs in the two housing systems was assessed by following the recovery process of the animals after submission to different successive challenges in the following order: a 21 h-isolation challenge (not detailed in this paper), a 2 h-transport challenge, an LPS injection to induce a sickness response, a 2 h-heat stress challenge and a wound. Figure 1 summarizes the order and time of the challenges described in this paper. Order of exposing pigs to the challenges was always balanced for housing system. Four animals per pen (Focals, two males and two females) were exposed to the experimental challenges, while two other pigs served as companions (i.e. the two pigs who most deviated from the average pen weight).

#### Transport challenge

At the age of 83.0 ± 1.7 days (weight: 41.6 ± 5.5 kg), 94 focal pigs experienced a 2 h transport. At this point, one of the 96 focal pigs was a tail biter (CONV housing). It was euthanized using an injection of pentobarbital (Euthasol® Vet) and replaced by a companion pig after the transport challenge. Neither of those two pigs were exposed to the transport challenge. Pigs were loaded in a trailer with a thin layer of sawdust on the floor and were transported from 8:00 to 10:00 h. All pigs from one batch (from both housing systems) were mixed in the same trailer, which created both a metabolic and social challenge. To follow the recovery of the animals, blood samples were drawn 24 h before the transport challenge (baseline), when they came back from the 2 h-transport (referred to as 0 h), and at 3 h, 24 h and 48 h after the end of transport.

The day before the transportation, pigs were marked with paint to individually track their behaviour in the trailer during the 2 h-transport challenge. Number of aggressive acts (either a push, a bite, a knock, a mount or a fight) and lying behaviour were continuously scored from video by a single trained observer using the software Observer 14.2 (Noldus Information Technology, Wageningen, The Netherlands). The percentage of time spent lying was averaged over 30-min intervals. The total number of aggressive acts and the total number of posture changes were summed over the 2 h period. Because of poor lighting in the trailer causing difficulties to observe the pigs before sunrise, the first half-hour of batch 1 was excluded from analysis.

#### LPS challenge

At the age of 104.4 ± 1.7 days (weight: 60.0 ± 7.5 kg), 92 focal pigs were injected in the ear vein with 2 μg of LPS/kg of body weight (LPS sigma L4391 Escherichia coli O111 B4). Four focal pigs were not subjected to the challenge as they were sick and on antibiotic treatment at the day of challenge. For time constraints, the pigs were challenged in two different groups on two consecutive days, balanced for housing system. Pigs were injected in their home pen in one of their ears. Blood samples were collected at 24 h before (baseline) and 1 h, 3 h, 5 h and 24 h after the injection. At each of these time points, rectal temperature was measured before taking a blood sample. Pigs were habituated to rectal temperature measurements in the weeks prior to the challenge to prevent a temperature change related to handling stress.

#### Heat stress challenge

At the age of 111.0 ± 1.9 days (weight: 68.3 ± 6.9 kg), the 96 focal pigs were group-wise subjected to a heat stress challenge. Due to time and space constraints, the pigs were tested on two different days. The focal animals were moved per pen to climatic respiration chambers (4.5 m × 2.5 m). This was done the day before the challenge at 13:00 h to habituate them to the new environment. The focal animals were housed in pens of 3.5 m × 1.8 m (1.6 m2 per pig) with a partly solid and partly slatted floor. Feed and water were available ad libitum. A ball on a chain was provided as a toy. The light schedule was similar to that of the regular room. The ambient temperature was set at 21 °C. During the heat challenge, the temperature raised in 2 h (from 8:30 to 10:30 h) from 21 to 35 °C, stayed at 35 °C for two extra hours and returned to 21 °C in the following 2 h. Humidity was set at 50%. The following day, at 8:00 h, the pigs were moved back to their original room.

From 13:00 h on the day before heat stress until 8:00 h on the day of the heat stress challenge, heat production (kJ/kg0.75/day), activity (counts), O2 consumption (L/kg0.75/day), CO2 production (L/kg0.75/day) and CH4 production (L/climate respiration chamber/day) were measured every 5 min as described before37. Average values per relevant time period (baseline during the day, baseline during the night from 20:00 to 8:00 h (based on 2 days, the day before and the day after the challenge), during the 2 h temperature increase, during the 2 h period of temperature at 35 °C, and during the 2 h temperature decrease were calculated. At the individual level, the respiration rate per min was measured by counting belly movements of each animal for a period of 30 s and by doubling the amount obtained. Respiratory rates were measured three times on the day before the challenge at 12:00 h in their home pen; at 15:00 h and 16:00 h in the climatic chambers; the day of the heat challenge every 30 min between 8:00 and 15:00 h and at 16:00 h; the day after the challenge at 8:00 h in the climatic chambers before leaving, and at 12:00 h in their home pen. Feed intake was determined for a period of 19 h from entering until leaving the respiratory chambers.

