Effect of different levels of essential oils (<i>Satureja hortensis</i>) in diet on improvement growth, blood biochemical and immunity of Angelfish (<i>Pterophyllum scalare</i> Schultze, 1823)

<p>The aim of this study was designed to analyse the effect of Savoury essential oil (<i>Satureja hortensis</i>) on growth performance, biochemical parameters and immunity of angelfish (<i>Pterophyllum scalare</i>). The angelfishes (average body weight of 5.12 ± 0.01 g) were treated with three effective dosage of Savoury essential oil 100, 200 and 400 mg/kg in three separated aquaria water with one control group for 60 days. At the end of experiment, the angelfishes treated with 400 mg/kg Satureja showed minimum Feed Conversion Ratio, maximum Specific Growth Rate and Survival Rate. There was significant difference (<i>p</i> < 0.05) between the treated and control fish in the serum total protein, albumin, globulin, triglyceride, cholesterol, glucose and cortisol. Maximum significant amount (<i>p</i> < 0.05) in immunoglobulin is found in both 200 and 400 mg/kg Savoury concentration group, while significant enhancement in serum lysozyme was only found in 200 mg/kg group. Overall, the results presented in the current study revealed that savoury is a beneficial dietary supplement to improve growth performance, stress resistance and innate immune response of angelfish and the best level of inclusion was 200 mg/kg.</p

Effects of GSK3 inhibitors SB216763 or GSK-XIII on K⁺ independent (NCX) and K⁺ dependent (NCKX) Na⁺/Ca²⁺ exchanger activity in DCs.

<p><b>A,B.</b> Representative original tracings showing [Ca²⁺]_i in Fura-2/AM loaded <i>gsk3^WT</i> without (control, open circles) or with (closed circles) SB216763 treatment (1 µM, 30 min) DCs prior to and following removal of external Na⁺ (0 Na⁺) at 0 mM K⁺ (A) and at 40 mM K⁺ (B). <b>C,D.</b> Arithmetic means ± SEM of the peak (left) and slope (right) values of [Ca²⁺]_i increase following removal of external Na⁺ at 0 mM K⁺ (<b>C</b>, n = 24–51) and at 40 mM K⁺ (<b>D</b>, n = 43–46) in <i>gsk3^WT</i> DCs, without (white bars) or with (black bars) SB216763 treatment (1 µM, 30 min). *(p<0.05), unpaired <i>t</i>-test. <b>E,F.</b> Arithmetic means ± SEM of the peak (left) and slope (right) values of [Ca²⁺]_i increase following removal of external Na⁺ at 0 mM K⁺ (<b>E</b>, n = 47–82) and at 40 mM K⁺ (<b>F</b>, n = 43–46) in <i>gsk3^WT</i> DCs without (white bars) or with (black bars) GSK-XIII treatment (10 µM, 30 min). *(p<0.05), unpaired <i>t</i>-test or Mann–Whitney U test.</p

K⁺ independent (NCX) and K⁺ dependent (NCKX) Na⁺/Ca²⁺ exchanger activity in DCs from <i>gsk3^KI</i> and <i>gsk3^WT</i> mice.

<p><b>A,B.</b> Representative original tracings showing [Ca²⁺]_i in Fura-2/AM loaded <i>gsk3^WT</i> (open diamonds) and <i>gsk3^KI</i> (closed circles) DCs prior to and following removal of external Na⁺ (0 Na⁺) at 0 mM K⁺ (<b>A</b>) and at 40 mM K⁺ (<b>B</b>). <b>C,D.</b> Arithmetic means ± SEM of the peak (left) and slope (right) values of [Ca²⁺]_i increase following removal of external Na⁺ at 0 mM K⁺ (<b>C</b>, n = 95–129) and at 40 mM K⁺ (<b>D</b>, n = 27–34) in <i>gsk3^WT</i> DCs (white bars) and <i>gsk3^KI</i> DCs (black bars). *(p<0.05), ***(p<0.001), unpaired <i>t</i>-test.</p

Effects of GSK3 inhibitor GSK-XIII on Orai1, STIM1, and STIM2 protein abundance in DCs.

<p><b>A.</b> Original western blot showing the protein abundance of Orai1, STIM2 and respective GAPDH, STIM1 and respective GAPDH, in DCs without (control) and with GSK-XIII treatment (10 µM, 4–24 h). Blots were stripped and reprobed with a GAPDH antibody to determine equal protein loading. <b>B.</b> Arithmetic means ± SEM (n = 4–5 independent experiments) of the relative (to GAPDH) protein abundance of Orai1, STIM1 and STIM2 in DCs without (white bar) and with (black bars) GSK-XIII treatment (10 µM, 4–24 h).</p

Effects of GSK3 inhibitor SB216763 on Orai1, STIM1, and STIM2 protein abundance in DCs.

<p><b>A.</b> Original western blot showing the protein abundance of Orai1 and respective GAPDH, STIM1 and respective GAPDH, STIM2 and respective GAPDH in DCs without (control) and with SB216763 treatment (1 µM, 4–24 h). Blots were stripped and reprobed with a GAPDH antibody to determine equal protein loading. <b>B.</b> Arithmetic means ± SEM (n = 4–5 independent experiments) of the relative (to GAPDH) protein abundance of Orai1, STIM1 and STIM2 in DCs without (white bar) and with SB216763 treatment (1 µM, 4–24 h, black bars).</p

Thapsigargin-induced intacellular Ca²⁺ release and subsequent SOCE in DCs from <i>gsk3^KI</i> and <i>gsk3^WT</i> mice.

