Growth performance, gut morphometry and innate immune profiles of common carp, Cyprinus carpio juveniles fed diet fortified with Mitracarpus scaber leaves extract and its susceptibility to pathogenic bacteria, Aeromonas hydrophila

Mitracarpus scaber , an endemic medicinal plant to Nigeria, Africa, has medicinal value. In the present study, Mitracarpus scaber leaves extract (MSLE) was fed to common carp, Cyprinus carpio to evaluate its effect on growth performance, nutrient utilization, gut morphometry, and innate immunity parameters. Four isonitrogenous diets (32% crude protein) containing 0.0, 5, 10, or 15 g MSLE/kg diet were fed to fish (7.52 ±0.23 g) for 12 weeks. After the feeding trial, fish were exposed to pathogenic bacteria ( Aeromonas hydrophila ) for 14 days. Growth performance, nutrient utilization, and feed intake were significantly improved with increasing MSLE levels up to 10 g/kg diet. Similarly, fish fed MSLE diets increased significantly intestinal villi length/width, and absorption area. Furthermore, activities of respiratory burst, lysozyme, catalase, and superoxide dismutase were significantly higher in fish fed diets containing MSLE levels, and their highest values were obtained at fish fed 15 g MSLE/kg diet. After bacterial challenge, fish mortality was lowest (8.45 ±1.30%) in fish fed 15 g MSLE/kg diet, whereas highest mortality (52.50 ±4.56%) was observed with fish fed the control diet. The present study conjured that MSLE inclusion in fish diets with optimum level of 10 g/kg diet stimulated significantly the performance, nutrient utilization, modified gut morphometry, and innate im- mune response of common carp. Also, its inclusion protected fish against pathogenic bacteria, hydrophila


Introduction
Fish are considered as ones of the highly nutritional sources for animal proteins. It has highquality protein, vitamins and minerals, and unsaturated fatty acids. The knowledge of nutritional, and health benefit of fish has resulted into its consumption (Adeshina, Jenyo-Oni, Emikpe, Ajani, Abdel-Tawwab, 2018b); therefore, creating mammoth gap between the demand, and supply for fish. In recent times, aquaculture has been augmenting shortage between the demand, and supply of fish, following the shortage of supply from captured fisheries. However, diseases are impeding the success of aquaculture industry.
Common carp is one of the cultured fish species in tropical Africa, and widely accepted . To large extent, it is important to develop feed supplements as a replacement to the use of synthetics drugs to promote, and sustain responsible aquaculture. Therefore, the present study was carried out to evaluate the application of Mitrascarpus scaber leaf extract (MSLE) in practical diets for common carp as growth, nutrient utilization, and immune parameters. Fish resistance to pathogenic bacteria, A. hydrophila, was also investigated.

Plants collection and identification
The fresh leaves of M. scaber were collected from Ile-Apa community, and authenticated in the Herbarium Unit, Forest Research Institute of Nigeria, Ibadan, Nigeria.

Experimental procedure and design
Common carp, C. carpio juveniles were obtained from a reputable farm, and acclimated to lab conditions for 14 days during which fish fed a commercial diet (32% crude protein). Fish (7.52 ±0.23 g) were randomly allotted into 12 aquaria (50 L) at a density of 20 fish/aquarium. The fish were fed on one of the formulated diets up to nearly satiation two times every day at 08:00 am and 06:00 pm for 84 days. Throughout the experiment, water from tanks was replaced every 3-day intervals. Water samples were collected from the rearing tanks to monitor water quality. Water temperature ranged from 23.23 to 25.17°C, dissolved oxygen from 5.80 to 6.66 mg/l, conductivity from 0.36 to 0.90 ms.cm -1 , nitrite from 0.95 to 1.00 ppm, nitrate ranged from 33.35 to 53.20 ppm, and pH from 7.15 to 7.89 measured with mercury thermometer, Winkler method, commercial kits (Colombo nitrate, nitrite, and pH test kit) respectively.

Growth performance and nutrient utilization
Fish per each tank were collected, counted, and group-weighed biweekly and at the end of the feeding trial on a digital ScoutPro sensitive scale (Model: KD-200-110, USA). Parameters of growth performance and nutrients utilization were calculated as: Where: W 1 = final mean weight; W 0 = initial mean weight; L 1 = final mean length; L 0 = initial mean length.