#### Wound healing challenge

At the age of 123.0 ± 1.7 days (weight: 88.5 ± 13.3 kg), the 96 focal pigs were subjected to a fat biopsy to study their wound healing. A wound of a diameter of 0.7 cm and a length of 3.5 cm was created in the fat from the neck by a penetrating gun as described by Baes et al.38. Briefly, the pigs were restrained with a nose sling during the procedure. The location sampled was cleaned with betadine soap before the biopsy. Between each pig, the needle of the gun was cleaned with a solution of 70% ethanol. After the biopsy was done, the wound was sprayed with betadine to prevent infection.

The wound healing process was followed by measuring the perimeter of the wound from photographs taken, without restraining the pigs, by a digital camera daily for 10 days and on day 15 after the biopsy. If needed, the wound was cleaned with water before taking a picture. Measurements were made by a single trained person, blind to treatment, using the ImageJ software39 to delimit the wound perimeter. The length of a sticker placed close to the wound was used as a scale to standardize the measurements.

### Indicators measured during the challenges

#### Weights

All pigs were weighed 24 h before and 24 h after the start of each challenge. Relative weight change was estimated as follows: $$\frac{\left(Final weight-Initial weight\right)}{Initial weight}$$.

#### Blood samples

The animals were restrained with a nose sling during the blood sampling procedures. Blood samples were collected from the jugular vein. The blood (10 ml) was distributed as follows: 4 ml in EDTA tubes, 4 ml in heparin tubes and 2 ml in serum tubes. The EDTA and heparin tubes were centrifuged at 1500g for 10 min at + 4 °C. The serum samples were kept at ambient temperature for 30 min and thereafter were centrifuged at 1500g for 10 min at 4 °C. Plasma and serum were stored at − 20 °C until laboratory analyses.

Cortisol assays were performed in EDTA samples using the cortisol RIA kit from Immunotech (Beckman-Coulter, ref IM1841, Czech Republic). Glucose concentrations were measured in EDTA samples using a GOD-PAP kit (Hoffmann-La Roche, Switzerland). Non-Esterified Fatty Acids (NEFA) concentrations were measured in serum samples using a NEFA kit (WAKO Chemicals GmbH, Germany). Haptoglobin levels in heparin plasma were measured using the kit PHASE TM Haptoglobin Assay from Tridelta Development Limited (ref TP-801, Ireland). Titers of IgG and IgM in heparin samples binding keyhole limpet hemocyanin (KLH), myelin basic protein (MBP) and phosphoryl choline-conjugated to Bovine Serum Albumin (PC-BSA) were measured as described previously40. For IgG and IgM determination, only the sampling points 24 h pre-challenge, 24 h post-challenge and 48 h post-challenge were used. Urea concentrations were measured in EDTA samples using the enzymatic colorimetric test (ref 10506, HUMAN Gesellschaft für Biochemica und Diagnostica mbH, Wiesbaden, Germany).

#### Hair samples

The animals were shaved at 11 weeks of age before the start of the period with challenges, and at 18 weeks of age simultaneously with the biopsy, to determine the accumulation of cortisol over this period. The shaving area of about 225 cm2 was located at the same spot for all piglets to avoid potential bias related to the body part. The location was close to the hip of the animals, at the connection between the abdominal area and the hind leg. A one-use surgical razor was used for each animal. The experimenter wore gloves to avoid contamination of the samples. At 11 weeks, only the right side of the animals was shaved, while two sides were shaved at 18 weeks. This enabled to distinguish the effect of the period of challenges between 11 and 18 weeks (regrowth of the right side between the two shaving points) from the full life of the animals from birth onwards (left side never shaved). Samples were stored in aluminium foils at room temperature in the dark until analysis.

Hairs were washed three times: once in PBS and twice in isopropanol to remove any dirt and dust and were dried for 96 h at 37 °C. To extract the cortisol from the hairs, the samples were first cut with scissors in small pieces before being grinded with a tissue lyser. The cortisol from the hair powder was then extracted with methanol for 24 h. The extract was dried in a speedvac for 3 h and dissolved in a phosphate buffer. Cortisol concentration was determined using the high sensitivity salivary cortisol ELISA kit (ref 1-3002) from Salimetrics (Pennsylvania, USA).

