Dietary Incorporation of Natural and Synthetic Reproductive Inhibitors: Exploring Their Impact on Sex Characteristics in Cyprinus carpio (Common Carp)

Abstract

The present study was designed to evaluate the effect of supplementation with papaya seeds (PSM), fish testes powder (FTP), and 17α-methyltestosterone (MT) on the reproductive parameters, growth performance, digestive enzymes, and histology of Cyprinus carpio. In the present study, fries (2–3 days old) were acclimatized for 2 days and then equally distributed into one control and six treatment groups and fed with one control and six experimental diets for 30 days, followed by a control diet for 60 days in each group with triplicates. These diets included control (T0), different levels of 17α-methyltestosterone (T1: 60 mg/kg; T2: 70 mg/kg), papaya seed powder (T3: 6 g/kg; T4: 7 g/kg), and fish (tilapia) testes powder (T5: 70%; T6: 80%). The treatment groups receiving papaya seed meal (PSM) showed significant improvements in growth performance, with a significant increase in final body weight. The best zootechnical performances (PER, SGR, and CF) were observed in fish fed with papaya seed compared to the control group. Reproductive analysis showed significant variations between treatment groups, with a large number of female C. carpio observed in the control group. Fish treated with T4 increased the sex percentage in favor of male fish by achieving 90% male phenotype followed by T6 with 88% male. Diets based on papaya seeds and fish testes powder significantly reduced the reproductive performance by reducing GSI, which affected the gonadal histology. The results revealed a visible effect of 17 α-MT and PSM feeding on the gonad structure. There were significant elevations in protease enzyme activity in T6 compared to the control (p < 0.05), and the highest amylase activity was observed in T3. Natural resources are not only more cost-effective but also environmentally friendly and readily available; they are a superior choice over synthetic alternatives for controlling the prolific breeding of C. carpio.

