Gut and intestinal biometrics of the giant trevally, Caranx ignobilis, fed an experimental diet with difference sources of activated charcoal

Background : The giant trevally, Caranx ignobilis, is a commercially important marine fish in Indonesia. This species was initially cultured in Aceh Province. Previous reports showed that charcoal has a positive effect on survival and feed utilization of the giant trevally. However, the effects of adding charcoal to the diet on gut and intestine biometrics has, to our knowledge, never been described. Methods : Four activated charcoal sources were tested in this study using a completely randomized experimental design; coconut shell charcoal, mangrove wood charcoal, rice husk charcoal, and kernel palm shell charcoal. All treatments were performed with four replications. Juvenile giant trevally (average body weight, 16.52 ± 3.12 g; and average total length, 10.26 ± 0.64 cm) were stocked into the experimental tank at a density of 15 fish per tank. The fish were fed an experimental diet twice daily at 7 AM and 5 PM ad satiation for 42 days. Results : Analysis of variance showed that adding charcoal to the diet had significant effects on the length and width of the foveola gastrica and villous intestine (P < 0.05). The greatest length and width of the foveola gastrica was recorded in fish fed an experimental diet of rice husk charcoal with average values of 311.811 ± 9.869 µm and 241.786 ± 10.394 µm, respectively. The greatest length of intestinal villous was found in fish fed the mangrove wood charcoal diet, with a value of 135.012 ± 5.147 µm, but this length was not significantly different to that in fish fed rice charcoal and kernel palm shell charcoal. However, the greatest width of intestinal villous was recorded in fish fed the control diet (without charcoal; P < 0.05). Conclusion: The optimal sizes of the foveola gastrica and villous intestine were found in fish fed an experimental diet with rice husk charcoal.


Introduction
Trevally fish are a commercially important group of marine fish in the family Carangidae. A total of 146 species of trevally have been recorded worldwide 1 . These fish are distributed in tropical, subtropical, and temperate waters 2-7 . In Indonesia, trevally fish are found in the Aceh waters 8,9 , East Borneo 10 , Papua and Wester Nusa Tenggara 11,12 , and Java 13 .
Giant trevally, Caranx ignobilis, is among the most popular trevally fish in Indonesia. The population of this species has declined over the years due to overfishing 7,14-16 . Culture of this fish has been initiated in Aceh Province, Indonesia. However, farmers are faced with a feeding obstacle. Giant trevally in culture systems are currently fed waste fish and a commercial diet (Hi-Pro-Vite, Central Proteina Prima Company). The commercial diet is costly and difficult to obtain in remote areas, and the waste fish supply is very seasonal. Trash fish are limited in nutrients, particularly the essential amino acid composition 17 . Therefore, it is crucial to formulate a diet for giant trevally using local raw materials with higher protein, that is inexpensive, easy to find, and digestible.
Activated charcoal is commonly added to the diet to increase digestibility and trigger growth in fish. For example, Jahan et al. 18 successfully used activated charcoal to increase the digestibility and growth performance of river catfish, Pangasiaodon sp. Other researchers have used charcoal in the diets of fish species, such as Nile tilapia, Oreochromis niloticus 19-21 , tiger pufferfish, Takifugu rubripes 22 , Japanese flounder, Paralichthys olivaceus 23 , African catfish, Clarias gariepinus 24,25 , gilthead seabream, Sparus aurata 26 , and sturgeon, Huso huso 27 . Firdus et al. 28 added rice husk charcoal to the diet of giant trevally. However, the effect of charcoal on the morphology of the gut and intestine has not been reported.
Organogenesis of the digestive system occurs as fish age, and this process is strongly dependent on the quantity and quality of food [29][30][31][32] , which is related to the development of mucosal cells, amplification of apical plasma membranes, and formation of the foveola gastrica and intestinal villi 33,34 . It has been hypothesized that adding activated charcoal to the diet triggers the digestive organogenesis system process 35,36 . In this study, we tested four charcoal sources in the diet to evaluate the morphology of the gut and intestine of giant trevally. Information on the gut and intestinal morphology is important to understand the absorption mechanism of nutrients from the diet.

Time and site
The study was conducted at the Center for Brackish Water Aquaculture, Ujung Batee, Aceh, Indonesia from February to July 2018. The activated charcoal was characterized at the Integrated Laboratory of Calibration, Universitas Gajah Mada, Yogyakarta, Indonesia. Histological samples were prepared at the Laboratory of Histology, Faculty of Mathematics and Natural Sciences, Universitas Syiah Kuala, Banda Aceh, Indonesia.

Experimental design
A completely randomized experimental design with five treatments consisting of control and four different charcoal sources was used in this study. The experimental groups were: (A) the experimental diet without charcoal, (B) the experimental diet with 2% charcoal from coconut shell, (C) the experimental diet with 2% charcoal from mangrove wood, (D) the experimental diet with 2% charcoal from rice husk, and (E) the experimental diet with 2% charcoal from kernel palm shell. All treatments were performed with four replications.

