Azaperone

Participating observers were 12 MSc students in Applied Animal Behaviour and Animal Welfare, who all had a general understanding of animal behaviour, but no specific expertise in pig behaviour. These observers scored pig behavioural expression in the OF and EPM following protocols as developed for Free Choice Profiling (FCP) methodology (Wemelsfelder et al., 2000, 2001 ). The first stage of the FCP method requires that observers generate their own descriptor lists for use in subsequent assessments. For this 16 demonstration clips (1 min long), deemed to be representative of the range of different behavioural styles and demeanours observed in the tests, were selected (eight for each apparatus) and shown to the observers. All demonstration clips and the subsequent study clips included sound. The 16 clips were shown to observers in two batches with a 10 min break between each batch. Each batch consisted of both OF and EPM footage but no more than two clips of the same apparatus were played in succession. The reason for showing recordings of both types of test in a single term generation session was that a single vocabulary would be produced that was applicable across both tests. After each individual clip, observers were given 2 min to write down as many descriptive terms for the observed pig as they thought were needed to adequately characterise that pig's behavioural expressions. All the terms generated in this way were then collated for each observer and used to create each observer's individual list of terms. When individual observers used both positive and negative antonyms (e.g. “confident” and “unconfident”, “comfortable” and “uncomfortable”), only the positive term was kept for use in subsequent scoring. The number of terms generated by each observer ranged from 11 to 43. For each observer's score sheet, the terms were arranged so that successive terms had contrasting meaning with (as far as possible) similar terms being listed further apart.
In the second stage of the FCP process, the observers scored the full set of clips, each using their individual list of previously generated terms. This scoring took place over four sessions on different days. On each day, observers were shown a batch (11 or 12 clips) of OF clips and a separate batch (13 or 14 clips) of EPM clips, with a break between batches. The order of OF and EPM batches was rotated each day. Clips were allocated to a session so that sessions were as far as possible balanced for whether pigs were drug-treated or not, by litter group, and recording time within the observation. The open field clips were also balanced for order of testing since the pigs were tested in the apparatus twice. Following viewing of each individual clip, observers were asked to quantify, for each term, the degree of expression shown by the pig, by marking a vertical line on a 125 mm visual analogue scale, ranging from minimum to maximum possible expression. Observers were unaware of any prior drug or other treatment applied to the pigs. They were instructed that the purpose of the experiment was to investigate and compare behavioural expression in the 2 different types of test.
Prior quantitative assessment of pig behaviour in these tests (Donald et al., 2010, 2011 ) identified three main factors that change with Azaperone treatment: activity, vocalisations and exploration. To provide a comparison with the qualitative measures of behaviour, these same quantitative measures of behaviour (see Table 1 for a summary Ethogram) were recorded (using event logging software: Observer 5.0, Noldus Information Technology) from the same 1 min long clips used for QBA.

Investigations were performed on 20 sexually immature female pigs of the Large White Polish breed (approximately 8 weeks old, weighing ca. 20 kg) divided into 4 groups: control animals (C group, n = 5), acetylsalicylic acid treated (ASA group, n = 5), the partial stomach resection group (RES group, n = 5), and animals with hydrochloric acid infusion (HCl group, n = 5). All animals were kept under standard laboratory conditions with admission to species-specific chow and water ad libitum. All surgical procedures were carried out in accordance with the Local Ethical Committee in Olsztyn (decision number 05/2010). All possible efforts were made to minimize animal suffering.
Animals were treated with azaperone (Stresnil, Janssen Pharmaceutica N.V., Belgium, 4 mg/kg of body weight, i.m.) 15 min before the injection of the main anaesthetic, sodium thiopental (thiopental, Sandoz, Kundl-Rakusko, Austria; 10 mg/kg of body weight, i.v.). Following median laparotomy, the stomachs were exposed and injected with 50 μl (1 μl per 1 injection) of a 5% aqueous solution of the fluorescent retrograde neuronal tracer Fast Blue (FB, EMS-CHEMIE, GmbH, Germany) into the diamond-shaped part (ca. 4 cm × 4 cm) of the stomach anterior prepyloric walls.
Then, ASA pigs were given acetylsalicylic acid (aspirin, BAYER; 100 mg/kg b.w.) orally 1 h before feeding for 21 days (from 7th day after FB injection). On day 22 of the experiment, during laparotomy the partial resection of the previously FB-injected stomach prepyloric areas was performed with an electrosurgical diathermy (ERBE, VIO 300S) in animals of the RES group. On day 23, animals of the HCl group were reintroduced into a state of general anaesthesia (as described above) and intragastrically given 5 ml/kg of body weight of a 0.25 M aqueous solution of hydrochloric acid using a stomach tube. Gastroscopic examinations (using a video-endoscope Olympus GIF 145 with a working length of 1,030 mm and a 9.8 mm diameter) were performed in animals constituting ASA and HCL groups on the first day of the experiment and on the last day of the study. On day 28, all pigs were euthanized by an overdose of anaesthetic and then transcardially perfused with 4% buffered paraformaldehyde (pH 7.4). Following perfusion, the coeliac-superior mesenteric ganglion (CSMG) complexes were collected and postfixed by immersion in the same fixative for 20 minutes, rinsed in phosphate buffer (pH 7.4) for three days, and then stored in a 30% buffered sucrose solution until sectioning. Gastritis in animals of ASA group was confirmed by histopathological examination of fragments of the prepyloric stomach wall collected after perfusion (using routine histopathological methods).