1. Introduction

As a sector of food production, aquaculture is fast-growing. The demand for aquaculture is expected to expand along with the population worldwide and account for 62% of all worldwide production by 2030 [1]. It is estimated that at least 845 million people worldwide depend on fish for their nutritional needs. Fish are a valuable source of nutrition for humans due to their rich nutritional profile, which includes essential macronutrients such as proteins, lipids, and ash, as well as a wide range of micronutrients including vitamins and minerals [2]. Cyprinus carpio, commonly known as the common carp, is one of the most significant freshwater fish worldwide in terms of trade, having contributed 4.41 million metric tons in 2021 or around about 5.2% of the total worldwide inland fish production. For the past 800 years, it has been ranked third in importance among freshwater fish. As an omnivorous species, it exhibits rapid growth and a high degree of environmental adaptability, making it a popular choice in Asia, Europe, and China [3]. C. carpio exhibits several fundamental issues. These include early maturation at a weight of 400–500 g within 5–6 months and undesired spawning in standing water biannually. Consequently, ponds become overcrowded due to overpopulation [4]. In comparison to females, male C. carpio and Oreochromis niloticus perform better in terms of growth [5]. Monosex stocks in aquaculture have been shown to offer several advantages, including control over unwanted reproduction, the minimization of conflicts, enhanced survival rates during harvesting, and potential growth benefits [6]. To produce monosex male populations, aquaculturists employ various methods, including the use of hormones. Several steroids have been identified as capable of inducing sex change, including fluoxymesterone, testosterone, 17-methyltestosterone (MT), 19-norethyltestosterone, androstenedione, ethyltestosterone, dihydrotestosterone (DHT), trenbolone acetate, 17-methyldihydrotestosterone, 17-ethyltestosterone, and mesterolone [7]. Although the use of synthetic hormones has yielded certain benefits, the associated drawbacks have prompted farmers to seek alternative methods that are safe for both consumers and the environment. Phytobiotics are plant-derived dietary additives containing distinct phytochemicals that contribute to enhancing growth status [8]. Utilizing natural alternatives, such as biodegradable and cost-effective phytochemicals (e.g., phytoestrogens or phytoandrogens) found in the seeds of Carica papaya (Papaya), may present a viable strategy for fish sex reversal due to their ability to mimic the effects of hormones and promote growth [9]. In recent years, researchers have been paying more attention to the potential health benefits and nutritional value of various natural products. Among these, papaya seeds and fish testes are often overlooked and discarded as waste, and have emerged as a subject of growing interest due to their potential bioactive compounds. They are biological, decomposable, and inexpensive reproductive inhibitory agents. The predominant biologically active constituent found in C. papaya seeds, known as Benzyl isothiocyanate (BITC), has demonstrated its role in inducing a contraceptive impact. Studies have revealed that the use of pawpaw seed powders can stimulate growth and induce sex reversal in various freshwater fish species [10]. The principal bioactive ingredients in pawpaw seeds with anti-fertility or sterility potential are saponin, oleanolec acid 3-glucoside. Pawpaw seed meal contains glucosides that can destroy gonadal cells in tilapia testes and ovaries [11]. Examination of desiccated pawpaw seeds reveals a composition comprising 97.27% of dry material, 30.08% of crude protein, 34.80% of essential fat, 1.67% of crude fiber, 7.11% of ash, and 23.67% nitrogen-free extract, which potentially enhance the growth of fish [12]. Fish testes (natural sources of testosterone) are also important in sex reversal [13,14,15]. Fish testes are cost-effective and locally available sources to incorporate in feed. Natural sources are potential substitutes for synthetic hormones in aquaculture because they bring valuable pharmacologically active metabolites with a variety of benefits such as immune-boosting, growth promotion, antioxidant enhancement, antidepressant, increasing survival rates, digestive enhancements, stimulation of appetite, and improved food conversion ratio [16]. It is a new trend in aquaculture to use animal by-products (fish testes). Only a few studies have evaluated the use of fish testes powder as a natural source for masculinization. Based on the aforementioned properties of animal and plant by-products, the objective of this research was to precisely investigate the effectiveness of papaya seed meal (PSM) and fish (tilapia) testes powder and synthetic (17α-methyltestosterone) androgen on growth performance, sex ratio, enzymatic activity, and gonadal histology.

2. Materials and Methods

2.1. Feed Preparation

Papaya was bought from the local market in Faisalabad and fresh papaya seeds were obtained from them. The plant was authenticated based on previous studies and a botanist. The seeds were removed, rinsed with tap water, and dried by spreading them at room temperature. Fine powder was prepared by drying the seeds under shade; this was kept in a dry, clean, airtight plastic container at room temperature until usage. Mature tilapia testes (fresh) were collected from the local fish market, chopped in to 1 mm² and sun-dried for 2–3 days. Dried testes were ground and converted into fine powder and kept in an airtight container at −20 °C until further use. 17 α-MT powder was purchased from Sigma-Aldrich (Hamburg, Germany).

2.2. Experimental Fish and Rearing Conditions

Two-day-old Cyprinus carpio fries (n = 560, initial weight: 1.28 g ± 0.002 g), after having absorbed the yolk sac, were randomly distributed into 7 glass aquariums each with triplicates. The fries were fed with treatment diets once a day, split into two time slots (morning and evening), for 30 days during the treatment phase. The trial continued for the next 60 days in the same aquarium (rearing phase) to ensure that the fries were large enough for gonad squashing. The physiochemical conditions, including pH (7.3–7.6) and DO (5–6 ppm), were maintained as per guidelines in [17]. Growth was monitored biweekly over the 90-day period by measuring total body length and weight, following the method in [18]. Before handling, the fish were starved for 24 h to minimize stress. The final weight was determined individually at the end of the trial.

2.3. Experimental Framework and Feed Formulation

A Complete Randomized Design (CRD) was employed in this study, consisting of six experimental diets and one control diet (32% CP), each treatment with triplicates as shown in

Table 1. The composition of ingredients in basal and treatment diets.

