Extract of Aloe vera (Aloe barbadensis Miller) Enhances the Growth, Protein Contents, and Gastrosomatic Index (GaSI) of Common Carp Cyprinus carpio

Aquaculture is a growing agribusiness, and large-scale microbial infection frequently leads to considerable economic losses, as there are very few approved drugs available to counteract such a problem. Aloe vera is a natural therapeutic plant with biological activities, for example, antimicrobial, anticancer, mitigating, and immunomodulatory properties. Thus, an experiment was conducted to understand the effects of dietary Aloe vera on survival, growth performance, protein utilization, gastrosomatic index, and gut histomorphological studies in common carp in laboratory conditions. Aloe vera leaves were collected, washed, shade-dried, and homogenized to get a filtrate, which was extracted in ethanol (70%) using the Soxhlet apparatus. The carp with an average weight of 1.73 ± 0.14 g was divided randomly into 5 treatments (control and other 4 treated groups) with the extract of 0.0%, 0.2%, 0.4%, 0.8%, and 1.6% in the diet with 3 replicates and the stocking density was 25 fish per each replicate. Sampling was done on the 30th, 60th, and 90th days, and the results show that percent weight gain, specific growth rate, protein efficiency ratio, and feed conversion efficiency were significantly high in the treated diet compared to the control (0.0%). Gastrosomatic index, condition factors, and gut histology found better performance in common carp group fed 0.4% and 0.8% of Aloe vera after 90 days. In conclusion, it was found that Aloe vera extract between the amount of 0.4% and 0.8% is better for fish growth for common carp.


Introduction
Immunostimulants are of unfamiliar interest in medical care and have gotten quite possibly the most dynamic zones of applied clinical exploration [1]. Immunostimulants are dietary added substances that upgrade guard instruments and increment protection from explicit microbes.
Moreover, practices in the use of immunostimulants as a choice in contrast to drugs, substances, and antibiotics are environment friendly and accessible in different places of the world. It improves the intrinsic (or vague) immune response, which has a more conspicuous part in fish immunity [2,3]. In this way, the utilization of immunostimulants for the counteraction of sicknesses in fish is viewed as another option and promising zone [2].
ere is an extending interest in the utilization of medicinal herbs as immune stimulants in aquaculture [4], and the immunostimulant impacts of natural drugs in different fish species have been revealed [5]. In fisheries, immunostimulants enact the immune system of aquatic creatures and upgrade their ability for disease resistance [2]. Aquaculture is the highly dynamic and fastest-growing food-producing sector. e contribution of aquaculture to fish production is steadily increasing. e increasing demand for fish products and the commercialization of aquaculture led to the intensification of aquaculture. ese conditions tend to adversely affect the health of the animals and produce a poor physiological environment and subsequently increase the susceptibility to infectious disease [6]. Diseases have become a significant constraint by causing mass mortality and severe economic loss. To overcome the disease problem, generally, antibiotics, chemotherapeutics, and disinfectants are used in aquatic systems, which in turn lead to drug resistance, human carry over, bioaccumulation, and pollution to the aquatic environment. Vaccination is also a useful prophylactic treatment, but due to its limited availability and pathogen-specific protective action, much attention has been diverted towards the application of immunostimulants [7].
Aloe vera, a spiky prickly plant like xerophyte, is a cluster framing lasting plant with a thick stringy root that produces enormous basal leaves, generally 12-16 for every plant, weighing up to 1.5 kg when fully grown. e plant develops when it is around 4 years of age and has a life expectancy of around 12 years. e leaves are long and opposite the base, tightening to a point with saw-like teeth along their edges. In a cross over the area, the plant shows a marginally curved appearance on the adaxial surface and an unmistakably raised appearance on the lower abaxial surface [8]. e leaves are covered with thick fingernail skin, underneath which epidermis and mesophylls are available, which is then separated into the upper chlorenchyma and lower parenchyma; as the rosettes develop, progressive leaves have less whitish spots and grey-greenish in color [9]. e medicinal plant Aloe vera has been known and used for centuries for its health, beauty, medicinal, and skincare properties [10,11]. Aloe vera consists primarily of water and polysaccharides (pectin, cellulose, hemicellulose, glucomannan, acemannan, and mannose derivatives) and is composed of a long chain of acetylated mannose [12]. e biological activity of Aloe vera's polysaccharides has been reported widely [13] and shown to act as an immunostimulant [14]. Acemannan has been reported to have antimicrobial properties, including antibacterial, antifungal, antiviral, and antiparasitic properties [15]. Aloe vera is recognized for its widespread use and reported healing powers [16], alleviating pain and treating a variety of ailments [17].
For millennia, residents have utilized the gel from Aloe vera leaves for mending and mellowing the skin. Aloe vera gel taken orally (by mouth) appears to help lower glucose levels in individuals with diabetes. It might likewise assist with bringing down cholesterol [18]. Common carp (Cyprinus carpio) is of the order Cypriniformes and the family Cyprinidae. It by and large occupies freshwater conditions, particularly lakes and streams, and seldom possesses saline water conditions [19]. e native carp is considered as vulnerable to extinction by the International Union for Conservation of Nature [20]. However, the species has also been domesticated and introduced in freshwater lakes and large rivers of Nepal, Asia, and worldwide. Recently, various mass mortalities have been reported in the Nepalese aquaculture system [21], and the use of immunostimulants is a suggestible alternative to antibiotics in this species.
ere is restricted data accessible on the immunostimulatory, antitoxicity, and development impacts of Aloe in some fish species [22][23][24][25]. Zanuzzo et al. in the year 2017 clarified that dietary Aloe vera for 10 days before transport stress and infection with heat-killed Aeromonas hydrophila either improved or prevented the deficiency of innate immune activity in pacu (Piaractus mesopotamicus) after unpleasant taking care of and bacterial infection [26].
In another study, the Aloe vera methanolic extracts in the daily diet significantly improved the growth of red hybrid tilapia (Oreochromis sp.) [27]. Mesbah and Mohammadian in the year 2016 have explained that the oral administration of Aloe vera enhances non-specific immune responses in the shirbot (Barbus grypus) [28].
ere was information in the literature on the role of dietary Aloe vera, in the counteraction of irresistible illnesses and histological changes to the skin and gastrointestinal tract in rainbow trout [29]. As far as we know, there is no data in the literature on the role of dietary Aloe vera on growth index, protein utilization, gastrosomatic index, etc., in common carp. e present study aimed to investigate the effect of the Aloe vera extracts on growth index, protein utilization, gastrosomatic index, and gut histology in juvenile common carp Cyprinus carpio during intensive aquaculture.