The study was performed on 12 juvenile (8–12 weeks old; 15–20 kg bodyweight (b.w.)) female Large White Polish pigs. The animals were kept under standard laboratory conditions. They were fed standard fodder (Grower Plus, Wipasz, Wadąg, Poland) and had free access to water. To ensure adequate acclimatization, the pigs were transported from the breeder to the animal quarters 5 days before the scheduled procedure. The pigs were divided into two groups. Six pigs served as controls (CTR) and were treated with intravesical instillation of a 5% aqueous solution of ethyl alcohol (60 mL). Another group of 6 pigs was treated with RTX (product code: AG-CN2-0534, AdipoGen Life Sciences, Hamburg, Germany) by intravesical instillation of the toxin (500 nmol per animal in 60 mL of 5% aqueous solution of ethyl alcohol) to mimic the dose and route of its administration used in humans. Both the alcohol and RTX solutions were given under anesthesia. First, all the animals were pretreated with atropine (Atropinum Sulfuricum, Polfa, Warsaw, Poland, 0.05 mg/kg b.w., s.c.) and azaperone (Stresnil, Janssen Pharmaceutica, Beerse, Belgium, 2.5 mg/kg b.w., i.m.). After that, buprenorphine (Bupaq, Richter Pharma AG, Wels, Austria, 20 µg/kg b.w., m.c., i.m.) was given to abolish the visceral pain sensation. Thirty minutes later, anesthesia and analgesia were induced by intramuscular injection of ketamine (Bioketan, Vetoquinol, Gorzów Wielkopolski, Poland, 10 mg/kg b.w.) and xylazine (VetaXyl, Vet Agro, Lublin, Poland, 0.15 mg/kg b.w.) and by propofol (Propofol-Lipuro, B. Braun Melsungen AG, Vienna, Austria, 10 mg/kg b.w.,) given intravenously in a slow, fractionated infusion. The depth of anesthesia was monitored by testing the corneal reflex.
One week after the administration of the aqueous solution of ethyl alcohol or RTX, all the pigs were once again premedicated and deeply anesthetized (the drugs, their doses, and routes of administration were analogous to those previously used), and a midline laparotomy was performed. The urinary bladder was gently exposed in each animal and cut out for transcriptome analysis (the samples of the urinary bladder wall used in this experiment were collected from the middle part of the ventral side of the bladder). The samples were taken in vivo to ensure the acquisition of high-quality and -purity RNA for transcriptome sequencing. Next, all the pigs were euthanized with an overdose of sodium pentobarbital (Euthasol, LeVet B.V., Oudewater, Holland, 140 mg/kg).

Identical anesthetic protocols were applied for all boars included in this study. Animals scheduled for castration were fasted for 12 h prior to the surgery and isolated from the rest of the pigs on the farm in order to facilitate capture and restraint at the time of the intervention. Boars were restrained using a snout snare passed caudally of the superior canine teeth and over the dorsal surface of the snout. Peripheral intravenous (IV) administration of sedative and anesthetic drugs was achieved through the auricular veins, as seen in Figure 1. The weight of the animals was measured using a professional electric scale with a hook (PCE-CS 500, Ziboni Technology S.r.l., Rogno (BG), Italy).
Sedative and anesthetic drugs were administered in a single IV bolus. Sedation was achieved with 1 mg/kg of Azaperone (Stresnil, Elanco S.p.A. Italia, Sesto Fiorentino (FI), Italy) and 0.01 mg/kg of detomidine (Detonervin, Ecuphar Italia S.r.l., Milano (MI), Italy); general anesthesia was induced with a dose of 5 mg/kg of tiletamine/zolazepam (Zoletil 50/50, Virbac Italia, Milano (MI), Italy). The sedative and anesthetic medication administered was sufficient to maintain an adequate level of anesthesia for the entire duration of the procedure (which did not exceed 5–10 min in all cases starting from the first incision of the pre-scrotal area and ending with the excision of the second testis). After sedation and the induction of anesthesia, animals were positioned in dorsal recumbency, and the surgical site was prepared, allowing some time for deep anesthesia to initiate. This was done by first washing the whole area with antibacterial soap (DOC SCRUB PVP-IODIO, GARDENING S.r.l., Genova (GE), Italy) to eliminate large contaminant particles, followed by scrubbing with povidone-iodine solution (10%) for a few minutes. The testis was pushed cranially into the inguinal area, and a variable dose of 5–20 mL (according to the length of the incision) of 2% lidocaine solution was injected subcutaneously (SC) along the incisional line to achieve local anesthesia. In no instance did the dose of lidocaine exceed the dose of 15 mg/kg [8 ].