Diet Ingredients (g/kg)	T0	T1	T2	T3	T4	T5	T6
	Control (without Hormone)	MT1 60 mg/kg	MT 70 mg/kg	PSM2 6 g/kg	PSM 7 g/kg	FTP3 70%	FTP 80%
Fish Meal	28	28	28	28	28	28	28
Rice Polish	16	16	16	16	16	16	16
Canola Meal	19	19	19	19	19	19	19
Wheat Bran	18	18	18	18	18	18	18
Rice Broken	12	12	12	12	12	12	12
Fish Oil	5	5	5	5	5	5	5
Vitamin Premix	2	2	2	2	2	2	2
Total	100	100	100	100	100	100	100
17α-methyltestosterone	-	60	70	-	-	-	-
Fish (Tilapia) Testes powder (%)	-	-	-	-	-	70	80
Papaya seeds powder	-	-	-	6	7	-	-
Proximate Composition of diet (%)
Moisture	3.00 ± 0.06 E	4.00 ± 0.17 C	4.40 ± 0.06 B	3.50 ± 0.12 D	4.00 ± 0.08 C	5.00 ± 0.12 A	4.00 ± 0.05 C
Ash	76.00 ± 0.93 D	80.00 ± 0.51 AB	81.00 ± 1.15 A	79.01 ± 0.77 ABC	80.00 ± 0.17 AB	77.00 ± 1.03 CD	78.00 ± 1.06 BCD
Protein	29.01 ± 0.35 C	31.34 ± 0.70 B	31.86 ± 0.45 B	32.00 ± 0.48 B	33.00 ± 1.14 AB	33.07 ± 0.44 AB	34.50 ± 0.69 A
Lipid	16.00 ± 0.42 D	17.00 ± 0.22 CD	18.00 ± 0.26 BC	19.01 ± 0.47 AB	19.50 ± 0.28 A	16.00 ± 0.43 D	18.00 ± 0.24 BC

MT¹: 17α-methyltestosterone; PSM²: papaya seed meal; FTP³: fish testes powder. Proximate composition of diet presented as mean ± S.E. Rows with different letters represent statistically significant differences (p < 0.05).

. These diets included control (T0), different levels of 17α-methyltestosterone (Sigma-Aldrich) (T1: 60 mg/kg; T2: 70 mg/kg), papaya seed powder (T3: 6 g/kg; T4: 7 g/kg), and fish (tilapia) testes powder (T5: 70%; T6: 80%). The diets were made by mixing the material with oil, adding water to moisten it, and creating a dough that was cut into pellets and sun-dried. All diets were stored in a cool, dry place until use.

2.4. Growth Parameters

At the end of the trial, fish were weighed using a digital balance, and the following growth parameters were calculated: initial weight (g); final weight (g); weight gain (g) = final weight − initial weight; protein efficiency rate = weight gain/protein intake; condition factor = (Bw/TL³) × 100; specific growth rate (% d⁻¹) = [(Ln final weight (g) − Ln initial weight (g)]/days) × 100.

2.5. Digestive Enzyme Assay

To evaluate the digestive enzyme activity, the gut and liver were carefully removed and rinsed with distilled water. Samples were cleaned twice with 0.9% NaCl solution before measuring the digestive enzyme activity. Ice was used during the whole procedure. Samples were homogenized in PBS for 1 min before centrifugation at 20,000× g for 45 min at 4 °C. The supernatant was collected and kept at −80 °C for further analysis.

After adding 250 μL of enzyme extract to the test tube to determine the amylase activity, it was incubated at 25 °C for three and four minutes to achieve equilibrium. After adding 250 μL of 1% starch, incubation was carried out for 3 min. Then, 0.5 mL of dynitrosalicylic acid dye was added to the tubes, before cooling to room temperature; it was then immersed in boiling water for five minutes. Then, 5 mL of distilled water was added to the tubes, and absorbance was measured at 540 nm.

