The Dutch law on animal experiments and established principles of laboratory animal care were followed. They conform to the European Directive 2010/63/EU for the protection and care of scientifically-used animals. Wageningen University’s Animal Care and Use Committee approved this experiment (AVD1040020186245). All methods used in the study were conducted in accordance to the ARRIVE guidelines. The treatment of animals in different housing systems meant that the investigators knew about the treatment when they collected samples. Blindly, however, the lab analyses of tear staining on photos, behavioural observations during transport challenge, metabolic chamber parameters during heat stress challenge, respiratory rate, metabolic chamber parameters during heat stress challenge, wound healing zones on pictures and scores relating to organs during dissections were performed. The rise in cortisol levels was calculated using data from pigs under transport stress.36 (=5%, power=80%, SD=2.42, =2.8).
Animals
A total of 144 TempoTopigs-20 pigs were used in the experiment. There were n=71 females and n=73 males. The experiment was spread over three batches (n=48 per batch). Piglets were offspring of 24 multiparous sows. Half of the sows were housed in a CONV (meanSD, sow parity=4.21.8) while the other half was in an AHS (parity=4.01.7), at The Swine Innovation Centre (Sterksel), The Netherlands. The piglets were not castrated and their tails were not trimmed. Both systems had similar birth weights: 1.460.28kg for CONV and 1.440.27kg AHS.
Housing systems
From birth to 9 weeks old
Two housing systems were used to raise piglets (similar to van Nieuwamerongen and al)..21). The AHS contained five farrowing pens measuring 3.22.2m (mixture of solid 2.22.2m & slatted 1.02.2m), along with a communal area measuring 11.12.80m. The communal area was surrounded by a dunging (2.83.3m) and a feeding area (42.23.3m), both on slatted floors. Four jute bags (11080cm), and a slice of straw were added to the farrowing pen (approximately 1.5kg per pen) provided enrichment. Five sows were placed in this system one week before the expected farrowing day. Two days prior to the expected date of farrowing, five sows per batch were moved to a farrowing pen. The sows were then kept in a farrowing container (1.90.6m). They were allowed to use the entire system again two days after farrowing. For 1 week, new born piglets were kept in their litter in the farrowing pen. They could then access the entire system and mix with the other litters. The farrowing pens provided piglets with a heated nest (0.71.6m) with temperatures of 3335C (day 1-7), 2931C (day 2-5) and 2326C (day 25-26). From 3 weeks of age, piglets were fed in round bowls. The piglets could also participate in the feeding of the sows by being fed in a large trough on the floor. The intermittent suckling, which was started at week 5 in order to stimulate solid food intake, was continued until the end of the year. AHS piglets were weaned at 62.61.9 days and a weight of 26.64.9kg. They were given a starter food from 35 days onwards.
From birth, piglets were kept in farrowing pen of 2.81.8m up to the time they were weaned. Sows were kept in a crate (1.90.6m). The floor was made up of metal slats inside the crate. There was a solid floor measuring 1.20.3m with a heating lamp for the animals. The remainder of the floor was made of plastic slats. From one week after their birth, piglets received additional creep food in their farrowing pens. CONV piglets reached the age of 27.41.2days and weighed 8.71.3kg. CONV piglets were weaned at 27.41.2 days of age and 8.71.3kg. They were then housed in nursery pens measuring 3.181.0m (0.40m).2Each piglet was provided with an enrichment of a jute bag and a chain for five more weeks. They were given a commercial diet for 10 days after weaning as well as a starter diet that was similar to that given to AHS-piglets starting at 35 days.
Both systems had lights on from 07:00h to 19:00h. This gave the sows and piglets 12h light with 115 Lux. The AHS also had natural daylight through two windows. In 10 minutes, the system switched between night and day light settings. Ambient temperature in both systems was 23C. Both systems had water available ad libitum.
From 9 weeks of age onwards
After being weaned from the AHS piglets around 9 weeks old, all the piglets were transferred to Wageningen’s Carus research facilities. They were mixed together in groups of six piglets from different systems. The pens were balanced on litter, sex, and weight. To ensure representative litters, piglets were chosen based on their sex (50/50% male and 50% female). 6 piglets were chosen per litter: 2 piglets whose birth weight was between the minimum weight of litter+10% and 1st weight quarterile (light); 2 with a weight between 1st and 3rd quarterile (medium); 2 with a weight between the 3rd and maximum weight 10% (heavy).
