World Aquacluture Magazine - September 2020

WWW.WA S.ORG • WORLD AQUACULTURE • SEP TEMBER 2020 47 the Nigerian Institute of Science Laboratory Technology, Ibadan, Oyo State (NISLT). Qualitative (Oloyede 2005) and quantitative (Harborne 1973) analyses of the extracts were carried out at the NISLT, Ibadan, Oyo State, to ascertain the presence of organic constituents in the ex- tracts. Qualitative (alkaloids, flavonoids, steroids, saponin, tannins, gly- cosides and cardiac glycosides) and quantitative (alkaloids, flavonoids, steroids and saponin) screening were conducted using the process and procedure described by Oloyede (2005) and Harborne (1973), respec- tively. Standard procedures were used to measure tannins and phytate (Pearson 1976), oxalate (AOAC 2010) and cyanide (Onwuka 2005). Neem leaf extract contained the following phytochemicals, with concentrations given as mg/g extract: alkaloids (1.34±0.04), flavo- noids (0.01±0.00), saponin (0.85±0.19), tannin (5.63±0.02) and phenol (1.47±0.01). In addition, the following phytochemicals were detected in neem leaf extracts: cardiac glycoside, anthraquinone, terpenoids, phylobatanin and steroid. Neem leaf extracts also contained phytic acid (2.65±0.05) and oxalate (1.44±0.83). Bacteria Challenge Staphylococcus aureus cultures were centrifuged at 1000 rpm for 10 min at 4 C. Supernatant was discarded and isolated bacteria were washed three times and placed in distilled water at a pH 7.4. Op- tical density of the solution was adjusted to 0.5McFarland standards at 456 nm, corresponding to 3×10 8 CFU/mL. Plant extracts were then used for sensitivity tests on isolated and identified fish bacteria and for phytochemical screening (Awe et al. 2019b). An experiment was carried out at Lagos State University Fisher- ies Laboratory. Plastic bowls (25 L capacity) were filled with 15 L of water supplied from hatchery storage tanks. African catfish Clarias gariepinus were cultured from fingerlings to sub-adults. Healthy fish (n=80) were stocked at ten fish in each bowl. During acclimation, fish were fed ad libitum twice daily with Coppens commercial feed. Ten mL of bacterial solution was added directly to water in plastic bowls. Water in each tank was exchanged with fresh water daily in the morn- ing. Remnant feed and fecal matters were siphoned on daily basis without stressing the fishes and mortality in each rearing tank was closely monitored. Neem leaf extracts were applied to water at 2.5 mg/mL, 5.0 mg/ mL, 7.5 mg/mL, 10.0 mg/mL. A positive control of 10.0 mg/mL of aquaceryl antibiotic was also applied. Hematology Blood was collected from the caudal peduncle of each fish and placed in Vaccutainers containing EDTA as an anticoagulant. Blood samples were refrigerated prior to hematological analyses at the Hematology Unit of the University of Lagos Teaching Hospital, Idi-Araba, Lagos. AMindray BC-3200 Auto Hematology Analyzer (ShenzhenMindray Bio-Medical Electronics Co., Ltd.) was used for quantitative determination of white blood cell (WBC), red blood cell (RBC), hemoglobin concentration (HGB), mean cell volume (MCV), mean cell hemoglobin (MCH), mean cell hemoglobin concentration (MCHC), coefficient of variation of red blood cell width (RDW-CV), standard deviation of red blood cell width (RDW-SD), hematocrit (HCT), platelet (PLT), mean platelet volume (MPV), platelet distribu- tion width (PDW) and plateletcrit (PCT) using the process and proce- dures of Klinkon et al. (2015) and Hammed et al. (2015 and 2017). Results of hematological examination of fish from different treatment groups (Table 1) showed significant difference (p<0.05) inWBC from control to post infection. Thereafter, WBC increased significantly (p<0.05) in treatment T3. White blood cells increase with the invasion of bacteria that alters the normal functioning of a food animal. White blood cells are on the front line to fight the foreign body invading the system. The white blood cell increased significantly from 9.5±0.05 in the control experiment to 11.0±0.5 post infection and 15.8±0.5 at a neem concentration of 7.5 mg/mL. The significant increase in lymphocyte level could result in resistance of the fish by enhancing the immunological actions of the white blood cells to the effect of bacteria. There were no significant (p>0.05) difference between control catfish and post-infection catfish in HGB, RBC, HCT, MPV and PDW. There were significant differences inMCV, MCH, MCHC and PCT. The PCV of the fish was not affected by the invasion of bacteria, in contrast to other studies that demonstrated that PCV is reduced in response to bacterial invasion (Sebastiao et al . 2011). This also sug- gests that bacterial infection does not cause destruction of heamato- ( C O N T I N U E D O N P A G E 4 8 ) treatment with antibiotics and neem leaf extracts at different concentrations against Staphylococcus aureus infected fish. MCHC g/dL RDW-CV % RDW -SD fL PLT 10 MPV fL PDW PCT % 35.55±0.0 c 14.65±0.05 bc 78.45±0.05 a 150.95±0.05 b 7.75±0.05 b 17.15±0.05 b 0.11±0.01 b 30.30±0.05 d 12.90±0.30 cd 63.20±0.05 b 69.00±0.50 c 7.80±0.10 b 17.40±0.05 b 0.02±0.01 a 49.20±0.05 a 44.20±0.05 a 0.00±0.05 c 299.0±0.05 a 8.40±0.05 b 18.20±0.05 a 0.25±0.00 a 31.20±0.25 d 13.20±0.55 c 64.00±0.00 b 70.00±0.15 c 8.10±0.05 ab 17.40±0.05 ab 0.66±0.00 c 28.30±0.05 d 9.60±0.05 d 62.50±0.05 b 11.00±0.05 f 10.60±0.05 a 17.40±0.05 b 0.01±0.00 d 34.60±0.20 c 17.90±0.05 b 0.00±0.05 c 60.00±0.50 d 7.80±0.10 b 17.50±0.35 b 0.05±0.00 c 41.40±0.05 b 12.40±0.05 cd 64.6±0.00 b 45.00±0.05 e 8.10±0.05 b 18.40±0.05 a 0.04±0.01 cd Variables: WBC = white blood cells (leukocytes), RBC = red blood cells (erythrocytes), HGB = hemoglobin concentration, MCV = mean corpuscular (erythrocyte) volume, MCH = mean cell (erythrocyte) hemoglobin, MCHC = mean cell (erythrocyte) hemoglobin concentration, RDW-CV = erythrocyte distribution width – coefficient of variation, RDW-SD = erythrocyte distribution width – standard deviation, HCT = hematocrit, PLT = platelet, MPV = mean platelet volume, PDW = platelet distribution width, PCT = plateletcrit.