Effect of various local natural ingredients fortified in fish feed on the gonad histology and water quality of tilapia (Oreochromis niloticus)

The addition of natural ingredients fortified in fish feed was developed to enhance good formulation engineering. Currently, there is a need to improve follow-up research strategies for designed feed formulations using natural ingredients in tilapia farming. It is hoped that a sustainable and eco-friendly fish farming system will be improved. This study aimed to evaluate the effect of local natural ingredients fortified in fish feed on the gonad histology and water quality of tilapia (O. niloticus). Following these results, the histology test of tilapia showed that more than 50% of the gonad tended to be male in all treatments. This showed that the use of natural ingredients in the appropriate dosage in feed would affect the masculinization of tilapia. Furthermore, the specific growth rate (SGR) with the addition of natural ingredients to the feed showed results comparable to that of synthetic hormones. It can be concluded that the use of natural ingredients such as honey, cow testes, and Tribulus fortified in fish feed revealed a positive effect on the masculinization of tilapia and water quality levels in the fish farming system.


Introduction
The application of sex reversal in fish is intended to produce monosexual fish by manipulating hormonal pathways [1].Sex reversal is a process of gonadal differentiation driven by genetic, behavioural, physiological, and environmental variables that affect the growth and maturation of somatic cells in the gonads [2].Somatic cells have a role in providing instructive signals that trigger sexual differentiation during oogenesis and spermatogenesis [3,4].Sex reversal technology reverses the sexual development to the other way during embryonic development.During sex determination, the testicular and ovarian pathways oppose each other to regulate the fish gonads [5].
Exogenous administration of steroid hormones could impact the development of gonads treated with feed containing methyltestosterone, thus inducing a genetic transition in female fish to male [6].Monosexual tilapia fish are produced through masculinization or feminization techniques [7,8].The use of high hormone formulations and concentrations as well as long duration of exposure are required in phenotypic sex reassignment [9].Male monosexual seeds could be produced through hormone administration and chromosome engineering.Cultivating male monosexual tilapia has the advantage of the same growth rate and preventing unwanted spawning [10] 17α-methyltestosterone is a synthetic hormone that can induce the development of male tilapia seeds [11].Providing a 60 mg MT/kg dosage in feed can enhance growth performance in monosexual fish [12,13].MT exposure can enhance egg yolk production by enlarging follicle diameter and serum 1328 (2024) 012021 IOP Publishing doi:10.1088/1755-1315/1328/1/012021 2 11-ketotestosterone (11-KT) levels.MT can interfere with the endocrine system when it enters fish.Furthermore, the hypothalamus and pituitary gland will interpret to nerve impulses from the brain and convert them into chemical signals [14].However, the usage of 17α-MT is restricted due to anabolic residues [15].
The use of natural ingredients such as cow testicles at a dose of 9%/kg of feed by the dipping method can produce a percentage of male fish of 88.56% [16].Other natural ingredients that show the effectiveness of fish monosex are sea cucumber extract containing steroids and honey containing chrysin compounds that act as aromatase inhibitors [17].Natural components contain bioactive that function as aromatase inhibitors and antagonists to nuclear estrogen receptors (ERs) in the gonads.It is important to consider the anabolization and virilization effects of these natural substances [18].This study aimed to evaluate the effect of local natural ingredients fortified in fish feed on the gonad histology and water quality of tilapia (O.niloticus).

Research Design
Tilapia fish larvae (O.niloticus) which remaining egg yolk were prepared first in a holding pond for the acclimatization.Tilapia larvae were obtained from parent sources by spawning.This research was carried out at the Fish Culture Laboratory, FPIK UB for maintenance and in vivo test.Tilapia larvae were reared in 5 treatments with three replications each in aquariums with a stocking density of 100 individuals per experimental unit.The larvae had a lifespan of around 8-10 days.The larvae were feed flour pellets with a frequency of four times per day.During the maintenance period of approximately 55 days, the fish growth performance was calculated for each experimental unit.

Preparation of Local Natural Ingredients
Local natural ingredients used included honey, cow testicles and Tribulus terrestris extract.The honey was prepared from fresh cow testicles that was first extracted to become cow testicle flour.Tribulus terrestris extract was prepared by boiling 18 grams of the extract in 1500 mL of distilled water for 30 minutes, then followed by filtration using Whatmann paper.This extraction method referred to the modified technique described by Omitoyin et al. [19].Meanwhile, for steroid hormones (17αmethyltestosterone), it was prepared at 30 mg and dissolved it into 50 ml of 95% ethanol.

