TITLE: Prebiotic research protocol
AUTHORS: Mohammad Abdus Salam

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>The dietary chitosan promotes growth, biochemical composition, hematological parameter and morphology of internal organs by improving gut microbiota status of juvenile </span><span style = "font-style:italic;">Barbonymus gonionotus.</span></div></div>

[STEPS]
?. [Euthanasia methods]
Pure clove oil was first dissolved in ethyl alcohol in 1:9 ratio (clove oil: ethyl alcohol)
?. [Euthanasia methods]
This solution then diluted in water in order to obtain concentrations of 0.05 mL (50 mg), and 0.20 mL (200 mg) of clove oil per 500 mL of water
?. [Euthanasia methods]
For hematological study, experimental fish were anesthetized by using 0.05 mL clove oil per 500 mL of water
?. [Euthanasia methods]
For histological, reproductive and intestinal microflora study, fish were euthanized by using 0.20 mL of clove oil per 500 mL of water, and death was confirmed by the destruction of the brain
?. [Chemical analysis of carcass composition of B. gonionotus]
The experimental fish were humanely killed by using clove oil (0.20 mL per 500 mL of water), and death was confirmed by the destruction of the brain
?. [Chemical analysis of carcass composition of B. gonionotus]
The fresh fish were weighed by an electric balance
?. [Chemical analysis of carcass composition of B. gonionotus]
Then the fishes were dried overnight using an oven at 105 ⁰C (MC2846SL, LG Company, India) for 12 h and the dried fish weight was measured again
?. [Chemical analysis of carcass composition of B. gonionotus]
Then moisture content was determined by subtracting weight of dried fish from the weight of fresh fish
?. [Chemical analysis of carcass composition of B. gonionotus]
Then those dried fish samples were ground using an electrical blender
?. [Chemical analysis of carcass composition of B. gonionotus]
To determine crude protein, nitrogen content of fish carcass was measured according to modified Kjeldhal method following H2SO4 - salicylic acid digestion, distillation and titration
?. [Chemical analysis of carcass composition of B. gonionotus]
Fat content in carcass was determined by Soxhlet extraction with diethyl ether. Ash content was determined by incineration of the samples at 550 ⁰C for 12 h in a muffle furnace (Hayashi Denko Co. Ltd, Japan).
?. [Analysis of mineral contents in muscles of B. gonionotus]
Nine fishes from each treatment were anesthetized with the clove oil and gradually sacrificed.
?. [Analysis of mineral contents in muscles of B. gonionotus]
The fish muscles were collected by removing head, fins, scales and intestine. 
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							Then the muscles were dried using an oven at 105 ⁰C (MC2846SL, LG Company, India) for 12 h and the dried muscles were ground using a blender. 
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?. [Analysis of mineral contents in muscles of B. gonionotus]
The powdered samples were digested in boiling nitric acid and perchloric acid mixture (5:1) by following standard methods.
?. [Analysis of mineral contents in muscles of B. gonionotus]
After appropriate dilution, the mineral contents such as K, Na, Ca, Mg, Zn, Fe and Mn of fish muscles were estimated using Atomic Absorption Spectrometer (170-30, serial 6268-001, Hitachi, Japan).
?. [Analysis of mineral contents in muscles of B. gonionotus]
Calibrated standards for mineral estimation were prepared from commercially available standards (Buck Scientific 1-800-562-5566, BS-AQ-PB, Single element AA standard, USA).
?. [Analysis of mineral contents in muscles of B. gonionotus]
The estimations of K, Na, Ca, Mg, Zn, Fe and Mn were done using a hollow cathode lamp (Hitachi, Japan).