Gut morphometry
Three fish from each tank were collected and anthesized with buffered tricaine methane sulfonate (30 mg/L). Fish intestine was taken for histological examination. The guts were prepared on slides as described by Drury, Wallington and Roy (1967). The guts were excised, and cut with a sharp scalpel blade into small pieces, and fixed by immersing in modified Bouin's fluid made up of picric acid (300 mL), formalin (100 mL), and 1% tricarboxylic acid (20 mL) according to the methods of Eyarefe, Emikpe and Arowolo (2008), followed by the dehydration of the tissues in 70, 80, 90, 95%, and absolute ethanol. The sections were prepared (4 mm thickness), followed by hydration in absolute ethanol, 95, 90, 80, and 70% ethanol. Villus length and width (mm), depth of the crypt (DC, mm), villus width (VW, mm), and area of absorption (AA, mm 2 ) were Measurements of taken using light microscope (HE x40) (Olympus CX21, Japan) in triplicates with a micrometer rule (Eyarefe et al., 2008;Fox et al., 1997;Bello et al., 2012).
Area of absorption mm Villus length mm Villus width mm = ×

Innate immunity assays
Three fish from each tank was collected, and anthesized with buffered tricaine methane sulfonate (30 mg/L). Blood collected from the caudal vein and used to determined innate immune parameters within the aid of Randox® Laboratories diagnostic kits (Crumlin, County Antrim, UK). Blood of fish from each experimental unit was pooled together. Superoxide dismutase (SOD) activity was determined by ferricytochrome-C method. Xanthine oxidase was used as the source of superoxide radicals. The quantity of enzyme required to produce 50% inhibition of ferricytochrome-C was taken as one activity unit (McCord, Trouslade, Ryu, 1984). The rate of H 2 O 2 at 240 nm was used to estimate the catalase (CAT) activity (Aebi, 1984). To determine the lysozyme activities in the fish sera, Micrococcus luteus (0.60 mg/mL) was spread in agarose gel (1%), and phosphate buffer (pH 6.2, 50 mM) (Difco BD Co, Franklin Lakes, NJ). Nutrient Agar prepared plates were dug to have wells of 6 mm diameter using cork-borer. The wells were filled with fish sera (25 μL/well), and incubated (25°C) for 20 hr. Then, the lysozyme activity was estimated from a standard curve prepared with lysozyme from chicken egg white (Abdel-Tawwab et al., 2018a).

Bacterial challenge test
The susceptibility of common carp, C. carpio was examined using pathogenic bacteria (A. hydrophila) after the 12-week feeding trial. The A. hydrophila (ATCC 4356) isolate was pobtained from Department of Microbiology Laboratory, University of Ibadan, Nigeria. The LD50 (lethal dose) of the pathogenic bacteria was determined. Bacterial isolates were grown on nutrient broth plus yeast extract, and incubated at 35°C for 24 h to revive the bacteria. Ten (10) fish from each experimental unit were exposed to A. hydrophila, which were grown on nutrient broth plus yeast extract for 24 hour at 35°C in an incubator. Bacterial cells were then centrifuged at 3000 g for 30 min to form pellets. The pellets were resuspended in 1.0 mL of 0.1% peptone water. For the challenge test, fish from each treatment were collected, and 20 fish (10 fish per tank) were transferred into two other tanks previously filled with dechlorinated freshwater as two replicates. Fish were challenged with a 0.1-mL dose of A. hydrophila (5 × 10 5 CFU/mL) by intraperitoneal injection, and were returned to the experimental setup (Adeshina et al., 2018b;Abdel-Tawwab et al., 2018a). Fish were fasted for 24 h before infection, and refeeding with the corresponding experimental diets 12 h later. All fish groups were kept under observation for 14 days to record any abnormal clinical signs and daily mortality.

Statistical Analysis
The data obtained were subjected to one-way analysis of variance (ANOVA) using IBM statistical package (SPSS version 20) to determine differences among the treatments in all parameters. Individual means were separated using Duncan multiple range test. Quadratic regression was used to determine the optimum level of extract for weight gain. All data were presented as means ± SD, and were declared significant at P < 0.05 according to Dytham (2011).

Growth performance
The growth and nutrients utilization of common carp, C. carpio, fed diets fortified with M. scaber leaves extract showed significant improvements when compared to the control group (P < 0.05; Table 2). Diets fortification with higher MSLE (10 g/kg diet) gave higher performance in terms of final weight, body weight gain, SGR, feed intake, and protein efficiency ratio, while least ones were recorded in fish fed the control diet. There were no significant difference was noticed in the fish survival (P > 0.05). Fish growth was significantly improved by dietary MSLE over that fed the control diet (P < 0.05).

Intestine morphometry
The inclusion of MSLE in diets for common carp, C. carpio increased significantly villi length/width and absorption area of the intestine (P < 0.05). The increase in the intestine morphometry was observed with the increase in MSLE levels in diets; however, fish fed the basal diet had lowest villi length/width and area of absorption. Similarly, there was a progressive increase in the cryptal depth of the fish fed diets fortified with MSLE (P < 0.05; Table 3). Different superscripts in the same column are statistically significant different between means at P < 0.05. Note: area of absorption (mm 2 ) = Villi length (mm) × Villi width (mm).