A substrate solution using 1.5% azocasein in 50 Mm Tris-HCl buffer was prepared to measure the protease activity. The enzyme extract was incubated with 0.5 mL of 1.5% azocasein in a Tris-HCl buffer for 10 min at 25 °C. For the termination of the reaction, 0.5 mL of TCA was added, and the reaction was centrifuged at 6500× g for 5 min. Ultimately, the uppermost layer was shifted to a purification plate, and the absorbance at 440 nm was determined using spectrophotometry.

2.6. Identification of Sex

Ten fish from each treatment were examined at the termination of the trial. Sample fish were carefully netted, anesthetized using clove oil at a concentration of 0.05 mL per 500 mL of water, and dissected to determine their sex by direct visual inspection of morphological characteristics. Fish were classified as female, male, or sterile based on whether they had visible ovaries, testes, or immature gonads (thread-like appearance), respectively. The gonads were removed and weighed, then put in a 10% neutralized formalin solution for more investigation. Indigo-carmine was used to stain randomly selected gonad samples [19,20]. A Leitz microscope was used to observe the stained tissue (Huma Scope Advanced ^LED 40X power with 0.5X CCD adapter) to evaluate the quality of sex-reversal fries [21].

$$\mathrm{G}\mathrm{S}\mathrm{I}={\displaystyle \frac{Gonadal weight}{Body weight-Gonadal weight}}\times 100$$

2.7. Histological Evaluation

The gonads were removed from the fish after they were sacrificed and washed with sterile saline solution (Figure 1). They were then directly fixed in a 10% neutral buffered formalin solution for 24 h. The paraffin embedding technique [22] was used to process the fixed specimens. In brief, the specimens were dehydrated in increasing ethanol concentrations, cleared in xylene, embedded in paraffin wax, cut into several sections of 5 μm thickness, and stained by the hematoxylin and eosin stain. The stained sections were photographed using a digital camera attached to an optical microscope and examined for histological changes.

2.8. Statistical Analysis

After finding the conceivable data on growth, enzymatic activity, and gonad histology, they were subjected to statistical analysis (ANOVA) and ± SE values were computed by utilizing SPSS version 20; differences between means (p ˂ 0.05) were calculated by an HSD test [23]. Sex ratio percentage was calculated by the chi-square method.

3. Results

3.1. Growth Performance

The growth performance indices of fish fed different diets are outlined in

Table 2. Growth performance parameters of C. carpio fed the experimental diets (mean ± SE).

Growth Index	Treatments
	T0	T1	T2	T3	T4	T5	T6
	Control	MT (60 mg/kg)	MT (70 mg/kg)	PSM (6 g/kg)	PSM (7 g/kg)	FTP (70%)	FTP (80%)
IW	1.29 ± 0.005 S	1.28 ± 0.015 S	1.28 ± 0.010 S	1.29 ± 0.005 S	1.28 ± 0.020 S	1.27 ± 0.000 S	1.26 ± 0.020 S
FW (g)	3.86 ± 0.060 EFG	4.17 ± 0.065 D	4.64 ± 0.035 C	4.98 ± 0.005 A	4.81 ± 0.105 B	4.24 ± 0.030 D	4.28 ± 0.020 D
WG 1 (g)	2.57 ± 0.643 E	2.89 ± 0.833 CD	3.35 ± 0.482 C	3.69 ± 0.643 B	3.53 ± 0.833A	2.97 ± 1.322 A	3.02 ± 0.583 B
CF	1.42 ± 0.040 AB	1.43 ± 0.041 AB	1.44 ± 0.039 A	1.44 ± 0.053 A	1.41 ± 0.050 B	1.32 ± 0.062 D	1.34 ± 0.061 C
PER 2	0.08 ± 0.002 C	0.09 ± 0.005 BC	0.10 ± 0.004 AB	0.11 ± 0.003 A	0.11 ± 0.002 A	0.09 ± 0.006 BC	0.09 ± 0.005 BC
SGR 3 (% day−1)	1.22 ± 0.015 E	1.31 ± 0.017 D	1.42 ± 0.020 BC	1.50 ± 0.023 A	1.46 ± 0.021 AB	1.34 ± 0.018 D	1.36 ± 0.038 CD

