New methods to objectively evaluate animal welfare in intensive pig farming.

Summary Animal welfare is assuming a great importance in intensive farming systems, to improve productive performances and to satisfy the consumers’ request of ethical animal management. Objective evaluation of animal welfare, both from a qualitative and a quantitative point of view, is essential in order to identify critical situations and the respective corrective measures. The assessment of the global oxidative stress of a subject is a good indicator of its welfare status. Oxidative stress results from a mismatch between the production of damaging reactive oxygen species (ROS) and the organism’s capacity to mitigate their damaging effects. The principal defenses against oxidative stress reside in antioxidant enzymes and chain-breaking antioxidant compounds. The antiradical activity of blood is a parameter suitable to evaluate the global oxidative stress of an animal organism. Total antiradical activity of blood is a good indicator to evaluate pig welfare (Brambilla et al., 2002). Several assays to evaluate total antiradical activity (TAC) of different substrates have been developed, they belongs to two categories: 1. hydrogen atom transfer (HAT) reaction based assays that monitor competitive reaction kinetics between a synthetic free radical generator and the antioxidant compounds of the substrate, revealing the reaction with an oxidizable molecular probe; 2. single electron transfer (ET) reaction based assays that involve one redox reaction with the oxidant (also as the probe for monitoring the reaction) as an indicator of the reaction endpoint. A more suitable assay to evaluate TAC of blood is the KRL test, developed by French researchers (Prost M., 1989). It is a HAT reaction based assay optimized for the use on blood, with a different reaction revelation system. KRL is a biological test which measures the time needed to hemolyze 50% of the red blood cells exposed to a controlled free radical attack. The principle of the biological test is to submit whole blood to a thermocontrolled free radical aggression in order to mobilize all families of free radical scavengers present in the blood to fight off the oxidant attack (Stocker et al., 2003; Pieri et al., 1996; Girodon et al., 1997). The KRL test have several applications in vivo, particularly tested on human studies, or in vitro. KRL test description Whole Blood and RBC samples diluted to 1/50 was submitted in isotonic saline solution to organic free radicals produced at 37°C under air atmosphere from the thermal decomposition of a 27 mmol/L solution of 2,2´-azobis (2-amidinopropane) dihydrochloride (Spiral, Dijon, France). Hemolysis was recorded using a 96-well microplate reader by measuring the optical density decay at 450 nm. Results was expressed as the time required to reach 50% of maximal hemolysis (half-hemolysis time - HT50 - in minutes), which refers to the whole blood resistance to free-radical attack. Performance of KRL instrument provided by the company, indicate a CV of the repeatability less than 2.5% and of reproducibility less than 4% (Laboratoires Spiral, France). Since haemolysis times vary in a linear way with the trolox concentration, it is possible to convert them in AE (antiradical efficiency) where 1 unit of AE/L of blood or RBC corresponds to the antiradical power of 1 mMol of Trolox /L of reference blood. The aim of this work is to apply KRL on pigs, evaluating the oxidative stress with regard to different animal categories, age, housing conditions and nutrition. In this frame, five different studies have been carried on: Trial 1 - Total antiradical activity in male castrated piglets blood: reference values; Trial 2 - Effect of weaning on total antiradical activity in piglets; Trial 3 - Antioxidant supplementation in post weaning piglets: effects on total blood antiradicalic activity and serum haptoglobin; Trial 4 - Use of Phenylpropanoids in pig diet: effects on total blood antiradicalic activity and some serum biochemical parameters; Trial 5 - KRL test to objective evaluation of welfare: sensibility to housing conditions and dietary supplements.