IgM antibodies and antigens reacted with each other in the sample and formed an antigen/antibody complex. Among various immuno-stimulants, medicinal herbs contain chemical constituents that enhance the immunity via specific or non-specific pathways, making the animal more resistant to external stressors12. Curcumin is an orange-yellow, hydrophobic, bioactive herbal ingredient and polyphenolic compound IL17RA extracted from turmeric (Fiber optics spectrophotometer. To study the surface morphology and size of the nanoparticles, a high-resolution transmission electron microscope (TEM), JEOL JEM-2100, was used. TEM imaging revealed the nearly spherical shape, smooth surface and uniform size distribution of nearly 50??5.5?nm of nano-curcumin (Fig.?1). Particle size distribution and zeta potential of nano-curcumin particles were determined by the Zetasizer Malvern instrument (Malvern, UK), version 7.13, serial number: MAL 1203718. Zeta potential (mV) and Zeta deviation (mV) were -25.0 and 4.16, respectively (Fig.?2). Open in a separate window Figure 1 Transmission electron microscope micrograph for as-prepared PVA capped nano-curcumin particles. Open in a separate window Figure 2 Apparent zeta potential and Zetasizer analysis of nano-curcumin particles. Productive performance All fish in each tank were collected and weighed before starting and at the terminal of the feeding trial. Growth, feed utilization and other biometric indices were calculated according to the following equations: to separate the serum, which was assigned for the biochemistry assay. Hematological assay Erythrocytes, hematocrit (HCT), hemoglobin (HGB), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelets (PLT), leukocytes and differential counts (neutrophils (NEUT), lymphocytes (LYMPH)), monocytes (MON), eosinophil (EO), basophils (BAS) and immature granulocyte (IG) were counted using automated hemo-cytometer XE 2100 (Advia 2120 & Sysmex, Siemens). All mentioned Pipobroman indices were assessed before and after the heat stress. Serum biochemistry Serum alanine aminotransferase (ALT), serum aspartate aminotransferase (AST), immunoglobulin M (IgM), complements 3 and 4 (C3 and C4, respectively), cortisol and glucose concentrations were analyzed in the serum of blood samples. Glucose was analyzed according to the hexokinase method35 using Cobas pack (reference number, Pipobroman 04404483 190) through the?Cobas-c 311 analyzer, system-ID 0768316. Cortisol was assayed based on the formation of the respective immune complex method36 through the electrochemiluminescence immunoassay ECLIA pack (reference number, 11875116 122) using the?Cobas-e immunoassay analyzer. ALT analysis was performed using Cobas c pack (reference number, 207649q57 322), Cobas-C 311 analyzer, system ID 0,764,957. ALT was detected according to Bergmeyer et al.37 and ECCLS38. ALT catalyzed the reaction between L-alanine and 2-oxoglutarate. The formed pyruvate is reduced by NADH in a reaction catalyzed by lactate dehydrogenase (LDH) to form L-lactate and NAD. The rate of the NADH oxidation is directly proportional to the catalytic ALT activity. It is determined by measuring the decrease in absorbance. AST in the sample catalyzed the transfer of an amino group between L-aspartate and 2-oxoglutarate to form oxaloacetate and L-glutamate. The oxaloacetate then reacted with NADH, in the presence of malate dehydrogenase (MDH), to form NAD. This assay followed the recommendations of the IFCC but was optimized according to Bergmeyer et al.37 and ECCLS38 using Cobas c pack (reference number, 20764949322) through the?Cobas-C 311 analyzer. System lD 076494I. IgM was determined based on the principle of immunological agglutination39 through the?Cobas c 311, system ID 0767883 using Cobas c pack (reference number, 03507190). IgM antibodies and antigens reacted with each other in the sample and formed an antigen/antibody complex. After agglutination, this was measured turbidimetrically at a sub/main wavelength: 700/340. C3 and C4 were determined by forming a precipitate through the addition of a specific antiserum and then?they were?determined turbidimetrically at a sub/main wavelength: 700/34039. C3 was measured through the Cobas?c 311, system ID 0765600 using Cobas c pack (reference number, 03001938322). C4 was analyzed through the?Cobas c 311, system ID 0765619 using Cobas c pack (reference number, 03001962322). Heat stress After the termination of the feeding experiment, five fish from each replicate were collected from the experimental glass aquaria and transferred into a 0.5-ton fiberglass tank containing 7 sub-rearing units (7-L perforated plastic cylindrical containers). Each container represents a replica of each treatment. Replicates A, B and C of each treatment have been exposed to the heat stress on?the first, second and third days, respectively. Thus, the heat stress challenge was repeated three times over three consecutive days. The water temperature was gradually raised from 25 to 40 C within 3?h. The water temperature was controlled by an artificial thermostat. After exposure to 40 C for 4?h, fish from each unit were anesthetized using clove oil. The Pipobroman blood samples were collected to?determine?ALT, AST, IgM, C3 and C4, glucose and cortisol levels, as well as the hematological count of fish. Statistical analysis Data of the?growth performance and feed utilization were processed.