Plant extraction and antibacterial screening
Phyllanthus amarus foliage were obtained, rinsed to remove dirt, air-dried for 7 days after which the stems and stalks were removed, while the leaves and flowers were grinded into fine powder form using kitchen blender. Ethanol, methanol or distilled water was used as solvents for extraction by mixing the blend plant and solvent at the ratio of 1:10 (weight/volume) and thereafter concentrated using rotary evaporator as described by Adeniyi et al. (2023)
The antibacterial activity of the plant extracts on Aeromonas hydrophila, Pseudomonas florescens, and P. aeroginosa isolates was determined in accordance with the agar-well diffusion method described by (CLSI, 2012). The isolates were grown on nutrient agar for 24 hours before use and standardized to 0.5 Mcfarland standards. About one hundred micro-liters of the standardize cell suspension was spread on Mueller-Hinton agar in plates, after which approximately 100 µL of each concentrated crude extract at 10 mg/ml was introduced into wells bored with sterile 6 mm diameter core borer and incubated at 37⁰C in triplicates for 24 hours. Controls were set up in parallel using the solvent that was used to reconstitute the extract as negative control, and Aquamedics (containing oxytetracycline and erythromycin) as positive control. The plates were observed for zones of inhibition (mm) after the incubation at 37⁰C.
Experimental diet
Five isonitrogenous (40% crude protein) and isocaloric (≈ 405 kcal/100g gross energy) diets were formulated using fish meal, ground nut cake, soya bean meal, maize flour, fish oil, and vitamin/mineral premix (Table 1). Phyllanthus amarus methanol extract (PAE), with the highest antibacterial activity, was added at 0.0 (control) 0.5, 1.0, 1.5 and 2.0 g/kg basal diet. The feed ingredients were thoroughly mixed, pelletized and dried using pelletizer machine (CAP, China).
Experimental set-up and feeding
Fingerlings of Clarias gariepinus were obtained and acclimated for two weeks, during which they were fed with a commercial feed (Skretting, 1.8mm). Thereafter, a total number of 450 fingerlings (4.37 ± 0.21g) were randomly stocked at 25 fish per tank in triplicate using indoor static water-renewal system during which culture water was renewed at 48-hour interval. The fish were starved overnight before the commencement of the feeding trial and thereafter fed to apparent satiation twice daily for 84 days. The fish in each tank was monitored for mortality, dead fish, if any, was removed and number recorded. The water quality was determined and monitored during the study and dissolved oxygen, temperature and pH were found to fall within 4.5-5.5 mg/L of water, 25.6- 27 7⁰C and 6.9-7.1, respectively.
Table 1 Gross composition of experimental diets containing Phyllanthus amarus extract (PAE)
Ingredients
|
Composition (g/kg diet)
|
|
0.0
|
0.5
|
1.0
|
1.5
|
2.0
|
Fish meal
|
200.0
|
200.0
|
200.0
|
200.0
|
200.0
|
Soya bean meal
|
260.0
|
260.0
|
260.0
|
260.0
|
260.0
|
Groundnut cake
|
300.0
|
300.0
|
300.0
|
300.0
|
300.0
|
Corn flour
|
206.0
|
205.5
|
205.0
|
204.5
|
204.0
|
Fish oil
|
20.0
|
20.0
|
20.0
|
20.0
|
20.0
|
PAE
|
0.0
|
0.5
|
1.0
|
1.5
|
2.0
|
Di calcium phosphate
|
2.5
|
2.5
|
2.5
|
2.5
|
2.5
|
aFish premix
|
2.5
|
2.5
|
2.5
|
2.5
|
2.5
|
Acidifier
|
1.0
|
1.0
|
1.0
|
1.0
|
1.0
|
Toxin binder
|
1.0
|
1.0
|
1.0
|
1.0
|
1.0
|
Methionine
|
1.0
|
1.0
|
1.0
|
1.0
|
1.0
|
Lysine
|
1.0
|
1.0
|
1.0
|
1.0
|
1.0
|
Common salt
|
5.0
|
5.0
|
5.0
|
5.0
|
5.0
|
Total
|
1000.0
|
1000.0
|
1000.0
|
1000.0
|
1000.0
|
Proximate Composition (g/kg)
|
Moisture
|
86.5
|
86.5
|
86.7
|
87.3
|
87.1
|
Ash
|
100.2
|
100.2
|
100.2
|
100.2
|
100.2
|
Ether extract
|
94.7
|
94.7
|
94.8
|
94.8
|
94.8
|
Crude fibre
|
45.0
|
44.8
|
44.8
|
45.3
|
45.0
|
Crude protein
|
400.2
|
400.2
|
400.2
|
400.2
|
400.2
|
bNitrogen free extract
|
359.8
|
360.2
|
360.0
|
359.6
|
359.8
|
cGross energy (kj/kg)
|
19309.9
|
19307.2
|
19308.5
|
19301.1
|
19305.7
|
aFish premix (Miconsult International Ltd; per kg premix): Vitamin A, 22,000IU; vitamin B1, 20mg; vitamin B2, 25mg; vitamin B6, 10mg; vitamin B12, 0.05mg; vitamin C, 300mg; vitamin D3, 5,000IU; vitamin E, 300mg; vitamin K3, 10mg; niacin, 120mg; folic acid, 5mg; biotin, 1mg; inositol, 50mg; calcium pantothenate, 60mg; choline chloride, 500mg; manganese, 30mg; iron, 35mg; zinc, 45mg; copper, 3mg; cobalt, 2mg; iodine, 5mg; selenium, 0.15mg
bNitrogen free extract = 1000 – (Crude protein + crude fibre + ether extract + ash)
cGross energy was calculated as 23.6, 39.5, and 17 kj/g for protein, lipid, and carbohydrates, respectively (NRC, 1993).