Experimental fish
A total of 300 giant trevally juveniles of mixed sex (average body weight, 16.52 ± 3.12 g; total length, 10.28 ± 0.64 cm) were purchased from a local farmer in Lancang Barat Village, Aceh Utara District, Aceh, Indonesia. The fish were acclimatized in ponds (ponds size 2 m x 1.8 m and temperature of around 29°C) at the Center for Brackish Water Aquaculture, Ujung Batee for 2 weeks. The fish were fed an experimental diet containing 50% crude protein twice daily at 7 AM and 5 PM at 3% of body weight per day (Table 1).

Charcoal preparation and activation
The raw coconut shells, mangrove wood, rice husks, and kernel palm shells were chopped and ground. Approximately 500 g of the ground materials were placed on aluminum foil and heated in a furnace at 400°C for 1 hour. Nitrogen gas was flowed into the furnace to remove the oxygen. Then, the temperature was decreased to 30°C gradually and held for 1 hour. After 1 hour, the charcoal was removed from the furnace, sieved through a No. 40 mesh, and held in a jar before

Amendments from Version 1
In response to comments from the reviewers, the following is our amendments:

Abstract
• We changed: 'This species was…' to be 'The species was…' • Four activated charcoal sources were tested in this study not include control, in the Methods has been explained with five different charcoal sources including control (no active charcoal source) • All mistake numbers in the Abstract have been adjusted based on reviewer comments

Methods and Experimental
• All the mistake numbers in the Methods have been adjusted based on the reviewer comments including type mistake in the Tables and Figures

Discussion
• Some sentences have been modified to make easy to understand such as " Hence, a longer foveola gastrica and larger intestinal villi were able to provide more nutrients to be absorbed due to a larger surface area of digestive organs" and also the contain amount of rice husk charcoal has been revised. Some words have been added to make easier to understand.
Any further responses from the reviewers can be found at the end of the article activating. A total of 100 g of sieved charcoal was taken and mixed with 400 ml of 0.2 M citric acid. The solution was stirred for 24 hours. After 24 hours, the solution was filtered through filter paper. The filtered charcoal was washed with distilled water and dried in an oven at 110°C for 24 hours.

Diet preparation
The experimental diet was formulated from both plant and animal-based protein sources, such as Ebi-shrimp meal, fish meal, blood meal, soybean meal, rice flour, and corn flour. All raw materials were subjected to a proximate analysis before use in the formulation. Three types of amino acids i.e. isoleucine, L-tryptophan, and DL-methionine were also added ( Table 1). A total of 2% of the tested charcoal sources was added to the formulation ( Table 1). The formulated diets were subjected to a proximate analysis before use in the experiment.

Stocking and feeding
The fish was captured randomly, measured for body weight and total length, and then distributed into 20 plastic containers (48 × 43 × 70 cm) at a stocking density of 15 fish per container. The water volume in the container was 75 L. The fish were fed an experimental diet twice daily at 7 AM and 5 PM to satiation for 42 days.

Histological sample preparation
Gastric and intestinal samples were collected at the end of the study. Three fish from each treatment were taken randomly from the experimental tanks. The fish were anesthetized with 30 mg L −1 clove oil 37 , and the abdomen of the fish was gently dissected following the procedure of Purushothaman et al. 38 . The stomach and intestines were removed with scalpel scissors and preserved in 4% formalin for 1 week. Histological sampling was carried using the paraffin method based on Osman and Caceci 39 . The samples were dehydrated through an alcohol series and cleared in xylol. Subsequently, the gut and intestine samples were embedded in paraffin. The paraffin block was sectioned to 6 µm, and the sections were stained with hematoxylin and eosin. The size (height and width) of villi was determined using a binocular microscope (Zeiss Primo Star, Carl Zeiss Suzhou Co., Ltd., Suzhou, China) which was connected to a CCD camera and computer monitor 19 . All efforts were made to lessen harm to the animals by complying to the guidelines of ethics animal use in research of Syiah Kuala University.

Data analysis
The qualitative gut and intestinal morphology data were subjected to one-way analysis of variance followed by Duncan's multiple range test. The analysis was performed using SPSS ver. 18.0 software. The qualitative (histological) gut and intestinal data were analyzed descriptively. A P-value < 0.05 was considered significant.

Results
Adding activated charcoal to the diet significantly affected the length and width of the foveola gastrica and intestinal villi (P < 0.05). In general, fish fed the activated charcoal diets produced better results than those not fed the charcoal ( Figure 1 and Figure 2). The best foveola gastrica morphology was obtained with the rice husk charcoal and the mean length and width of the foveola gastrica were 311.811 µm and 241.786 µm, respectively; followed by coconut shell charcoal (257.040 µm and 183.816 µm), kernel palm charcoal (229.969 µm and 169.131 µm µm), and mangrove wood charcoal (229.595 µm and 166.509 µm).
The greatest length of the villous intestine was recorded in fish fed a diet with activated charcoal than those not fed the activated charcoal ( Figure 3). The greatest growth of intestinal villi was determined in the mangrove active charcoal (mean, 135.012 µm) group, but this value was not significantly different from the rice husk or kernel palm shell charcoals ( Figure 4). However, the greatest intestinal villi width was obtained in the treatment without activated charcoal (38.341 µm), and this value was significantly different from the other treatments.

