Introduction

Heavy metals are becoming one of the most serious environmental hazards due to their persistence, biological toxicity, non-degradability, and ability to infiltrate the food chain. Furthermore, heavy metal pollutants have been shown to react with some organic compounds, converting them into even more deadly metal–organic complex pollutants under specific conditions (Maceda-Veiga et al. 2013; Chowdhury et al. 2016). Heavy metals can be accumulated and concentrated to some extent by aquatic species. When heavy metal concentrations and toxicity levels surpass aquatic animals’ tolerance, they have significant toxic impacts on their related indicators or even essential aspects of life. Heavy metals such as copper (Cu), iron (Fe), and zinc (Zn) are necessary for fish metabolism (Schmitt et al. 2005; Has-Schon et al. 2007). The effects of different dietary zinc sources (mineral, nanoparticulate, and organic) onquantitative and qualitative semen rainbow trout (Oncorhynchus mykiss) was investigated (Kazemia et al. 2020). Various studies have shown that heavy metal accumulation in tissues is primarily influenced by metal concentrations in water and exposure time, while other environmental variables such as salinity, pH, hardness, and temperature also play a role in metal accumulation (Has-Schon et al. 2007). The ability of a fish to sustain itself is determined by its reproductive mechanism and fish exposed to contaminants, particularly heavy metals such as Cd, Fe, Pb, and Cu, may develop acute or chronic poisoning, affecting all biological processes, including reproduction (Zulfahmi et al. 2018).

The effects of environmental toxicants on fish are being studied using several methodologies; however, histology improves the quality of research by clearly showing cellular responses (Van Dyk et al. 2007). Histopathological evaluations of fish are a tool to reflective of the overall quality of the aquatic environment and can be linked to levels of pollutants such as heavy metal contamination. Fish develop pathological changes because of these pollutants. The histological examination of fish tissue is useful as a bio-assessment technique and may be used to identify biochemical and physiological changes induced by pollution exposure (Short and Meyers 2001). Histopathological changes in the African catfish liver following exposure to a pollutant have been reported in several studies (Karami et al. 2016). Antioxidants, both enzymatic and non-enzymatic, are essential defensive mechanisms that protect animals against environmental pro-oxidants by mitigating the severity of reactive oxygen species (Tabrez and Ahmad 2009). Reactive oxygen species, such as superoxide anion (O2−), hydroxyl radicals (OH), and hydrogen peroxide (H2O2), cause extensive cellular damage, including lipoperoxidation of polyunsaturated membrane lipids (Nishiyama et al. 1998). Potential biomarkers like antioxidant parameters such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase, and oxidative stress indices are widely employed as screening tools to determine the effects of environmental stress (Kutluyer et al. 2016). Fish are subject to stressors in their habitat. Antioxidants, both enzymatic and non-enzymatic, help the body maintain oxidative homeostasis and respond to oxidative stress. Reactive oxygen species (ROS) are slowed down or neutralized by these molecules. It has been revealed that sperm cells include antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX). Antioxidants protect the lipid peroxidation sperm membrane by reducing oxidative stress (Cocchia et al. 2011). Antioxidant enzymes like SOD and CAT in the gonads experienced significant changes due to heavy metal accumulation. These enzymes may become inactive and exhibit inhibitory effects as a result of heavy metals exposure (El-Hak et al. 2022).

Steroid hormones are extremely vital and perform important roles in reproductive health (Kime et al. 1996; Rurangwa et al. 1998). Heavy metal contamination in the environment is known to alter the steroid hormone profile function in animals, including teleost fishes (Hontela 2005; Oliveira et al. 2008). Cadmium reported as an endocrine disruptor has been found to interfere with the production of steroids, eggs, and sperm in fish, as well as alter hormone synthesis in the testes (Vetillard and Bailhache 2005).