?. [Assessment of chitosan on the intestinal digestive enzyme activities of B. gonionotus]
Nine fish of each treatment were randomly sampled to assay the intestinal digestive enzyme activity of B. gonionotus
?. [Assessment of chitosan on the intestinal digestive enzyme activities of B. gonionotus]
Fish were sacrificed by the overdose of clove oiland the surface of each fish was sterilized using 70% ethanol
?. [Assessment of chitosan on the intestinal digestive enzyme activities of B. gonionotus]
The peritoneal cavity was opened aseptically with a sterile scalpel
?. [Assessment of chitosan on the intestinal digestive enzyme activities of B. gonionotus]
The intestine between the pyloric caeca and approximately 1cm anterior to the anus of the fish was excised and the feces with mucus were stripped off with sterile forceps
?. [Assessment of chitosan on the intestinal digestive enzyme activities of B. gonionotus]
The intestine of 9 fish from each treatment were removed and frozen in liquid nitrogen then stored at -80 ⁰C until subsequent analysis
?. [Assessment of chitosan on the intestinal digestive enzyme activities of B. gonionotus]
The intestinal samples were homogenized in 10 vol (v/w) of ice cold PBS and centrifuged at 3000 g for 20 min at 4 oC. The supernatant was conserved and used to determine the amylase activity, protease activity and lipase activity using ELISA kits (Thermo Fisher Scientific, USA)
?. [Histological analyses of intestine, liver and kidney of the dietary chitosan treated silver barb]
The experimental fish were humanely killed by using clove oil (0.20 mL per 500 mL of water)
?. [Histological analyses of intestine, liver and kidney of the dietary chitosan treated silver barb]
Death was confirmed by the destruction of the brain
?. [Histological analyses of intestine, liver and kidney of the dietary chitosan treated silver barb]
The whole liver, kidney and part of the intestine from each fish were dissected carefully, cut to separate each other, and stored in bouins solution for 24 h
?. [Histological analyses of intestine, liver and kidney of the dietary chitosan treated silver barb]
These samples were dehydrated in ascending grades of alcohol and cleared in xylene
?. [Histological analyses of intestine, liver and kidney of the dietary chitosan treated silver barb]
The fixed tissues were embedded in histoparaffin (Paraplast plus; Sigma-Aldrich) and sections (7 µm) were cut using a microtome (CUT-5602, Germany)
?. [Histological analyses of intestine, liver and kidney of the dietary chitosan treated silver barb]
Then the sections of intestinal villi, kidney and liver were selected and stained with Delafield’s hematoxylin-eosin for observation under a light microscope (DM 100; Leica, Wetzlar, Germany).
?. [Histological analyses of intestine, liver and kidney of the dietary chitosan treated silver barb]
Ten slides were prepared from the intestine of each fish through histological method.
?. [Histological analyses of intestine, liver and kidney of the dietary chitosan treated silver barb]
Then the slides were observed under a trinocular microscope.
?. [Histological analyses of intestine, liver and kidney of the dietary chitosan treated silver barb]
Images were captured using a digital camera (DFC 290, Leica) and the villi length of the intestine was measured using AmScope software (Version 3.7; Carl Zeiss Primo Star, Germany)
?. [Measurement of Hematological Parameters]
A total of 90 fish from each treatment were anesthetized with the clove oil (0.05 mL per 500 mL of water) for hematological analysis
?. [Measurement of Hematological Parameters]
Blood was collected from fish using a 3 cc syringe containing 10% blood anti-coagulant (EDTA) inserted into the caudal peduncle region to drag out blood.
?. [Measurement of Hematological Parameters]
The blood was transferred to a test tube coated with EDTA, and stored at -30 oC until use.
?. [Measurement of Hematological Parameters]
Red blood cells (RBCs) and white blood cells (WBCs) were counted using an improved Neubauer hemocytometer (MarienFeld Company Germany) under the light microscope (DM 100; Leica, Wetzlar, Germany)
?. [Measurement of Hematological Parameters]
To measure hemoglobin, fresh blood was collected from fish from each treatment and was poured in the edge of a strip of hemoglobin meter before the coagulation of blood.
?. [Measurement of Hematological Parameters]
The glucose level of blood was measured through a glucose meter from the sample.
?. [Measurement of Hematological Parameters]
To measure packed cell volume (PCV) (%), blood was taken in a capillary tube at the marked level and sealed with gum.