OPTIMIZATION OF PROCESSING FOR BLOOD SAMPLES EVALUATION OF STRESS BIOMARKERS

ODEON, MM; CAPUCCIO, J; ROMERO, S; ROMERA, SA

Congreso; V Congreso Nacional- V Reunión Científica Regional- II Simposio de Métodos alternativos- AACyTAL; 2016

In recent years it is being given to increasing animal welfare standards importance due to the confluence of several factors. The objective measurement of welfare is a complex process, plasma levels of glucocorticoids and behavioral changes have been used as measures of stress, but we need to expand the search for biomarkers in order to control and monitor this state, both production systems and research. In order to evaluate cellular and molecular parameters as indicators of well-being in different species, we set out to develop a efficient method of obtaining sample for such assessment. We worked with samples of lama, swine and chicken. Blood samples were taken with citrate 3.8 % (1:10) as an anticoagulant. Then they processed with three different protocols for future evaluation of obtaining white blood cells with each of them in order to maximize the number of white cells obtained, avoiding contamination of red blood cells. It began with 500 ul of blood in each protocol, were performed in duplicate. The difference between protocols mainly consists of the method of red blood cell lysis, as it is the main variable to adjust due to the diversity between species. To analyze the efficiency of each cell counts protocol was performed with trypan blue in Neubauer chamber. To assess purity of the samples obtained an extended cells was performed, they were stained with Giemsa and observed under optical microscope. The general protocol follows this steps:1-Centrifuge 1000g 30min to separate plasma. 2-LYSIS 3-Centrifuge 1200g 5min 4-LYSIS 5-Centrifuge 1200g 5min. 6-Wash with RPMI. 7- Centrifuge 1200g 5min 8- Resuspend cells in RPMI10 % FBS. After analyzing the number of cells obtained and the contamination of RBCs is concluded that: in swine the lysis that optimized processing was: NH4Cl lysis Buffer 5 min , 4 ° C. In lama : NH4Cl lysis buffer 15 min , 37 ° C and in the case of chickens no red blood cell lysis was performed but a protocol was followed with ficoll gradient (1.077 g / ml) blood:ficoll 1:2. It was achieved, efficient purification and a good method of cryopreservation of cells at -80 ° C for each species.