AHS pigs were kept in a pen measuring 2.404.67m, i.e. Double the size of a traditional pen (1.87m).2Per pig), and enriched with sawdust, peat, and straw bedding. These were replenished daily (2.5kg of straw every day, 30 L of seedust every other day, 22.5L peat every other week). AHS pigs received a handful of hay and alfalfa, or cardboard egg trays, once a week, as well as a chain, rope, jute bag, or rope (rotation every other week). They were also provided with an extra toy: a chewing toy for dogs, a chewing toy for dogs, and a biting ball on the chain.Toy, a green MS Schippers Bite tube, or a MS Schippers Cross in greenTo preserve the toys’ attractiveness, they were changed every 2 days. CONV pigs were kept in standard pens measuring 1.204.67m and with conventional space allowance (0.93m).2per pig) with a partially solid and partly slatted flooring without substrate. CONV piglets were given a ball, a chain with screws and a key with keys. These items were not altered. AHS and CONV pens could be placed in separate rooms. The same feed was used to raise pigs.
The light regime was the same as before 9 weeks of age. The pigs were given 115 Lux in their pens during daylight (from 7:00h until 19:00h; 5000K UV A at an intensity of 42, 2700K, at 60), and 30 Lux at night (5000K UV A at an intensity of 3, 2700K, at 0). The 10-minute transition between night and day was gradual. There was no natural daylight. For the first 2 days, temperatures were maintained at 23C. Then, they dropped to 22C for the next 2 days. At 21C, the temperature continued to rise.
Challenges
The two housing systems were used to assess the resilience of the pigs. This was done by following the recovery of the animals after they had been subjected to successive challenges: a 21h-isolation (not detailed here), a 2h transport challenge, an LPS injection in order to induce sickness response, a 2h heat stress challenge, and a wound. Figure1 shows the order and duration of the challenges as described in this paper. The housing system required that the pigs were exposed to the challenges in a balanced order. Four animals per pen were subjected to the experimental challenges (Focals – two males, two females – and two males – while two other pigs acted as companions (i.e. The two pigs with the highest deviation from the average penweight
Schematic view showing the experiment from birth until dissection of pigs kept in either an alternative (AHS), conventional (CONV), or both.
Transport challenge
At the age 83.01.7 days (weight 41.65.5kg), 94 of the 94 focal pigs were transported for 2 hours. One of the 96 focal porks was a tailbiter (CONV housing). It was put to sleep with pentobarbital (Euthasol) and euthanized.Vet) and then replaced by a companion porc after the transport challenge. The transport challenge was not presented to either of these two pigs. The pigs were loaded into a trailer with a thin layer on the floor of sawdust and transported from 8:00 to 10:00 h. All pigs from the same batch (from both housing system) were loaded in a trailer with a thin layer of sawdust on the floor. They were transported from 8:00 to 10:00h. This created both a social and metabolic challenge. To track the animals’ recovery, blood samples were taken 24h before (or baseline) the transport challenge. The samples were taken at the animals’ return from the 2h transport (referred to as 0h), 3h, 24h, 48h, and 48h after the end.
The day before transportation, pigs were painted with paint to track their behaviour in the trailer over the 2h-transport challenge. A single trained observer used the software Observer 14.2 (Noldus Information Technology), Wageningen, The Netherlands, to continuously score aggressive behaviours (either a push or a bite, a knock or a fight) as well as lying behaviour. Over 30-min intervals, the average time spent lying was calculated. Over the 2h period, both the total number dreadful acts and the total number obstructive postures were averaged. Poor lighting in the trailer made it difficult for the pigs to be observed before sunrise. Therefore, batch 1’s first hour was not included in the analysis.
LPS challenge
At the age 104.41.7 days (weight: 60.07.5kg), 92 focalpigs were injected into the ear vein with 2g LPS/kg body weight (LPS Sigma L4391). Escherichia coli O111 B4). Four focal pigs were excluded from the challenge because they were sick or on antibiotic treatment at the time of the challenge. The pigs were divided into two groups, each on a separate day, to allow for housing system balance. Injections were done in one pig’s ears in their home pen. Blood samples were taken 24 hours before (baseline), and 1h, 3, 5, 5h, and 24-hours after the injection. Before taking a blood sample, rectal temperature was measured at each time point. To prevent any temperature changes related to stress handling, the pigs were trained to take rectal temperature measurements for several weeks.
