WWW.WAS.ORG • WORLD AQUACULTURE • SEPTEMBER 2019 67 quality is a potential factor in changing hemostasis as it prompts many physiological responses (De Verdal et al. 2014, Miranda-Baeza et al. 2017). Furthermore, additives such as probiotics can be used to modulate the microbial community to benefit host organisms. However, microbial diversity within different aquatic production systems is an inherent characteristic of aquaculture, making research necessary to establish criteria for selection and use of probiotics (D’Abramo 2018). This article describes the results of research to evaluate the physiological adjustments of red tilapia juveniles farmed in seawater with biofloc and probiotics. Study Methods Experimental facilities and experimental design. The experiment was carried out in the Aquaculture Laboratory of the Facultad de Ciencias del Mar, Universidad Autónoma de Sinaloa, México. The experiment had four treatments: low density (114 fish/ m3) and high density (228 fish/m3) of red tilapia juveniles (6.7 ± 0.2 g initial weight) reared in a biofloc system (B1 and B2, respectively), each density with or without addition of a commercial probiotic (B+P1 and B+P2). Control tanks at each density (C1 and C2) used seawater without biofloc and probiotics. The experiment had a completely randomized design with three replicates (Fig. 2). Tanks (n=18) were filled with 350 L of seawater with an average salinity of 35 g/L. The study was carried out in outdoor conditions with a natural photoperiod (12 h light, 12 h dark) and water levels were maintained by additions of fresh water. Prior to the start of the bioassay, biofloc was established at a carbon/nitrogen ratio of 15 using a mix of cornmeal, molasses and commercial tilapia feed that was fermented for 18 h before adding to respective tanks. The commercial probiotic (Eco-Technology Solutions S.A. de C.V.) was added to the mix of cornmeal and molasses for each respective tank according to the experimental design. Fish were fed three times per day (0900, 1300 and 1700 h) with a commercial tilapia feed (Nutripec, 40 percent crude protein, 8 percent crude lipid) at 5 percent of average body weight daily. The duration of the experiment was seven weeks. Blood sampling and analysis. At the end of the culture period, fish were starved for 24 h and then eight fish per tank were sampled. Immediately after fish were captured with a net, blood samples were collected by cardiac puncture (Fig. 3) using a 1-mL syringe containing 0.16 mL of EDTA anticoagulant solution (Valenzuela et al. 2002). Each blood sample was analyzed individually. Total erythrocyte count was determined by the modified methods of Blaxhall and Daisley (1973). Hemoglobin concentration was measured using the methaemoglobine method of Pointe Scientific kits. Packed cell volume was determined by micro-capillary glass tube and centrifugation (10,000 rpm for 5 min). Those values were used to calculate the red blood cell indices: mean corpuscular volume, mean concentration of hemoglobin and mean corpuscular hemoglobin concentration. The remaining blood samples were centrifuged at 12,000 rpm for 5 min at 4 C to obtain plasma (Atencio-García et al. 2007). Glucose, total protein, albumin and total cholesterol were measured using a standardized procedure of Pointe Scientific kits. Statistical analysis. Results are presented as means ± SD. Normality and homoscedasticity were evaluated by the Kolmogorov– Smirnov and Levene test, respectively. ANOVA one-way analysis was performed to compare means among groups. An two-way ANOVA was used to determine the significance of main effects and the interaction between densities and the different cultre systems (C, B and B+P). Data were analyzed by SPSS 19. FIGURE 4. Mean ± standard deviation (represented by the error bars) from red blood cells indices of red tilapia cultured at low (114 fish/m3) and high (228 fish/m3) densities with sea water. The X axis represents different systems: control (C), biofloc (B) and probiotics (P). The Y axis represents A) total erythrocytes count (EC), B) hemoglobin (HB) concentration, C) packed cell volume (PCV), D) mean corpuscular volume (MCV), E) mean corpuscular hemoglobin (MCH) and F) mean corpuscular hemoglobin concentration (MCHC). Different superscript letters mean significantly different groups (P<0.05) from one-way ANOVA. FIGURE 5. Mean ± standard deviation (represented by the error bars) blood biochemistry of red tilapia cultured at low (114 fish/m3) and high (228 fish/ m3) densities in seawater. The X axis represents different systems: control (C), biofloc (B) and probiotics (P). The Y axis represents A) glucose (GLU), B) total cholesterol (CHOL), C) total protein (TP) and D) albumin (AL). Different superscript letters mean significantly different groups (P<0.05) from one-way ANOVA. (CONTINUED ON PAGE 68) D. A. B. E. C. F. C. A. B. D.
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