#### Skin lesions

Skin lesions were scored 24 h before and 24 h after the start of each challenge according to the Welfare Quality Assessment Protocol® for pigs41, except for the wound healing challenge, when it was done simultaneously to the biopsy. Briefly, the body of the pig was divided into 5 separate regions: 1—ears, 2—front (head to the back of the shoulder), 3—middle (back of the shoulder to the hind quarters, 4—hind quarters, and 5—legs (from the accessory digit upwards). A scratch longer than 2 cm was considered as one lesion and two parallel scratches with up to 0.5 cm space in between were considered to be one lesion. Wounds smaller than 2 cm were considered as one lesion. Bleeding wounds, healed wounds of more than 5 cm as well as deep and open wounds of more than 5 cm were never observed. The number of lesions from the different body regions were summed to create a global score.

#### Tear staining

Photographs of the left eye of each focal pig were taken 8 times during the experiment to measure tear-staining. This was done 24 h before and 24 h after transport, LPS and heat stress challenges, right before the biopsy and before slaughter. Measurements were made on photographs by a single experienced person, blind to treatment, using the ImageJ software39 to delimit the tear perimeter. The length of the iris was used as a scale to standardize the measurements. All the brownish areas on the direct periphery of the eye (bottom of the upper eyelid, top of the lower eyelid, internal and external corners) were recorded42. The variable analysed was the cumulative area covered by the stain.

#### Pathological examination at slaughter

At the age of 140.0 ± 1.9 days (weight: 95.2 ± 8.5 kg), the 96 focal pigs were exsanguinated after an electrical stunning for dissection purpose. The judging of pathological changes was done blind to the housing treatment of the pigs. The heart was examined for pericarditis, the lungs for pneumonia and pleurisy, and the stomach for lesions and ulcers. The heart of the pigs was assessed for pericarditis with a score of 0 (no pericarditis) to 3 (severe pericarditis)43. Lung lesions for pneumonia were scored with a scale ranging from 0 (no lesion) to 28 (total pulmonary consolidation)44. Pleurisy was scored for the entire lungs ranging from 0 (no adhesion between the lobes) to 4 (lungs fully adherent to the thoracic cavity). Stomach wall damage at the pars oesophagus was assessed with a score of 0 (normal pars oesophagea, no hyperkeratosis or lesions) to 5 (hyperkeratosis and more than 10 lesions, ulcer or occlusion of the oesophagus into the stomach) (adapted from Hessing et al.45).

### Statistical analyses

Statistical analyses were performed with the software R 4.0.3.46. The variables tear staining, serum NEFA concentration and hair cortisol concentration were normalized by logarithmic transformation. Areas under the recovery curves (AUC) were approximated from repeated measurements using the trapezoidal rule $${\sum }_{k=0}^{N}\left(\frac{f\left({x}_{k-1}\right)+f\left({x}_{k}\right)}{2}\right)\times \Delta xk$$, where f(x) is a function and $$\Delta {x}_{k}$$ is the length of the k-th subinterval. The resilience indicator (ln(variance) based on body weight deviations was estimated using the same formulas as in the paper of Berghof et al.6.

On focal pigs only, linear mixed models were used for all variables measured once (relative growth, tear and skin lesions measured 24 h after the biopsy and before slaughter, post-mortem examinations, AUC, latencies of behaviours, total numbers of aggressive acts and posture changes during the transport challenge) with the function lmer from the R package “lme4”, except for frequencies of behaviours and scores of skin lesions and post-mortem measurements. For these variables, generalized mixed models with a Poisson distribution and Log link function were used and the function glmer from the R package “lme4”. In these models, Housing and Sex (boar versus gilt) were fixed effects, and Pen and Batch were random effects. For the repeated variables, repeated linear mixed models or repeated generalized mixed models with Housing, Sex, Time and the housing × time interaction as fixed effects, Pen and Batch as random effects, and Pig as a repeated variable were used. For hair cortisol, the Side of shaving and the Status of the animal (focal versus companion) were also included as fixed effects.

For the measurements of the heat challenge at the group level, Housing, Time period and housing × time period were used as fixed effects and Batch as a random effect. For the feed intake in the climatic respiration chambers, Housing was used as a fixed effect and Batch as a random effect.

p values below 0.05 were considered as significant effects and below 0.1 as tendencies. When a significant effect was found, pairwise comparisons between groups were made with the emmeans function of the emmeans package from R, including a Tukey correction. Data are presented as means ± SEM.

Source of this news: https://www.nature.com/articles/s41598-022-10745-4

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