Heat stress challenge
The 96 focal pigs, who were 111.01.9 days old and weighed 68.36.9kg, were subject to a heat stress challenge group-wise. The pigs were tested on two days because of space and time constraints. The focal animals were moved from one pen to climatic respiratory chambers (4.5m2.5m). To get them used to the new environment, this was done at 13:00 the day before the challenge. The focal animals were kept in pens measuring 3.5m1.8m (1.6m)2Each pig will have a floor that is partly solid and partially slatted. Water and feed were available ad-libitum. As a toy, a ball attached to a chain was offered. The light schedule was the same as that for a regular room. The ambient temperature was set to 21C. The heat challenge saw the temperature rise in 2h (from 8:30-10:30h) from 21C to 35C. It remained at 35C for 2 hours, then returned to 21C within 2h. The humidity was set at 50%. The pigs were returned to their original room at 8:00 the next morning.
Between 13:00h on day before heat stress and 8:00h on day of heat stress challenge, heat output (kJ/kg).0.75/day), Activity (Counts), O2Consumption (L/kg)0.75CO (/day),2Production (L/kg0.75/day) or CH4Production (L/climate respiratory chamber/day) were measured every 5min, as described previously37. Average values for the relevant time period (baseline during day, baseline during night) were calculated. They were based on 2 days (the day before and the following day), during the 2h temperature rise, during the 2h period at 35C and during 2h of temperature decline. The individual respiration rate per minute was determined by counting the animal’s belly movements for 30 seconds and then doubling the amount. The day before the challenge, at 12:00h in the home pen, at 15:00h in climatic rooms and at 16:00h respectively; on the heat challenge day at 16:00h every 30 minutes between 8:00 and 15:00h; on the day following the challenge at 8:00h before leaving and at 12:00h in home pen. From the time they entered to the exit of their respiratory chambers, feed intake was measured for 19 hours.
Wound healing challenge
The 96 focal pigs reached the age of 123.01.7days and were weighing 88.513.3kg. A fat biopsy was performed to examine their wound healing. Baes and colleagues described how a penetrating guns created a wound in the neck fat with a diameter 0.7 cm and a depth of 3.5 cm.38. The procedure was brief. Before the biopsy, the location was cleaned with betadine soap. The needle of the gun was cleaned between each pig with a 70% ethanol solution. To prevent infection, the wound was sprayed using betadine after the biopsy.
The wound healing process was completed by measuring the perimeter using photographs taken by a digital camera every day for 10 days and 15 days after the biopsy. The wound was cleaned with water if necessary before taking a photograph. A single trained person was responsible for measuring the wound using ImageJ software.39To delimit the wound perimeter. The measurement was standardized by a sticker that was placed close to the wound.
During the challenges, indicators are measured
Weights
All pigs were weighed before and after each challenge. The following was an estimate of the relative weight change: (frac{left(Final weight-Initial weightright)}{Initial weight}).
Blood samples
During the blood sampling procedures, the animals were secured with a nose sling. Blood samples were taken from the jugular blood vessel. The blood (10ml) was divided as follows: 4ml for EDTA tubes, 4ml for heparin tubes, 2ml for serum tubes. The heparin tubes and EDTA were centrifuged at 1500gfor 10 minutes at+4C The serum samples were kept at ambient temperatures for 30min, then centrifuged at 1500gFor 10 minutes at 4C Between laboratory analyses, plasma & serum were kept at 20C.
Cortisol assays were conducted in EDTA samples using a cortisol RIA kit (Beckman-Coulter – ref IM1841, Czech Republic). Glucose concentrations in EDTA samples were measured using a GOD-PAP (Hoffmann-La Roche), Switzerland. Non-Esterified Fatty Acids concentrations (NEFAs) were measured in serum samples using a NEFA Kit (WAKO Chemicals GmbH Germany). The kit PHASETM haptoglobin assay from Tridelta Development Limited was used to measure the hemoglobin levels in plasma containing heparin (ref.TP-801, Ireland). As described previously, the IgG and IgM levels in heparin samples binding keyhole hemocyanin (KLH), Myelin Basic Protein (MBP), and phosphoryl Choline-conjugated To Bovine Serum Albumins (PC-BSA), were measured.40. Only the 24h pre-challenge sampling points, 24h postchallenge sampling points, and 48h afterchallenge were used for IgG or IgM determination. The enzymatic colourimetric test was used to determine the concentration of uranium in EDTA samples (ref 10506 HUMAN Gesellschaft Fr Biochemica und Diagnostica mbH Wiesbaden).