Selection of Site.
Affiliated to Tribhuvan University, Institute of Science and Technology, Amrit Campus, situated in the heart of Kathmandu Valley ( amel), is one of the pure science campuses of the country. e aquaculture research lab (27.7172°N, 85.3240°E) of this campus is considered a Center of Excellence for Fisheries and Aquaculture Research; hence, this feeding trial was conducted to understand the feeding and growth pattern of carp concerning Aloe vera extracted diets.

Selection of Fish.
Common carp (Cyprinus carpio) was selected for this experiment, and around one thousand juvenile carps with an average body weight of 1.73 ± 0.14 g were obtained from the hatchery of Janakpur (a fish super-zone), Nepal, to the Aquaculture Research Lab of Tribhuvan University, Amrit Campus, Kathmandu. Carps were acclimatized for three weeks in natural conditions prior to the beginning of the experiment. Water quality factors were recorded during the experiment as temperature 27 ± 13°C; pH 7.9 to 8.5, dissolved oxygen 5.8 to 6.5 ppm; NO 2 < 0.1 mg/L, NH 3 < 0.01 mg/L. e regular diet was fed during the acclimatization period (Table 1).

Preparation of Aloe vera Extract.
Fresh Aloe vera plants, a spiky cactus-like xerophyte [8,9], were collected from the local nursery within the campus, and the Department of Botany makes taxonomic identification. Briefly, 437 g of freshly cut leaves of Aloe vera was mixed with 100 ml of 70% ethanol and squeezed in a Whizzer, agitated on a shaker incubator for 12 hours, filtered through filter paper (Albert ® , Pores 7-11, size 185 mm, England), and then lyophilized in a lyophilizer (Vacuum Gauge, USA). e extract for alcohol extract was 1.5%. e isolates were stored in tightly sealed dark containers in a freezer at −20°C for later use. Desiccations of the leaf determined the dry matter of Aloe vera at 50°C for 7 days. e humidity content of Aloe vera was 92%.