Because of their importance in fish metabolism, lipids, particularly fatty acid accumulation, are essential for reproduction and larval development throughout maturation (Ghaedi et al. 2016). Lipid and fatty acid fluctuations occur primarily in the ovaries, testis, and liver, likely to result in differences in gonadosomatic (GSI) and hepatosomatic indices (HSI) (Love 1980). Long-chain polyunsaturated fatty acids (LC-PUFAs) such as eicosapentaenoic (EPA), docosahexaenoic (DHA), and arachidonic (ARA) acids are essential for fish growth and function (Turkmen et al. 2017). Dietary lipids and fatty acids (FAs) are geared toward gonadogenesis throughout maturation, at the expense of somatic tissue investment. Saturated fatty acids (SFAs) are preferentially and mobilized away from muscle tissue during sexual development in farmed fish, according to prior findings (Cleveland et al. 2012; Manor et al. 2014; Fazio et al. 2021).

The Jhelum River is Kashmir’s major water source which flows through Kashmir. It is the main source of irrigation in the Kashmir valley. It is the primary source of the Verinag rainbow trout hatchery and is free of contamination. There is no anthropogenic activity on this river at the Verinag site, the Jhelum River has made a substantial contribution to Kashmir’s economic growth. The communities of fishermen rely heavily on this river for their livelihood; the river is well known for trout fish. In contradiction to it, community sewage, agricultural runoff, and effluent discharges pollute the Panzath spring. Unfortunately, the alarming increase in anthropogenic activity and effluent discharges in the Panzath spring has resulted in a significant decrease in water quality. Because fishes are more sensitive to changes in their environment, environmental contaminants or their metabolites can cause oxidative stress (Lopez-Lopez et al. 2011; Sattari et al. 2020; Müller et al. 2021). The rainbow trout (Oncorhynchus mykiss) is a well-evolved global fish and one of the most common commercial species (Abbate et al. 2020). The aim of the present study was to compare the histological, biochemical, and fatty acid levels of a representative species of rainbow trout collected from two locations, Verinag hatchery, least polluted (S1), and Panzath hatchery, more polluted (S2) Jammu and Kashmir (India). To our knowledge, this is the first investigation into how heavy metal exposure effects on reproduction of rainbow trout at the histological, biochemical, and fatty acid levels. The obtained information will offer novel insights on fish toxicity heavy metals.

Materials and methods

Fish and tissue collection

Rainbow trout (Oncorhynchus mykiss) is a commercially significant fish that has been designated as a pollution sentinel species because can provide information regarding the types, quantities, availability, and effects of environmental contaminants (Benedetto et al. 2016). Between August 2017 and April 2019, 60 adult rainbow trouts (30 from each site) with body weights ranging from 400 to 650 g were collected from two locations, Verinag hatchery, which was less polluted (S1), and Panzath hatchery, which was more polluted (S2) (Fig. 1). Immediately after capture, the fish were euthanized with a lethal dose of clove oil (0.20 mL of clove oil per 500 mL of water). The gonads were then removed and transported to the laboratory at 4 °C in 0.9% saline solution and subsequently processed for histological, biochemical, and fatty acid analyses.

Fig. 1
figure 1

Rainbow trout (Oncorhynchus mykiss) caught at sites S1 (a) and S2 (b)

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Chemical analysis of water

Polyethylene bottles were used to collect samples from the S1 and S2 sites to test the presence of heavy metals; copper (Cu), lead (Pb), zinc (Zn), cadmium (Cd), arsenic (Ar), and iron (Fe) in the water using an atomic absorption spectrophotometer (Analyst 400, PerkinElmer, Inc. 940 Winter Street Waltham, MA 02,451 USA). After being treated with 5% nitric acid, the bottles underwent a complete deionized distilled water rinse. The water samples were then preserved by acidification with diluted hydrochloric acid (HCl, 37 percent, Merck, Germany). The solution was then put into a conical flask and evaporated to 20 ml by heating it to 110 °C on a hot plate. Then it was transferred into a pre-cleaned bottle for atomic adsorption spectrophotometer (AAS) examination. All analyses were done in triplicate.