Growth performance and nutrient utilization study
At the end of twelve weeks of feeding trial the following parameters on fish growth performance and feed utilization were calculated from the mean data obtained for each treatment during the experiment:
Weight gain (WG, g) = Final weight (FW) – Initial weight (IW)
Weight gain (%) = 100 (WG / IW)
Specific growth rate (%/day) = 100 (Ln FW- Ln IW) / Duration of experiment (days)
Feed intake (g)= Sum of feed fed during the experiment
Feed conversion ratio = Feed intake / WG
Protein efficiency ratio = WG / Protein intake
Fish Survival (%) = 100 x (Number of survived fish / Initial number of fish stocked)
Condition Factor (CF) = 100 x (FW / Final standard lenght3).
Collection of blood and analyses
Blood were collected from sampled anesthetized fish in each treatment after the feeding trial through the caudal vein using a 2mL disposable plastic syringe and 21-gauge disposable hypodermic needle. Two sets of blood samples were dispensed into both ethylenediaminetetraacetic acid (EDTA and non-EDTA bottles for haematological and serum biochemical analyses. The haematological parameters haematocrit (also known as packed cell volume, PCV), haemoglobin concentration, Red blood cell (RBC), White blood cell (WBC), lymphocyte, neutrophil, eosinophil, monocyte and platelet counts) were analyzed with an automated hematological analyzer. Thereafter, the following erythrocytic indices were calculated from the data obtained:
1. Mean Corpuscular Volume (MCV, fl) = 10 (PCV / RBC)
2. Mean Corpuscular Hemoglobin (MCH, pg) = 10 (Heamoglobin / RBC)
3. Mean Corpuscular Hemoglobin Concentration (MCHC, %) = 100 (Heamoglobin / PCV)
The blood samples in non-heparinized were allowed to coagulate, centrifuge at 3000rpm ambient temperature of 28oC to obtain the sera samples. The samples were stored in freezer and later analyzed total protein, albumin, total blood cholesterol, blood bilirubin, creatinine, urea nitrogen, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) were analyzed using Randox kit, following the standard procedure of the manufacturer. Globulin (Total protein - albumin), albumin globulin ratio (AGR, albumin / globulin) were calculated. Concentrations of Malondialdehyde (MDA) (Varshney and Kale, 1990) and reduced glutathione (GSH) (Jollow et al., 1974) as well as activities of glutathione‐S‐transferase (GST), glutathione peroxidase (GPx), and superoxide dismutase (SOD) (Rotruck et al., 1973), were measured spectrophotometrically.
Fish Challenge protocols and non-specific immune responses
Fish (n = 8/replicate) were randomly selected, aseptically challenged through intraperitoneal injection with 0.2 mL (1 x 108 / mL) A. hydroplila (ATCC 23211) by (Wang et al., 2014) and observed for 14 days for survival. Thereafter, blood samples were obtained from the fish and examined for phagocytic (Yoshida and Kitao, 1991), respiratory burst (Secomebs, 1990) and lysozyme (Ellis, 1990) activities spectrophotometrically (UV/VIS, Mettler Toledo, USA) as described by (Adeniyi et al., 2023). Briefly, the number of phagocytic cells was counted and expressed as the percentage of cell with phagocytic activity; respiratory burst activity was expressed as the NBT reduction of the sample, while one unit of lysozyme activity was defined as the amount of the enzyme producing a decrease in absorbance of 0.001/minute.