Preparation of Diets and Experiment Design.
Altogether, four Aloe vera supplemented diets were prepared, as well as the diet without Aloe vera, controlled diet A (0.0%), while the other treated diets were as B, C, D, and E in which Aloe vera was supplemented as 0.2%, 0.4%, 0.8%, and 1.6%, respectively (Table 1). e proximate composition of a basal diet based on the formulation was prepared (Table 1). e experimental diets were prepared according to the method explained in the literature [7]. Other ingredients were used as per the standard norm of a laboratory. A total of 270 selected juvenile carp (1.73 ± 0.14 g) were circulated randomly at the rate of 15 fish per aquarium into 18 glass tanks (30" × 24" × 8"). Fish were fed twice daily (09 : 30 am and 3 : 30 pm) on their 3% of body weight. Sampling was on 30, 60, and 90 days, and during specific sampling; five fish were sampled from each tank and anesthetized using 0.03% tricaine methanesulfonate (MS-222) as per the guideline of the Canadian Council on Animal Care (CCAC 2005) [30] and then the fork length (cm) and body weight (g) of fish were recorded using a measuring tape and digital balance. For the gastrosomatic index (GaSI), three fish were sacrificed from each tank, and the gut of the fish were collected separately, weighed on 30, 60, and 90 days using the proper dissection procedure.

Growth Performances.
In this study, fish growth parameters were assessed in terms of percentage weight gain (PWG), specific growth ratio (SGR), food conversion ratio (FCR), feed efficiency rate (FER), protein efficiency ratio (PER), and condition factor (CF) calculated according to the following equations on 30th, 60th, and 90th days of feeding followed by percentage survival on regular monitoring. Gastrosomatic index (GaSI) was also monitored during the growth studies. All the fish weights were calculated in gram units.

Protein Profiles.
Before sampling for tissue collection, all fish were starved for 24 h and then five fish per treatment were sampled and anesthetized with tricaine methanesulfonate (5 mg l −1 ) for 2-3 min, and then the liver tissues were collected through proper dissection weighed carefully. A 5% homogenate was prepared as per the method reported in the literature [7]. e total protein, albumin, globulin, and the ratio of albumin and globulin were estimated through Bio-Rad Bradford (1976) [33] protein assay reagents and available standard kits.

Gut Histomorphology.
e gut histomorphological examination was done at the end of the feeding trial on the 90th day and for it, the intestine was collected along with liver from the same anesthetized fish used during protein estimation. e proximal intestine was selected because it is where most of the nutrient absorption takes place [34]. During the process, the intestine tissue tests were fixed in 10% formaldehyde and handled by standard histological procedures (dried out in ethanol arrangement, inserted in paraffin, and sequentially segmented at 4 to 5 μm) and stained with hematoxylin and eosin (H&E) [35]. Segments were assessed aimlessly under a light magnifying lens. Photos of the segments were made with a Nikon Eclipse 80i magnifying instrument (Nikon, Tokyo, Japan) furnished with a Nikon Digital Sight SD-MS camera and the Nikon programming NIS-Elements. Adobe Photoshop CS3 Extended was utilized for the last photographic readiness without adjusting the first uprightness of the photos. Morphometry consisted of measurements at 500x magnification of five randomly selected fields per intestinal tract.

Statistical Analysis.
Analysis of data was done by utilizing a one-way analysis of variance (ANOVA) with Aloe vera consideration levels as a factor, utilizing the measurable bundle for the social sciences (SPSS) program (variant 22). Duncan's various reach tests at a critical degree of 95% were utilized to decide huge contrasts between treatments. e data introduced in the content, figures, and Table 1 implies means ± standard error, and P < 0.05 was considered as significant.

Growth
Performance. Percent weight gain (PWG) (Figure 1), specific growth rate (SGR) (Figure 2), feed conversion ratio (FCR) (Figure 3), feed efficiency ratio (FER) (Figure 4), protein efficiency ratio (PER) (Figure 5), and condition factor (CF) (Figure 6) of common carp after the 30 th , 60 th , and 90 th days of feeding on diets containing different levels of Aloe vera extracts experienced a significantly high (P < 0.05) growth performance among groups C (0.4%) and D (0.8%) compared to control A (0%) group. e PWG and SGR of C. carpio were significantly affected in treated groups by dietary ethanolic Aloe vera extract (P < 0.05). Decreasing trends were observed in the FCR among the treated groups, and the lowest FCR was recorded in C (0.4%) diet-fed group (Figure 3). CF improved significantly in the treated groups and the lowest was found in the control diet-fed group. e survival percentage (S%) was higher among the treated groups (Figure 7).
It was observed that as the dose of Aloe vera increased in the diet, the GaSI level also increased, and GaSI was significantly higher (P < 0.05) in common carp fed with D diet (0.8%) compared to control and other diet-fed groups (Figure 8). During the whole experimental period, the common carp fed with experimental diets tended to have better growth performance compared with the control.

