*2.1. Animals*

Blood collection at the farm was carried out by a veterinarian in conjunction with routine sampling for sanitary controls. Transportation and slaughter of all pigs were carried out in compliance with EC regulation No 1/2005 and EC regulation No 1099/2013, respectively.

Fifty-one Apulo-Calabrese pigs registered in the herd book of ANAS and 52 crossbreeds [Duroc × (Landrace × Large White)] were used. Apulo-Calabrese pigs were born in this farm from the mating of 13 sows by seven boars of Apulo-Calabrese whilst crossbreeds were bought at about 30 kg live weight from another piggery in the same region. All the pigs were fattened in the same finish facility in separate pens (7–10 pigs per pen) according to their genetic type and were fed the same commercial diet (14,644 KJ DE/kg, 155 g crude protein/kg, 22 g crude fat/kg, 80 g lysine/kg, 58 g ash/kg) in a liquid feeding system with dry feed and water mixed in a 1:4 ratio. All the pigs were identified by a numbered plastic ear tags. Apulo-Calabrese pigs were slaughtered when they reached 135 kg live weight (364 ± 58 days old) due to their slow growth whilst crossbreeds were slaughtered at approximately 155 kg live weight (300–330 days old).

## *2.2. Pre-Slaughter and Slaughter*

Approximately 12 h prior to transport, feed was withdrawn. Loading was carried out at 7 a.m. using a mobile ramp (length 4.5 m, width 0.7 m, with solid side walls of 1.0 m and adjustable height) available at the farm. Pigs were delivered through three consignments to the slaughterhouse. At each delivery, pigs from the four pens were herded by electric prods and walked the same distance (25 m) to reach the ramp which was positioned in correspondence with the facility door. The lorry was a hydraulic three tier equipped with internal partition and mechanical ventilation on the left side. Pigs were transported for approximately 1 h to a local processing plant (Piano Lago, Cosenza, Italy) on two decks with a space allowance of about 0.50 m2/100 kg live weight. Unloading was done using the ramp of the lorry and pigs were driven for 10 m to the lairage pens where they rested for 30 min. Outdoor temperature and relative humidity were recorded for each journey by a thermo-hygrometer (mod. HI9065, Hanna, Padua, Italy) during loading and unloading at the entrance of the ramp and at the entrance of the resting pen, respectively (Table 1). During transport and lairage, mixing between pens was avoided. The pigs were stunned by electrical tongs (head only; 220 V, 1.3 A). After stunning, exsanguination blood was collected from each pig (Table 1).

**Table 1.** Outdoor temperature (Temp), relative humidity (RH), and durations of loading, transport and unloading recorded in pigs in the three deliveries.


1 From the opening of the farm gate until the last pig entered the lorry. 2 From the opening of the gate of the lorry until the last pig entered the lairage pen.
