Interactive effects of digestible protein levels on thermal and physical stress responses in Nile tilapia

- The effects of dietary digestible protein (DP) levels (22, 26, 29, 32, and 34%) and different stressors (cold-induced stress, CIS; heat/dissolved oxygen-induced stress, HDOIS; transport-induced stress, TIS; and size-sorting-induced stress, SSIS) on hemato-biochemical parameters were evaluated. Four hundred and forty Nile tilapia fingerlings were distributed into 40-250 L aquaria and fed experimental diets for 110 days, and fed each of the five experimental diets, that were randomly distributed to eight replicates per treatment. Then, different groups of fish were subjected to one type of stress. Groups of 40 fish were used on CIS (17 °C), HDOIS (32 °C), and TIS (4 h), and a group of 140 fish on SSIS (15 min air exposure and 60 s handling). There was no effect on hemato-biochemical profile when DP levels were compared, neither before nor after stress; however, there was a significant stress effect. Digestible protein did not mitigate stress response under SSIS and CIS; lymphopenia and neutrophilia were the main cell-mediated immune response; dietary 22 and 26% DP impaired oxygenation on SSIS and TIS; fish under HDOIS and SSIS demanded more energy using triglycerides as an energy source; the diet formulated to contain 22% DP was not adequate to keep homeostasis under temperature stress. Cluster analysis showed that, for DP levels below the requirement for growth, SSIS and CIS were considered the most stressful conditions. At 34% DP level, HDOIS response was comparable to that of non-stressing conditions. Red blood cell (RBC) and leukocyte counts were determined by dilution and enumeration using a hemocytometer. Leukocyte differentiation was performed using blood extension stained with May-Grunwald-Giemsa stain according to Jain (1986). Differential counting was performed under a microscope at 100X in immersion oil. To establish the percentage of each cellular component, 200 cells were counted on each extension. Hemoglobin (Hb) was determined by the cyanomethemoglobin colorimetric method using a commercial kit (Gold Analisa Diagnostica, Belo Horizonte, MG, Brazil) according to Collier (1944). Hematocrit (Htc) percentage was determined using the microhematocrit method described by Goldenfarb et al. (1971). Mean corpuscular volume [MCV = (Htc × 10)/RBC] and mean corpuscular hemoglobin concentration [MCHC = (Hb × 100)/Htc] were calculated according to Wintrobe (1934). Total plasma protein (TPP) level was measured using a manual Goldberg refractometer (Model SPR – N, Atago CO LTD, Japan) after the blood was centrifuged at 5000 rpm for 15 min. For the biochemical analyses, blood was collected from the caudal vein, as aforementioned, using a tuberculin syringe with no anticoagulant. After that, blood samples were centrifuged at 5000 rpm for 20 min, and supernatant serum was collected. Albumin concentration (ALB) was determined by the bromocresol method using the commercial kit Albumina Analisa Diagnostica ® for colorimetric determination. Albumin:globulin ratio (A:G) was determined using the ALB and TPP values [Globulin = TPP − ALB; A:G = ALB:Globulin]. Glucose (GLU), triacylglycerol (TGL), and cholesterol (CHOL) concentration were determined by enzymatic method