Weight gain ¹ (g) = mean final weight − mean initial weight; condition factor = (Bw/TL³) × 100; protein efficiency ratio ² = weight gain/total protein intake; specific growth rate ³ (SGR% day⁻¹) = (ln weight final − ln weight initial)/feeding trial duration. Rows with different letters show a significant variation (p < 0.05). IW—initial weight; FW—final weight; WG—weight gain; CF—condition factor; PER—protein efficiency ratio; SGR—specific growth rate.

. The growth performance of fish fed with plant supplementation showed the best results. Maximum weight gain was noticed in T3 followed by T4 and T2 (4.98 ± 0.005a, 4.81 ± 0.105b, and 4.64 ± 0.035c, respectively; Figure 2). After the 90-day trial, treatment-group fish gained weight by 5-fold, whereas the basal-diet fish only gained weight by 3-fold. Also, papaya seed supplementation significantly improved all growth parameters, as these fish had the highest condition factor (CF), protein efficiency ratio (PER), and specific growth rate (SGR). In the case of the hepatosomatic index (HSI), no significant difference was observed in papaya seed (PSM) and fish testes powder (FTP), but there was a highly significant difference (p  <  0.05) from the fish from the control group.

3.2. Sex Ratio and Gonad Index

As shown in

Table 3. Sex percentage, sex ratio, and gonadal index of male and female C. carpio fed different diets.

Treatments
Variables	CR		MT 60 mg/kg		MT 70 mg/kg		PSM 6 g/kg		PSM 7 g/kg		FTP (70%)		FTP (80%)
	Control		T1		T2		T3		T4		T5		T6
	M*	F*	M	F	M	F	M	F	M	F	M	F	M	F
Sex (%)	54 ± 0.7 C	46 ± 1.4 A	76 ± 0.4 B	24 ± 0.5 B	85 ± 0.89 C	15 ± 0.85 C	85 ± 0.892 C	15 ± 0.854 C	90 ± 1.042 A	10 ± 0.744 E	84.00 ± 2.08 A	16.00 ± 1.83 C	88.00 ± 1.16 A	12.00 ± 1.01 CD
Sex ratio	1.17:1 A		3.16:1 B		5.66:1 C		5.25:1 CD		9:1 E		5.25:1 CD			7.33:1 F
GSI values of male and female Cyprinus carpio
GSI* (Male)	1.54 ± 0.025 B		1.76 ± 0.035 A		1.82 ± 0.015 A		1.00 ± 0.010 E		1.08 ± 0.010 D		1.18 ± 0.030 C		1.10 ± 0.020 D
GSI (Female)	1.67 ± 0.005 C		1.94 ± 0.010 B		2.12 ± 0.015 A		1.18 ± 0.015 E		1.32 ± 0.010 D		1.24 ± 0.015 E		1.38 ± 0.050 D

Rows with different letters show a significant variation (p < 0.05). GSI*—gonadosomatic index; F*—female; M*—male.

, the reproductive analysis of Cyprinus carpio revealed significant differences (p < 0.05) between the control and the other treatment groups. The highest percentage of female (46%) Cyprinus carpio was observed in the control group. T5 and T6 had significantly higher male percentages (90% and 88%, respectively), followed by T3 and T4 (85%). In general, the use of appropriate doses of plant seed and fish testes powder is more effective and safe than 17α-MT for inducing male Cyprinus carpio.

The expected ratio of males to females in the control group (T0) that were only fed the basic diet was 1:1 (M:F), and the actual ratio of 1.17:1 was not significantly different from that. However, when the fish were fed with PS, they became more male. The more PSM in the diet, the more males there were. The male-to-female ratio was 9:1 (M:F) in the group fed PSM at 7 g/kg. In the fish testes powder group (FTP2), the male to female ratio was 7.33:1. There was no significant difference between the PSM1 and FTP1 male to female ratio. GSI showed significant variations among control and treatment groups. The control and MT-treated groups had the highest GSI values, while the plant- and testes-treated groups had the lowest.