Feed treatments
The feed formula used in this study was made with a protein content of 38% for tilapia larvae and was fortified by various local natural ingredients.Five feed fortifications were applied consisting of (A) a combination of natural ingredients (honey 20 mg/kg, cow testes 13 mg/kg and Tribulus 0.75 g/kg feed).Other treatments were feed with fortification (B) honey with a concentration of 60 mg/kg, (C) cow testes 40 mg/kg feed, (D) fortification with Tribulus terrestris extract 2.25 g/kg feed, and (E) fortification 17α-methyltestosterone hormone 60 mg/kg feed.

Gonad Histology
The gonads resulting from masculinization in each treatment were subjected to histological testing with hematoxylin and eosin (H&E) staining.Micro anatomy was tested to determine the structure of fish gonads and differences in the level of gonad maturity that occurred between treatments.The work started with tissue fixation, tissue selection and cleaning, tissue blocking, slicing, staining, and observation using the microscope.

Water Quality
The water sample was collected as much as 25 ml in each week to measure the ammonia level in the fish rearing media.Then, 0.5 ml of Nesler was added to the sample and was homogenized at 30 minutes.Water quality (ammonia) was assessed using the spectrometer method at a wavelength of 425 nm.For dissolved oxygen (DO) was observed every day using a dissolved oxygen meter.

Statistical analysis
Data of specific growth rate (SGR) and water quality were analyzed by one-way ANOVA using statistical software.The difference between treatments can be seen from the DMRT (Duncan's Multiple Range Test) (p<0.05).

Research Ethics Using Animal Experiments
The research execution follows fundamental research ethical principles and adheres to animal welfare standards.Experimental animals were treated with respect and provided adequate care in the ideal living conditions of fish, including maintaining high standards of physical and chemical water quality.No animals were abused during this research.During the maintenance, fish were acclimatized to some factors such as temperature, pH, and dissolved oxygen in the rearing environment.This study altered the criteria by treating fish feed with various amounts of enriched local components to ensure it does not hinder fish development.Effective handling procedures could help minimize stress and disruptions to health check schedules during maintenance.This research follows the ethical norms and guidelines of the authorized Universitas Brawijaya Review Board (Protocol Number: 220-KEP-UB-2023).

Gonad Histology
The results of gonad histology observations were seen using a microscope with the aim of observing the gonads of male or female fish.The results of gonad histology are presented in Figure 1 which explains the differences between male and female gonads.Whereas, in the Figure 2 showed the results of gonad histological observation of all treatments.The observations of the gonads revealed that there were differences between male and female gonads.Male gonads possess spermatozoa cells that were smaller than egg cells, numerous, and resemble equally scattered scarlet spots.Female gonads have spherical egg cells with a central cell nucleus surrounded by cytoplasm [20].The sperm cells were depicted as small dot-like structure inside the centrosome, whereas egg cells were represented as huge circles with a paler centre surrounded by transparent cytoplasm.The histological structure of fish gonads may differ based on the fish species and its reproductive development phase [21].Providing hormones to masculinize fish was a method employed in fish farming as a sexual manipulation to produce a higher percentage of male fish.The formation of male gonads in fish was stimulated by the presence of androgen hormones such as 17α-methyltestosterone (MT) which were mixed orally into the feed.The mechanism of action of the androgen hormone was that the administration of the hormone would cause changes in the environment.Hence, it would be received by the senses, then it would be conveyed to the central nervous system.Afterwards, the information was sent to the hypothalamus and the pituitary gland would release the hormone gonadotropin (Gth).These hormones were carried by the blood, then transported to the gonads as instructions for starting the formation of male gonads [22].

a b
The use of natural ingredients in this study indicated that an increase in the percentage of male fish by more than 50% was demonstrated through histological examination.This showed that the use of natural ingredients in the appropriate dosage in feed would have an effect on the masculinization of tilapia.Natural compounds have bioactive properties that act as aromatase inhibitors and antagonists to nuclear estrogen receptors (ERs) in the reproductive glands.It was crucial to take into process the anabolic and virilizing effects of these natural compounds [18].The large proportion of males with a high levels of androgen hormones.Sex differentiation in fish was often controlled by exposing sexually undifferentiated fish to the right dose of external sex steroids [23].Senior et al. [24] reported that the chemicals employed for inducing sex reversal did not have any significant impact on survival.However, they indicated detrimental effects on gonadal structures.The production of masculinization in this study also supported the farmers to achieve the high growth rate.Magdeldin et al. [25] stated that the masculinized fish had a higher growth rate than both groups of phenotypic females, particularly after reaching sexual maturity.Sex-reversed pseudo-males exhibit behaviors and characteristics similar to genetic males such as producing normal sperm.This has been documented in many studies [8,26].