<p><b>A.</b> Representative original tracings showing [Ca²⁺]_i in Fura-2/AM loaded <i>gsk3^WT</i> (open circles) and <i>gsk3^KI</i> (closed triangels) DCs prior to and following removal of extracellular Ca²⁺, addition of SERCA inhibitor thapsigargin (1 µM) and readdition of extracellular Ca²⁺. <b>B.</b> Arithmetic means ± SEM (n = 44–59) of the peak (left) and slope (right) values of [Ca²⁺]_i increase upon Ca²⁺ release from intracellular stores (upper bars) and upon SOCE (lower bars) in <i>gsk3^WT</i> DCs (white bars) and <i>gsk3^KI</i> DCs (black bars). ***(p<0.001), unpaired <i>t</i>-test.</p

Effect of GSK3 inhibitors SB216763 or GSK-XIII on thapsigargin-induced intacellular Ca²⁺ release and subsequent SOCE in DCs.

<p>A. Representative original tracings showing intracellular Ca²⁺ concentrations ([Ca²⁺]_i) in Fura-2/AM loaded wild type (<i>gsk3^WT</i>) dendritic cells (DCs) prior to and following removal of extracellular Ca²⁺, addition of the sarco-endoplasmic Ca²⁺ ATPase (SERCA) inhibitor thapsigargin (1 µM) and readdition of extracellular Ca²⁺, all in the absence (open circles) and presence (closed circles) of GSK3 inhibitor SB216763 (3-[2,4-Dichlorophenyl]-4-[1-methyl-1H-indol-3-yl]-1H-pyrrole-2,5-dione, added 30 min before the experiment) 1 µM (left) or 10 µM (right). <b>B.</b> Representative original tracings showing [Ca²⁺]_i in Fura-2/AM loaded <i>gsk3^WT</i> without (open circles) and with (closed triangles) presence of GSK3 inhibitor GSK-XIII (10 µM, 30 min) DCs prior to and following removal of extracellular Ca²⁺, addition of SERCA inhibitor thapsigargin (1 µM) and readdition of extracellular Ca²⁺. <b>C.</b> Arithmetic means ± SEM (n = 16–83) of the peak (left) and slope (right) values of [Ca²⁺]_i increase following addition of thapsigargin reflecting Ca²⁺ release from intracellular stores (upper bars) and of [Ca²⁺]_i increase following readdition of extracellular Ca²⁺ reflecting store operated Ca²⁺ entry (SOCE, lower bars) in <i>gsk3^WT</i> DCs incubated in the presence and absence of GSK3 inhibitor SB216763 (100 nM, 1 µM, 10 µM, 30 min). *(p<0.05), ***(p<0.001), ANOVA Kruskal-Wallis Test, #(p<0.05), Mann–Whitney U test. <b>D.</b> Arithmetic means ± SEM (n = 41–53) of the peak (left) and slope (right) values of [Ca²⁺]_i increase upon Ca²⁺ release from intracellular stores (upper bars) and upon SOCE (lower bars) in <i>gsk3^WT</i> DCs incubated in the presence and absence of GSK3 inhibitor GSK-XIII (10 µM, 30 min). *(p<0.05), **(p<0.001), unpaired <i>t</i>-test or Mann–Whitney U test.</p

Orai1, STIM1, STIM2 and calbindin-D28k mRNA and protein abundance in DCs from <i>gsk3^KI</i> and <i>gsk3^WT</i> mice.

<p><b>A.</b> Original western blot showing the protein abundance of phosphorylated (p, Ser21/9) GSK3α,β, total GSK3α,β and respective GAPDH, STIM1, Orai1 and respective GAPDH, STIM2 and respective GAPDH in DCs derived from bone marrow of <i>gsk3^KI</i> and <i>gsk3^WT</i> mice. Blots were stripped and reprobed with a GAPDH antibody to determine equal protein loading. Also the blot of p-GSK3 was stripped and reprobed with GSK3 antibody. <b>B.</b> Arithmetic means ± SEM (n = 4 independent experiments) of the relative (to GAPDH) protein abundance of Orai1, STIM1 and STIM2 in <i>gsk3^WT</i> DCs (white bars) and <i>gsk3^KI</i> DCs (black bars). *(p<0.05), unpaired <i>t</i>-test. <b>C.</b> Original western blot showing the protein abundance of calbindin-D28k (first lane) and GAPDH (2^nd lane), in DCs derived from bone marrow of <i>gsk3^KI</i> and <i>gsk3^WT</i> mice. <b>D.</b> Arithmetic means ± SEM (n = 8 independent experiments) of the relative (to GAPDH) protein abundance of calbindin-D28k in <i>gsk3^WT</i> DCs (white bar) and <i>gsk3^KI</i> DCs (black bar). *(p<0.05), unpaired <i>t</i>-test. <b>E.</b> Arithmetic means (± SEM, n = 5–9) of the abundance of mRNA encoding Orai1, STIM1, STIM2, and calbindin-D28k in <i>gsk3^WT</i> DCs (white bar) and <i>gsk3^KI</i> DCs (black bar) as assessed by real-time PCR using TBP mRNA as a reference gene. **(p<0.01), unpaired <i>t</i>-test.</p