3.3. Enzymatic Assay

Regarding digestive enzymes, the activity of protease was significantly affected in T6 compared to other treatments (p < 0.05), both in terms of the liver and gut, and declined in the control group (Figure 3). The highest amylase activity in the liver was observed in T3, while the lowest was in T4. In the case of the gut, the highest amylase activity was observed in the MT-treated groups.

3.4. Histological Examination

The results revealed a significant visible effect of 17 α-MT and PSM feeding on the gonad structure, while normal stages of oocyte development were observed in females of the control groups (Figure 4). The testes of the 70 mg/kg dose-treated fish after the trial period showed disperse spermatozoa and deformation in both the seminiferous lobule and interlobular tissue (Figure 5). However, regarding the testis histology of the group fed the 7 g/kg papaya dose level, significant abnormalities were observed in the gonads, showing lesions in the seminiferous tubules (Figure 6).

4. Discussion

Cyprinus carpio are widely cultured and known for their high adaptability, reproductive capacity, and stable genetic traits. However, precocious maturity and the prolific breeding nature of this fish limit its aquaculture potential. Common carp become mature at the early age of 5 to 6 months and start breeding, which limits their growth to a small size. Excessive breeding behavior causes overcrowded conditions and elevates competition for food. Female common carp reproduce at an early age, resulting in stunted somatic growth, low yield, and unmarketable fish size. That is why males are preferred in monosex cultures because they grow faster than females [24]. Different synthetic and natural sources are available for rearing common carp (male) to market size to fulfill the demand of consumers by increasing their somatic growth. In aquaculture, monosex populations are caused by both natural and synthetic reproductive inhibitors. They are important for controlling maturity and breeding in fish species to understand the physiological and endocrine processes involved in gonadal development and sex differentiation. Recent research highlighted the potential positive impacts of incorporating plant by-products and animal by-products into aquaculture. These sources have been found to contribute to the advancement of aquaculture practices. The potential of plant by-products and animal by-products as alternative agents to synthetic drugs in aquaculture is noteworthy. They provide biologically active metabolites with a multitude of advantages, including growth enhancement, immune modulation, antioxidant enrichment, improved digestion, antidepressant properties, appetite stimulation, and hepatoprotective effects. This research emphasized the advantageous outcomes associated with the utilization of plant seeds and fish testes in feed formulations. The utilization of seed meal and testes powder in feed had a significant impact on both the growth attributes and sex ratio of common carp fingerlings. In this study, there is a comparison of the efficiency of three different treatments for growth, sex reversal, enzymatic activity, and histology of fish: papaya seed (PS), fish testes powder (FTP), and 17α-methyltestosterone (17α-MT).

4.1. Growth Parameters

The results showed that all three treatments were effective in the growth and sex reversal of Cyprinus carpio, with PSM showing the greatest growth followed by 17α-methyltestosterone. These findings agree with earlier investigations by [9] in Oreochromis niloticus, which have consistently highlighted the effectiveness of papaya seeds. In the context of this study, it is noteworthy that the incorporation of 6 g/kg of papaya seeds into the fish’s diet led to a substantial enhancement in growth rates. This growth promotion can be attributed to several factors, including the presence of crude protein, essential fats, crude fiber, and a plethora of beneficial phytochemicals/phytoestrogenics such as alkaloids, flavonoids, phenolic acids, carotenoids, tannins, lectins, terpenoids, and saponins within these seeds [25,26,27]. After the plant-based diet of PSM, a synthetic androgen, 17α-methyltestosterone, at an inclusion level of 70 g/kg promotes the growth of Cyprinus carpio. Additionally, the growth performance of the fish was more improved by the hormonal treatments (70 mg/kg feed) compared to the control group due to the anabolic effect of this hormone. This finding was consistent with the previous study by [28]. All of these findings may be related to the fact that the percentages of males in the treatment groups increased. Hence, most of the treated groups were not accompanied by sexual partners (females), so their energy was channeled towards growth instead of reproduction. Ref. [29] stated that as well as the metabolic cost occurring in mixed-sex culture, courtship behavior and gamete formation devour energy that ought to be used for growth into reproduction, resulting in small-sized fish. The liver, a key metabolic organ in fish, is significantly influenced by the body’s metabolism. Consequently, the liver index (HSI) serves as a valuable biomarker for identifying the detrimental impacts of environmental stressors. The current study revealed that fish subjected to PSM exhibited reduced HSI compared to the control and MT groups. Similar conclusions drawn by [26,30] indicated no compromised health status of Cyprinus carpio with the inclusion of papaya seeds in the diet.