Protein Profiles.
e total protein ( Figure 9) level was found significantly high (P < 0.05) in fish fed with 0.4% Aloe vera in C group and 0.8% D group of carp as compared to other treated and control diet-fed groups. Similar results were observed in globulin ( Figure 10) and albumin (Figure 11) of common carp fed with C (0.4%) diet of Aloe vera after 90 days of feeding trial. e highest ratio of albumin and globulin has been recorded in the D diet (0.8%) fed group compared to the control and another diet-fed group ( Figure 12).

Gut Histomorphology.
e crypts of Lieberkühn (regularly referred to just as crypts) are pits between villi as in Figure 13. ey are like the gastric pits in the stomach. e crypts contain stem cells that can create various diverse cell types, including enterocytes [36].
After 90 days into the feeding trial, gut histology from all Aloe vera treated and control diet-fed carp was studied (Figures 14(a)-14(e)). Mucosa, muscular mucosa, and villi were spotted in the anterior gut of all groups, but the quality of villi was found better in diet-fed groups (Figures 14(b)-14(e)).

Discussion
e common carp Cyprinus carpio is a freshwater fish of lakes and rivers in Nepal and is believed to be in danger of extinction by the International Union for Conservation of Nature (IUCN), but its varieties have also been domesticated and distributed worldwide [38]. e omnivorous Cyprinus carpio eats a herbivorous diet of aquatic plants and benthic worms [39]. e ethanol extracts of plants signify a continuous effort to find new compounds against pathogens, and about 20% of these submitted to the pharmacological or biological test [40]. A substantial number of new antibiotics available in the market are obtained from natural resources [41]. e medicinal plant Aloe vera is known for its healing properties for long years [42]. 4 Journal of Nutrition and Metabolism          In the present study, the effects of crude extract of Aloe vera on percent survival, growth condition factors, protein profile, gastrosomatic index, and gut histomorphology were studied for 90 days of common carp fed Aloe vera in laboratory conditions. Plants like onion, garlic, and black cumin seed have been tested for growth-promoting activities, feed conversion, and expansion of protein digestibility in aquatic animals [43]. e present study showed a significantly higher percentage (99.87%) of survival in the D diet-fed group compared to the control (93.17%). e results for growth performance in percentage weight gain (PWG) and specific growth ratio (SGR) in common carp fed groups Aloe vera supplemented with 0.4% and 0.8% of C and D groups look much better than the control group A. e present results are in harmony with the literature [44]. e latter reported 0.5% and 2.5% ethanolic dietary A. vera inclusion levels and they were able to improve growth performance significantly in common carp. Heidarieh et al. in the year 2013 revealed 0.1% and 1% Aloe vera inclusion level increased growth performance in rainbow trout (O. mykiss) [29] and significant 3% growth performance (GP) found in percent weight gain (PWG), feed conversion efficiency (FCE), protein efficiency ratio (PER), and specific growth rate (SGR) of Nile tilapia explained by Shalaby et al. in the year 2006 [45].
e present results are also in agreement with the literature [46]. Khattab et al. [46] found that the diet of Biogen increased feed intake and improved feed conversion ratio (FCR), and protein efficiency ratio (PER) in Pangasius.
In this experiment, total protein, globulin, and albumin have been quantified from the liver tissues of treated and control diet-fed carp. It was surprising that carp fed with Aloe vera supplemented diet perform drastically higher (P < 0.05) in all the treated groups compared to the control diet-fed group A. Similar results were observed in globulin and albumin levels. e total protein, globulin, and albumin levels were higher in Aloe vera 0.4% and 0.8% diet-fed group. e growth and immune response of silver striped catfish (Pangasianodon hypophthalmus) took care of lapsi fruits (Choerospondias axillaris) and discovered that absolute Journal of Nutrition and Metabolism protein, albumin, and globulin level was higher in fish taking care of 0.4% lapsi fruits diet [7]. In agreement with the present findings, the use of Allium sativum and Zingiber officinale increased total protein concentration significantly (P < 0.05) and concluded that they protect the liver against harmful agents and free radical-mediated toxic damages to the liver cells explained by Ajeel and