Tissue sampling

Tissue samples from fish testis were dissected into two portions, one for histopathological studies and another for the determination of heavy metals in tissues. Fish tissues of (1 g) were placed into polyethylene tubes and then treated with 8 mL nitric acid and 4 mL perchloric acid. All the chemicals and standard solutions that were used in the present study were purchased from Merck (Darmstadt, Germany), and they were all analytical quality. The samples were digested at 100 °C with ultra-pure nitric acid and perchloric acid till the solution became clear. The tubed sample was allowed to boil until all tissues had been dissolved. After cooling and filtering, they were put in plastic bottles. The determination of heavy metals (Cu, Pb, Zn, Cd, Ar, and Fe) was measured using an atomic absorption spectrophotometer, (Analyst 400, PerkinElmer, Inc. 940 Winter Street Waltham, MA 02,451 USA). The International Atomic Agency’s standard reference materials (MA-A-2/TM) were used to verify the precision and accuracy (IAEA). All analyses were done in triplicate.

Histopathological analysis of testes

The testes (n = 20, 10 each from the site) were fixed and dehydrated in graded ethanol (Merck, Darmstadt, Germany) before being cleared in xylene (Merck, Darmstadt, Germany). The samples were then fixed in paraffin blocks and dissected using a rotary microtome (Weswox Optik Model MT-1090A 15,125, India, Haryana). Hematoxylin and Eosin/Toluidine blue were used to stain the sections. Under a light microscope (Olympus CX31, Japan), tissue sections were examined and photographed.

Apoptotic assay (acridine orange staining)

The method of Kalia and Bansal (2009) for acridine orange staining was followed. A droplet (50 µl) of acridine orange was used to stain around 10 µL of cell solution on a micro slide. Under a fluorescence microscope (Olympus LS, Japan) using 500–525 nm filters, the slides were examined. Cells that appeared green were normal, yellow/orange cells were pre-apoptotic, and red cells were apoptotic.

Biochemical analysis

Antioxidant enzymes, malondialdehyde (MDA), catalase, SOD, and glutathione peroxidase (GPx) were investigated using the following techniques.

Malondialdehyde (MDA)

In a test tube, 0.1 ml tissue homogenate was prepared by mixing with phosphate-buffered saline (PBS, pH 7.2), 0.1 ml Tris–HCl buffer, 0.1 ml FeSO4, and 0.1 ml ascorbic acid (Fisher Chemical™), and 0.6 ml dH2O (deionized water) (Merck, Darmstadt, Germany) was added, making the volume 1.0 ml (Santamaría et al. 1997). For 15 min, the reaction mixture was incubated at 37 °C. One milliliter of 2,4,6-trichloroanisole (TCA) and 2 ml of 2,4,6-tribromoanisole (TBA) (HiMedia) were added and thoroughly mixed in. The solution was heated in a boiling water bath for 15 min before being centrifuged at 1000 rpm for 10 min to remove the flocculent adduct. The pink color produced by Malondialdehyde (MDA) formation was measured using NanoPhotometer™ UV/Vis Spectrophotometer From Implen GmBH, Germany, at 532 nm against a blank.

Catalase

A total of 0.5 ml of testicular cell suspension was incubated for 30 min at 4 °C with 50 mM phosphate buffer saline for catalase estimation. NanoPhotometer™ UV/Vis Spectrophotometer from Implen GmBH, Germany, was used to record the absorbance at 240 nm for 3 min at an interval of 20 s following the addition of newly produced 6 mM H2O2 (Merck, Darmstadt, Germany). The measure of catalase activity was directly related to the reduction in H2O2 absorbance per unit time (Aebi 1984).

Superoxide dismutase

The method stated by Marklund and Marklund (1974) was followed to measure superoxide dismutase. SOD catalyzes the dismutation of the superoxide radical into (O2−) hydrogen peroxide H2O2 and elemental oxygen (O2) and therefore offers a vital defense against the superoxide radical’s toxicity.

Glutathione peroxidase (GPx)

The dithiobis nitrobenzoic acid procedure by Rotruck et al. (1973) was used to measure GPx activity, which is based on the interaction between residual glutathione after GPx action and 5,5′-dithio bis-(2-nitro benzoic acid) to create a complex that absorbs maximum at 412 nm.