4.2. Sex Ratio

The key purpose of this study was to illustrate how papaya seed and fish testes powder act as a reproductive inhibitor as a substitute for synthetic hormones. The enhanced growth performance and feed efficiency observed in C. carpio fed PSM, FTP, and 17 alpha-MT may be linked to their prominent male population, making up 90%, 88%, and 85%, respectively [31,32], delivering relevant findings to this research that the catfish fed exclusively on pawpaw seed meal exhibited a complete presence of males, with not a single female in sight, may be attributed to the effect of the phytochemicals in papaya seeds such as saponin and papain, which act as sex reversal agent. Ref. [33] also reported that pawpaw seed had a masculinizing effect on fish. Fish testes powder also increases the male population; this observation is consistent with the findings of [13,14,34]. They illustrated that increments of testis in fish feed increase the male population. Ref. [35] reported that using solely catfish testes produced high percentages of phenotypic males, and these findings are in line with our results. The failure to obtain 100% sex reversal using testes might be a result of the method of preparation of the testes that were added to the diet. It may have been because some of the testosterone in the testes was unavailable for absorption due to the tough and fibrous septa inside testes, which are difficult to digest. As a result, it may have been possible that some of the testosterone in the testes was not available for assimilation [32]. Moreover, Refs. [36,37] supported 17α-methyltestosterone at a high dose level for the masculinization of Oreochromis niloticus. MT is a male androgen that stimulates male characteristics as well as significantly promoting the growth of tilapia due to the anabolic effect of the androgen. These results agree with [37,38], who claimed to obtain a 90% male population at high doses of 17 α-MT.

A low Gonadal Somatic Index (GSI) denotes gonads that are not fully developed. In the current study, it was discovered that the GSI of fish that were treated with PSM and FTP was lower than those in the control and MT groups. It has been reported by [29] that plants significantly inhibit prolific breeding. PSM and fish testes also have active compounds that have more pronounced hyperplasia and degenerative effects on germ cells than MT. A similar decreasing trend in GSI was observed in PSM-, FTP-, and 17 α MT-treated fish in [9,26,39].