Innate immune parameters
The innate immune profile of fish fed diets supplemented with MSLE is shown in Table 4. The results indicated that supplementation of MSLE to common carp, C. carpio enhanced significantly the immunity parameters (P < 0.05). It was noticed that fish fed diet containing 10 g MSLE/kg diet had highest RBA (154.31 ±8.01), lysozyme activity (10.41 ±1.45), catalase (1.32 ±0.10 mg/g protein), and SOD (1.37 ±0.06 mg/g protein), and their least values were observe with fish fed the control diet (121.02 ±8.24, 9.53 ±1.20, 1.24 ±0.02 mg/g protein, and 0.43 ±0.01 mg/g protein, respectively). Furthermore, fish resistance against bacterial infection was improved. The post-challenge mortality of the fish infected with A. hydrophila is also shown in Table 4. There was a significant reduction in the post-challenge mortality (P < 0.05). Highest mortality (52.50 ±4.56%) was recorded in fish fed the control diet, while least one (8.45 ±1.30%) was observed in fish fed a diet containing 10 g MSLE/kg diet. Values are represented as mean ± standard deviation of triplicates; different superscripts in the same column are statistically significant different between means at P < 0.05.

Discussion
The inclusion of medicinal plants in fish diets has been studied, and positive results have been documented (Abdel-Tawwab, Ahmad, 2009;Abdel-Tawwab, Ahmad, Seden, Sakr, 2010;Abdel-Tawwab, 2018a;Abdel-Tawwab, 2012, 2015, 2016Guardiola et al., 2016;Abdel-Tawwab, Abbass, 2017;Adeshina et al., 2017;Hoseinifar et al., 2017aHoseinifar et al., , 2017bVan Doan et al., 2017;Adeshina et al., 2018b). Their phytobiotics activity has made them suitable substitutes for antibiotics, growth promoters, and immune boosters. The present study, fish fed diets fortified with MSLE showed improved growth, and utilized the given diets efficiently when compared to the groups fed the basal diet. The higher growth observed herein might be associated with the higher feed consumed by fish fed MSLE-supplemented diets. The presence of antimicrobial, and antimycotic properties such as gallic acid, 3,4,5-trimethoxybenzoic acid, 4-Methoxyacetophenone, 3,4,5-trimethoxyacetophenone, n-octane, 2-Hexanol, p-Cymene, α and β pinene, eugenol etc might assist in reducing pathogenic organisms in fish gut; hence, promoting the colonization of beneficial bacteria that enhanced diets digestibility. The presence of the secondary metabolites MSLE may be stimulated its immunostimulant properties. The present of eugenol, and p-cymene in the plants could be responsible for it acceptability by the fish (Adeshina et al., 2018b). The results of the present study are in agreement with previous studies, which fed fish on phytobiotics additives (Abdel-Tawwab et al., 2018a;Sogbesan, Ahmed, Ajijola, 2017;Offor et al., 2014;Adewole, Faturoti, 2017;Harikrishnan, Balasundaram, Heo, 2011). Other studies (Adeshina et al., 2018b;Abdel-Tawwab et al., 2018a;Sogbesan et al., 2017) reported that fish fed varying inclusion levels of plants extracts had higher growth performance that the growth fed the basal diet. The present study revealed that the increase in fish weight with the increase in the MSLE levels in diets more than the control diet. Thus, such increase in the body weight of the fish fed MSLE-based diets suggests better ingestion, digestion activity, and nutrient absorption.
One of the tools that researchers have adopted in assessing the nutrients absorption in fish is the examination of gut morphometry. The improvements observed in villi length/width, cryptal depth, and area of absorption of common carp, C. carpio juveniles fed MSLE-based diets revealed the improvements of nutrients utilization by fish. These increases in gut morphometry in the present study supported the increase in fish weights better than observation in the control group especially fish fed 10 g MSLE/kg diet. These results are in concomitant with previous studies (Zhou et al., 2010;Zahran, Risha, AbdelHamid, Mahgoub, Ibrahim, 2014;Zhang et al., 2010). Furthermore, it has been reported that inducement of intestinal morphology increases nutrient utilization ability of the fish leading to increasing fish growth, and feed utilization (Dimitroglou et al., 2010;Zhou, Buentello, Gatlin, 2010;Zahran et al., 2014). In similar studies, Abdel-Tawwab et al. (2018a) and Adeshina et al. (2018b) found significant increases in villi length/width and areas of absorption of African catfish, Clarias gariepinus fed diets fortified with plants extracts.

Conclusions
Growth performance and nutrient utilization of common carp fed MSLE-fortified diets were significantly improved over the control diet with optimum level of 10 g MSLE/kg diet. The present study proffers a new outlook for the use of M. scaber as phytobiotics to boost fish growth, and immunity. Furthermore, MSLE inclusion in the diet of common carp, C. carpio, could be used to enhance intestinal morphology, and resistance against bacterial infection with A. hydrophila.

Ethical statement
The protocol of the study was subjected to ethical consideration and was approved by Animal Care Use and Research Ethics Committee, University of Ibadan, Ibadan, Oyo state, Nigeria with reference number UI-ACUREC/App/03/2017/008.