Protein estimation

The total protein content was calculated using the Lowry et al. (1951) method, which used bovine serum albumin as a reference.

Extraction of lipid

The lipids from the fish (n = 20, 10 from each site; during the reproductive season) were extracted using Folch et al. (1957) method. In the concentration of 10 g of sodium sulfate anhydrous (Na2SO4) (Merck, Darmstadt, Germany), 5 g of tissue (testis) was crushed. The total lipids were obtained from paste using a 1:15 (w/v) ratio of chloroform: methanol (2:1 v/v) (Merck, Darmstadt, Germany). G-3 sintered funnel was used to filter the extract. The filtrate was placed in a separating tunnel and washed with 0.9% normal saline (5:1 v/v). The difference between the initial and final weights of the crucible yielded the total lipid content. The overall lipids then were dissolved in 5 mL of chloroform (Merck, Darmstadt, Germany), and stored at 4 °C before being quantified.

Fatty acid analysis

The gas chromatograph mass spectrometer (Shimadzu Mass Spectrometer-2010 series, Italia) was used to analyze the fatty acids. Boron trifluoride was used to carry out the transesterification reaction of fatty acids. Gas chromatography was used to analyze fatty acid m column GC ethyl esters (FAME) using a 30 m × 0.25 mm fused silica capillary column and a flame ionization detector (Lochmann and Gatlin 1993). The extracted lipids were subsequently analyzed by GC MS with injection temperatures of 280 °C, quadruple temperatures of 150 °C, helium flow rates of 1.5 ml/min, and ion source temperatures of 230 °C. The injection was carried out in a split-less manner with a volume of 1 μL. The device was set to a temperature of 90 °C and kept there for 5 min. Following that, the temperature was raised at a pace of 10 °C each minute. Electron ionization at 70 eV was used to produce mass spectra of chemicals in tissue samples, and the detector was set to scan from 60 to 800 atomic mass units (AMU). Fatty acids were analyzed by comparing the retention periods with those of known standards. Heptadecanoic acid (17:0) was used as an internal standard to measure fatty acids, and the results were presented as mg/g of gonad wet weight.

Hormonal analysis: plasma testosterone and 11-ketotestosterone assay

The blood samples (n = 20, 10 from each site) were collected from the caudal vein and centrifuged for 15 min at 3000 g, following which the serum was kept at − 20 °C until ELISA analysis. Plasma was separated by centrifugation at 3000 rpm for 5 min at 4 °C. ELISA immunoenzymatic kits were used to detect testosterone and 11-ketotestesterone levels. This was done with commercially available enzyme-linked immunosorbent test kits from Cayman Chemical Company, USA. Protocols of Cuisset et al. (1994) and Nash et al. (2000) were used to determine sex steroid hormones. Each sample contains 50 µl of plasma, which is used to extract hormones. To extract testosterone and 11-ketotestosterone from plasma, diethyl ether, ethyl acetate/hexane 50:50 (v:v), and methylene chloride were used. Testosterone and 11-ketotestosterone assay standards were developed. The microplate reader (Spectramax 190, Beckman Coullter, Canada) was used to measure sample and reference absorbance at a wavelength of 412 nm once the plate was set up.

Statistical analysis

Data were expressed as the mean ± standard deviation and analyzed with one-way ANOVA performed with SPSS 19.0 for Windows. Pearson’s correlation analysis was used to examine the relationship between the sex steroid hormones. Tukey’s test was used to compare the average gonad fatty-acid composition across maturation stages. The level of significance was P < 0.05.

Results

Heavy metal bioaccumulation

The concentrations of Cu, Pb, Zn, Cd, Ar, and Fe were quantified in sampling water as well as in the testis. The findings revealed significant differences between the water samples taken from S1 (less polluted) and S2 (more polluted) sites (Table 1), as well as significant variations within the tissues (Table 1). Metals in water as well as in fish testes sampled from both S1 and S2 sites decreased in the order of Fe > Zn > Pb > Cu > Cd > Ar (Table 2). Overall, metal levels in fish collected from S2 were higher significantly than in samples collected from the S1 site. The results of the analysis of variance revealed that the mean concentrations of heavy metals differed significantly between the S1 and S2 water and testis samples (P < 0.05).