4.3. Enzymatic Activity

Digestive enzymes (such as protease, amylase) are required for digestion in fish. It may be difficult to absorb nutrients when there is low activity of digestive enzymes because in the absence of digestive enzyme activity, diets cannot be hydrolyzed, which prevents nutrients from being absorbed. Fish testes showed the highest protease activity as testes are a good source of protein, and these outcomes are in line with [40], who reported that protease activity increased significantly in fish when fed a blood meal-based diet, owing to the fact that blood meal contains high protein levels. Further studies are recommended/required to evaluate the effect of testes (animal by-product) on fish digestive enzymes. Papaya seeds lead to positive protease results as the level was elevated at 7 g/kg, but amylase activity declined, which may be due to antinutritional factors in plants. For protease activity, the outcomes of our research are similar with [9]. Papaya seeds may contain phytochemicals that increase appetite by stimulating enzymes and altering the bacterial community in fish intestines. Studies by [41,42] have explored this phenomenon. The activities of enzymes like amylase and protease were investigated, revealing notable findings. The presence of papaya leaf extract had an impact on amylase activity in red tilapia but decreased it in African catfish. Notably, protease activity showed changes with a 4% inclusion level of papaya leaf extract. While research suggests that papain can affect proteases in Heteroclarias and sterlet, the observed alterations did not follow a pattern as reported by [41,42]. Ref. [43] found that adding enzymes to juvenile tilapia meals led to increased total protease activity in their intestines, contributing to improved weight gain. The activities of digestive enzymes (amylase and protease) were the main factors that helped fish digest plant-based feed efficiently without dropping the feed intake. Results exposed that amylase activity decreases as protease activity increases. Similar results were observed in GIFT juveniles and in Pangasiodon pangasius; as crude protein content increased in the diet, amylase activity decreased linearly and exponentially, which may be attributed to reduced carbohydrate availability. The influence of 17α-methyltestosterone (MT) on the enzymes of fish, specifically amylase and protease, has been widely studied. Research indicates that MT treatment affects aspects of fish well-being. Exposure to MT induced stress in tilapia, impacting the antioxidant enzymes and gene expression in their livers [44]. Consequently, some protective antioxidant parameters and enzymes experienced a decrease. It seems that MT has an impact on the enzymes of fish, working through metabolic and oxidative pathways [45].

4.4. Histology

A lot of studies in the field of aquaculture have explored the impact of phytoestrogen extracts from various plants on the growth, digestive, and immune systems of many animals [46]. Only a handful of studies have delved into the effects of phytoestrogens on the histology of fish that have been fed. Phytoestrogens, which are endocrine-disrupting compounds (EDCs) found in plants, have been linked to the impairment of animal reproduction and can affect gonad differentiation or delay maturation. The findings revealed varying degrees of abnormalities in the gonads of male C. carpio from different groups that were fed PSM and MT. Furthermore, degenerative seminiferous tubules were observed in the group of fish treated with 7 g/kg PSM. These findings shed light on the fact that PSM’s disruptive effects could manifest either directly or indirectly, possibly through its impact on the pituitary–gonadal axis. It is also plausible that androgens, which play a crucial role in seminiferous tubule function, could exert their influence either directly or indirectly, given the necessity of maintaining high androgen concentrations within the tubules for proper cell development and function. These observations align with previous research conducted by [47,48]. In vertebrates, androgens are hormones (natural and synthetic) that bind to androgen receptors to regulate the development and maintenance of male characteristics. Recent research revealed that 17α-MT causes gonadal alternations in fish. MT induced dispersion of spermatozoa and disintegration of interlobular septa in the ductus deferens. This research corroborates the report of [49] since the application of MT resulted in stunting of growth and deterioration of mature oocytes in O. niloticus, as evidenced by a reduction in the oocytes’ diameter. According to a study by [46], Nile tilapia fed with diets containing PSM at 120 gkg⁻¹ showed similar findings.

5. Conclusions

The findings of this study indicate that the dietary inclusion of 17 alpha-methyltestosterone, Carica papaya, and fish testes powder significantly influenced the reproductive capabilities of Cyprinus carpio. By producing males, C. carpio will be able to reduce prolific breeding as result their size will increase, improving their market value. Plant by-products are widely being used in aquaculture, but animal by-products (testes powder) have not yet been commercialized, nor have they been adopted by farmers. Any technology that uses locally available materials or ingredients (cost-effective) should be adopted by farmers or practitioners. 17 alpha MT is a synthetic androgen, expensive, and has adverse side effects such as genotoxicity and fetotoxicity. Biological resources such as testes and Carica papaya are preferable over artificial means for controlling the uncontrolled proliferation of Cyprinus carpio due to their affordability, biodegradability, and availability. Hopefully, this study will provide valuable insights to the scientific community and provide a guiding light for fishery management when both synthetic and natural sources give the same findings. Further studies on the hematology and enzymatic activity of testes powder are recommended to understand the impact on fish health.