Growth Performances
The increasing specific growth rate (SGR) of tilapia could be an indicator of the success of supplementing natural ingredients in fish feed with the aim of sex reversal (Figure 3).This was linear with the water quality results obtained that the use of various natural ingredients showed optimal water conditions for tilapia (Figure 4).Enhancing the development rate with the use of natural substances and hormones demonstrated that fish could efficiently consumed feed and converted it into meat.The specific growth rate referred to the pace at which the weight of an individual tilapia changes during a certain timeframe.Based on the result of this study indicated that high specific growth rate values were achieved in the fortified feed with honey, Tribulus, and synthetic hormone treatments.The results showed that the proportion of male fish was greater in this treatment than in other treatments with low specific growth rate (SGR).Implementing single-sex (monosex) culture was suitable for obtaining tilapia with rapid development, as male fish grow faster than female fish.Beardmore et al. [27] noted that male tilapia is preferred in several fish species across different production methods due to their accelerated development rates.Dan and Little [28] who observed the growth performance of mono-sex and mixed-sex culture in three distinct strain of Nile tilapia.They found that the mono-sex strains developed substantially faster than the mixed-sex strains.
The addition of fortified natural ingredients to fish feed in this study as a bio-hormone testosterone was a simple method compared to others.This condition was caused by androgens exhibiting sex reversal and anabolic effects.Testosterone hormone administered from an external source would influence the hypothalamus to stimulate the release of luteinizing hormone (LH) and follicle stimulating hormone (FSH) in fish.Reversed sex tilapia fish exhibited better growth performance compared to normal tilapia fish [29].These findings indicated that the testosterone concentration absorbed by the larva through osmosis and diffusion mechanisms was adequate to change the larva's sex to male.Oral administration of male sex steroid hormones orally before primordial gonadal cell development could lead to a reversal of phenotypic sex.The effective production of 100% sex-reversed males with relies on the feeding strategy, hormone frequency, and meticulous regulation of all relevant elements during the sex reversal process in sexually undifferentiated fry [30].extract, and (E) 17α-methyltestosterone hormone) Several variables affected the acceleration of fish development consist of water quality, fish size, and species.High-quality feed can promote the growth of tilapia fish.Nutrient balance in the diet also affected the growth developments of tilapia.The tilapia growth would increase when there was surplus energy that could be utilized for bodily upkeep and metabolic functions [31].To achieve Treatments maximum development, it is crucial to consider several elements such as the quantity and quality of feed, form and size, attractiveness, durability, and stability feed in the water during feeding.This could enhance the fish's reaction to the meal given.An aquatic habitat that provided support would stimulate the fish's appetite, leading to increased growth in tilapia.Tilapia fish exhibited a growth rate that aligned with efficient feed consumption.Sukoco, et al. [32] stated that a high concentration of organic matter would elevate the growth performance values, therefore emphasizing the need of maintaining water quality to decrease organic matter in fish rearing environments.The growth rate of tilapia was affected by the protein content in their meal.Additionally, the growth rate may be increased by adjusting the amount of feed provided.Less feed resulted in more efficient feeding and faster development.Water quality, fish size, and fish species were also crucial factors in feed conversion and specific growth rate.

Water Quality
For the water quality of Tilapia rearing during the experiments, the range of all chemical factors in this study greatly influenced the behaviour and physiological condition of tilapia (Figure 4).In consequence, it would influence the metabolic rate of fish which affected the feed consumption, growth process, and survival of tilapia.The sources of ammonia in fish farming derived from fish feed waste and fish feces.Ammonia level was kept in tolerance concentration for fish rearing thus it does not cause toxicity to cultivated species.This is in accordance with the stated of Bhatnagar & Devi [33], the range of ammonia quality standard values for aquaculture does not exceed 0.2 ppm.Ammonia levels in waters were produced by decomposition processes, nitrate reduction by bacteria, fertilization activities and excretions of organisms in them.The low ammonia value indicates that the decomposition process of organic material takes place aerobically.The ammonia levels in the fish rearing media were closely related to pH and temperature.
The dissolved oxygen levels of this study showed there were no significant different for all treatments.It explained that the fortified feed with local natural ingredients did not influence the oxygen concentration in the fish rearing of tilapia.The presence of dissolved oxygen in tilapia fish culture is an essential factor for fish feed consumption because oxygen is needed to convert food into energy.Unbalanced dissolved oxygen would cause stress in fish because the brain does not receive an adequate oxygen supply.In addition, low dissolved oxygen (DO) would affect the growth rate of tilapia to decrease.

Figure 3 .
Figure 3. Specific Growth Rate of Nile Tilapia in All Treatments (Feed fortifications: (A) a combination of natural ingredients, (B) honey, (C) cow testes, (D) Tribulus terrestrisextract, and (E) 17α-methyltestosterone hormone) Several variables affected the acceleration of fish development consist of water quality, fish size, and species.High-quality feed can promote the growth of tilapia fish.Nutrient balance in the diet also affected the growth developments of tilapia.The tilapia growth would increase when there was surplus energy that could be utilized for bodily upkeep and metabolic functions[31].To achieve