Discussion
The results show that adding activated charcoal to the diet of C. ignobilis significantly affected favoela gastrica and intestinal villi biometrics. According to Pirarat et al. 19 , activated charcoal plays a significant role stimulating the development of epithelial cells of the digestive organs. Activated charcoal in the diet functions as a decontaminating agent to eliminate pathogenic organisms and toxic compounds, such as mycotoxins 20 . Hence, a longer foveola gastrica and larger intestinal villi were able to provide more nutrients to be absorbed due to a larger surface area of digestive organs 43 .  49 reported that the concentration and quality of charcoal depend on the composition of hemicellulose, cellulose, and lignin. The quality of the activated charcoal is higher    when these three components increase. According to Jasman 50 , rice husk contains 85-95% activated charcoal, while mangrove wood has 76% activated charcoal 51 , kernel palm shell 65% activated charcoal 47 , and coconut shell has 60% activated charcoal 46 .
The microscopic observations showed that the intestinal villi of the fish fed the diet with activated rice husk charcoal had a more pointed shape compared to other treatments, in which the villi tended to be round and blunt. According to Guo et al. 52 , blunt or rounded villi probably occur due to inflammation in the intestinal mucosa, which is characterized by infiltration of neutrophils into the lamina propria. An increase of intestinal villus size is related to nutrient absorption capacity. According to Nafis et al. 53 , long mucosal folds increase nutrient absorption and reduce food flow movement due to reduced peristaltic contractions, which provides sufficient time to optimally absorb nutrients. The increase in intestinal villi size is strongly related to the activities of digestive enzymes, such as lactase, sucrase, alkaline phosphatase, and disaccharidase 54-57 .
The morphology of the intestinal villi of fish fed a diet without activated charcoal was wider and shorter than that of fish fed the diets with activated charcoal. This was probably due to impaired intestinal mucosal integrity, causing interference in nutrient absorption. According to Choct 58 , shortening of the intestinal villi is related to the accumulation of intestinal pathogenic bacteria, resulting in increased susceptibility to infection in the intestinal mucosal layer. This causes the digestive organs to form more secretory cells than absorbent cells, which reduces nutrient uptake 59,60 . The active charcoal likely acts as an adsorbent of metabolic pathogens in the intestine in the form of endotoxins and ammonia, therefore, it was able to improve intestinal function 61 .

Conclusions
The application of activated charcoal in the diet significantly affected the length and width of the foveola gastrica and intestinal villi of giant trevally, C. ignobilis. The optimal biometrics of the foveola gastrica and intestinal villi were observed in fish fed the experimental diet with activated rice husk charcoal.

Bangladesh
It seems the manuscript has potentials in the scientific world, and work was done and formatted with care. The abstract is concise but well written.

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The introduction has supported with up to date literature and aim and objectives are well described.

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Results supported with details and tables and figures were provided.

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The discussion was also well described and literature supported.

If applicable, is the statistical analysis and its interpretation appropriate? Yes
Are all the source data underlying the results available to ensure full reproducibility? Yes

Are the conclusions drawn adequately supported by the results? Yes
Principal Scientist & Head (Acting) Riverine Ecology and Fisheries Division, ICAR-Central Inland Fisheries Research Institute, Barrackpore, India The paper has communicated a short but interesting study on the beneficial effects of adding different types of charcoal as a dietary component of giant trevally. The addition of charcoal has significantly improved the gastro-intestinal microstructures including foveola gastrica & villous intestine and thereby, has the potentiality to improve the fish nutrition and health. The rice husk was recorded as the best source of charcoal with respect to 3 other sources including coconut shell, mangrove wood, and kernel palm shell. However, some deficiencies have been noticed which needs rectification. In Abstract : This species was---: Better to use The species was---1.
Four activated charcoal sources were tested in this study -----but in Experimental Design mentioned ----A completely randomized experimental design with five different charcoal sources----2.
ponds size 2 x 1.8 m. No unit given after 2. It may be 2 m X 1.8 m 4.
(see Table 1) : see may be deleted 5. In Abstract : and 241.786 μm ---In Figure 1 241.768 7. Sentence to be modified : Hence, a longer foveola gastrica and larger intestinal villi provide a larger surface area to absorb nutrients43.

9.
Sentence to be modified : A previous report indicated that rice husk charcoal contains 39% hemicellulose, 44% cellulose, and 30% lignin44 : Since the addition of 3 parameters are giving values >100%, you may mention "up to" otherwise people will not understand.

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Sentence to be modified/changed : The active charcoal likely acts as an adsorbent of metabolic pathogens in the intestine in the form of endotoxins and ammonia to improve intestinal function61. The sentence to be rewritten to increase clarity.

11.
It has been noticed that the SE values have gone out of the bar area. Please check about it and reason behind. For the paper, the fish growth parameters may be given as supplements for better clarity.

Is the study design appropriate and is the work technically sound?
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