Hair samples
The animals were shaved at 11 weeks of age, before the start of the period with difficulties, and at 18 weeks of age simultaneously with the biopsy to determine the amount of cortisol that had built up over the period. The shaving area was approximately 225 cm2To avoid any biases regarding the body part, the location was the same for all piglets. The location was located close to the hips of the animals, at connection between the abdominal region and the hind legs. Each animal received a single-use surgical razor. To avoid contamination of the samples, the experimenter wore gloves. At 11 weeks, only the right side was shaved. At 18 weeks, two sides were shaved. This allowed us to distinguish the effects of the 11-18 week period of challenges (regrowth on the right side between two shaving points) and the full life span of the animals (left sides were not shaved). The samples were kept at room temperatures in the dark until analysis in aluminium foils.
The hairs were washed twice with PBS and twice with isopropanol to remove dirt and dust. They were then dried for 96h at 37C. The hair samples were first cut in small pieces with scissors to extract cortisol. Next, they were ground with a tissue grinder. The hair powder was then extracted using methanol for 24 hours. The extract was then dried on a speedvac for three hours and then dissolved in a buffer of phosphate. The high sensitivity salivary cortisol ELISA kit (ref 13002) was used to determine the concentration of cortisol. It is available from Salimetrics (Pennsylvania), USA.
Skin lesions
According to the Welfare Quality Assessment Protocol, skin lesions were recorded 24 hours before and 24 hours after each challenge.Pigs41Except for the wound healing challenge which was performed simultaneously with the biopsy, it was not. The body of the pig was divided into five distinct regions: 1ears to the front (head to back of shoulder), 2front to the head, 3middle to the hind quarters, fourhind quarters, 5legs to the accessory digits. A single lesion was defined as a scratch that is longer than 2cm. Two parallel scratches with a maximum of 0.5cm between them were also considered one lesion. One lesion was considered to be a wound less than 2cm. Bleeding wounds, wounds that have been treated and healed of greater than 5cm in length, as well as open and deep wounds of greater than 5cm in depth, were not observed. To create a global score, the number of lesions in each body region was added together.
Tear staining
Eight photographs of each focal porc’s left eye were taken during the experiment to measure tear staining. This was done 24h prior to and 24h after transport, LPS, heat stress challenges, and right before the biopsy. The ImageJ software was used to measure the patient’s body using photographs.39To delimit the tear perimeter. To standardize measurements, the length of the iris was used. All brownish areas around the eye’s direct periphery (bottom of upper eyelid, top lower eyelid) were recorded42. The variable analysed was defined as the cumulative area covered in stain.
Pathological examination at slaughter
The 96 focal pigs, who were 140.01.9 days old and weighed 95.28.5kg, were exsanguinated following an electrical stunning to dissect them. The housing treatment of the pigs was not used to judge the severity of any pathological changes. The heart was examined to determine if there was pericarditis. Also, the lungs were examined for pneumonia and pleurisy. The pericarditis score ranged from 0 (no pericarditis), to 3 (severe).43. Lung lesions due to pneumonia were scored using a scale that ranged from 0 (no lung lesion) up to 28 (total consolidation).44. Pleurisy was scored for all lungs. Scores ranged from 0 (no adhesion among the lobes to lungs) to 4 (4 lungs fully adherent the thoracic cavity to lungs). Stomach wall damage at pars oesophagus was scored with a score ranging from 0 (normal pars, no hyperkeratosis, lesions or lesions) up to 5 (hyperkeratosis, more than 10 lesions, ulcer, or occlusion into the stomach). (Adapted from Hessing and al.45).
Statistical analyses
With the software R 4.0.3, statistical analyses were done.46. Logarithmic transformation was used to normalize the variables tear staining and serum NEFA concentration as well as hair cortisol levels. The areas under the recovery curves (AUCs) were calculated from repeated measurements using a trapezoidal rule. ({sum }_{k=0}^{N}left(frac{fleft({x}_{k-1}right)+fleft({x}_{k}right)}