Al-Faragiin 2013 [47]. Similar results were also found in the literature [48], and Hamza et al. in the year 2008 [49] assessed the protein expression profile in the liver of 34-day-old Pikeperch larvae fed with three isotropic and isolipidic formulated diets. Hassan and Javed in the year 2000 [50] also found that a higher concentration of sarcoplasmic protein in Catla catla corresponded to a decrease of myofibrillar protein. By adding an extract of ginger to fish, the diet improved the protein level; the highest protein was observed in carp fed with 1% ginger extract [51]. e relationship between the weight of the alimentary canal and the weight of fish helps in determining the feeding condition in different months and seasons denoted by gastrosomatic index, and GaSI of common carp in the present study was found higher on the 30th, 60th, and 90th days of Aloe vera treated diets compared to control diet-fed group. e highest value, 5.22 ± 0.04, of the gastrosomatic index (GaSI) was recorded in Acanthopagrus latus by Sourinejad et al. [52] and Pramanik and Mohanty [53] recorded the highest 8.1% GaSI value of Rutilus crisis collected from Iranian waters. Histological monitoring of the structure of the alimentary canal in carp fed new ingredients provides essential information [54], but so many studies have not been carried out with carp [55].
After 90 days into the feeding trial, gut histology from all the Aloe vera treated and control diet-fed carp found villi spotted in the anterior gut of all groups. Such changes are because of Aloe vera present in the tissues. e taller, narrower, and routinely molded villi and the higher number of villi per unit zone are pointers that the capacity of the intestinal villi is initiated [56,57]. In general, these villi gave a more prominent surface area for absorption of accessible supplements. ese realities recommend that the villus capacity may be initiated after taking care of dietary Aloe vera in rainbow trout [29].
Similarly, morphological changes in the distal intestine are responsible for the growth that was observed in response to Aloe vera supplemented diets [58]. Moreover, we can compare our findings with the literature [59] that Ziziphus mauritiana leaf powder (ZLP) was used to see the growth performance, intestinal histomorphology, and growth-related gene expression of Nile tilapia (Oreochromis niloticus). e stomach demonstrated an expansion in the thickness of the gastric mucosa and muscularis in totally treated gatherings with the most elevated qualities in 10 g/kg took care of gathering. e intestinal mucosal folds, statures, widths, zone, and edge of the villi and thickness of strong layers were fundamentally (P < 0.05) higher in gatherings took care of with ZLP, which was affirmed by utilizing scanning electron microscopy. In this manner, ZLP can be utilized viably in tilapia consuming fewer calories for improving the development and intestinal wellbeing. ese days, the use of bioactive compounds through herbal supplementation in the aquafeed is growing interest for scientists and aquafarmers. erefore, the application of Aloe vera is very common in other animals but it is a new attraction from the aquaculture perspective.

Conclusion
e results of Aloe vera studies for other medical conditions have been less clear. is paramount attitude of this research was to understand the effects of Aloe vera on the growth and protein profiles of carp, and in the results, we can observe that Aloe vera extract is useful for fish growth by enhancing protein contents in the liver tissues. Survival and growth performance followed by gastrosomatic index and condition factors studied indicate the use of Aloe vera in aquaculture carp growth. In contrast, the protein profile and the ratio of albumin and globulin indicate the immunity level in carp fed Aloe vera supplemented diets. Gut histomorphology study is further proof, and the performance of the growth of villi in the treated group, especially in C (0.4%) and D (0.8%) dietfed group, boosts the morphophysiology of carp compared to the control A (0%) group. Finally, this experiment is an initial trial using Aloe vera extract supplemented diets in the Nepalese aquaculture system, which inspires new researchers to work further using the medicinal herb in aquaculture as an alternate dose of vaccine for carp growth. From the economic point of view, it solves food security for aquafarmers.

Data Availability
e data used to support the findings of this study are included within the article.

Conflicts of Interest
e authors declare no conflicts of interest.