Table 1 Heavy metal content (mean ± SD, mg/L) in water and (mean ± SD, µg/g) in testis of Verinag (S1) and Panzath hatchery (S2)
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Table 2 Fatty acid profiles (mg/g) in the testes of rainbow trout captured from S1 and S2
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Testicular histopathology

Histological analysis revealed that the fish testes captured from S1 waters had normal structure, as demonstrated by the well-organized distribution of cells in the seminiferous epithelium. The testes were comparable to those of other teleosts in terms of internal structure. Numerous germ cells at various stages of meiosis, differentiation, and maturation filled the germinal cysts. In the cysts, there were many primary spermatogonia (PSG), secondary spermatogonia (SSG), spermatocytes (SC) (Fig. 2a and b), and spermatozoa (SZ) (Fig. 2c) in the lobular lumen. Spermatogonia cells (SG) were round and deeply stained in Toluidine blue. Meiotic divisions produced the SCs from the SG. The nucleus of each SC was stained with hematoxylin. Toluidine blue was used to stain the spermatids (SPDs). They got reduced in size as they became mature, and chromatin became more evenly condensed. The SPDs were eventually converted into SZ, which are the smallest cells found in clusters in the testes. SZ were abundantly filled in the lumen of seminiferous lobules and stained strongly (Fig. 2c). While S2 fish were found to have testicular abnormalities. In comparison to S1 fishes, S2 fishes showed different alterations. Disorganization of seminiferous lobule structures, reduction in the number of germ cells (PSG, SSG, and SZ), vacuolization, and large empty areas in the seminiferous epithelium (Fig. 2d, e, and f) were among the abnormalities observed in S2 fishes. Significant differences were observed in the GSI values of the fishes collected from S1 and S2 sites (P < 0.05). S1 fishes had a GSI of 3.6%, whereas S2 fishes had a GSI of 3.4% (Fig. 3a).

Fig. 2
figure 2

Light micrograph of the testis of rainbow trout from S1 site depicting normal testicular structure with primary spermatogonia (PSG), secondary spermatogonia (SSG), spermatocyte (SC) (a, b); (arrowhead) organized seminiferous tubules with lumen filled with spermatozoa (SZ) (c). Light micrograph of the testis of rainbow trout from the S2 site depicting large empty spaces (double arrowhead), fewer germ cells (d), vacuolization (asterisk) (e); unorganized epithelium (triangle) (f)

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Fig. 3
figure 3

a The pattern of changes in GSI from S1 and S2 captured fishes. The values were expressed as mean ± SD (n = 20, 10 from each site). Asterisk value significantly higher than the ones from the other sites P < 0.05. b, c, d, e, f, g The pattern of changes in MDA, Catalase, SOD, and GPx from S1 and S2 captured fishes. The values were expressed as mean ± SD (n = 20, 10 from each site). Asterisk value significantly higher than the ones from the other sites P < 0.05. The pattern of changes in testosterone and 11-ketotestosterone from S1 and S2 captured fishes. The values were expressed as mean ± SD (n = 20, 10 from each site). Asterisk value significantly higher than the ones from the other sites P < 0.05

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Apoptotic cell frequency

Changes in apoptotic cell frequency were analyzed. Acridine orange staining revealed testicular cells that fluoresced green, indicating that they were alive, whereas those that fluoresced orange and red were apoptotic. The frequency of apoptotic cells captured from S2 water increased significantly (P < 0.05) in testicular cells (Fig. 4c and d) as compared to S1 captured fish (Fig. 4a and b).

Fig. 4
figure 4

Photograph of testicular cells revealing live cells (green) (a, b), compared with the S1 group; the number of apoptotic cells was significantly increased with S2 captured fish (c, d)

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Enzymatic antioxidants

As shown in Fig. 3b, MDA levels were (0.33 ± 0.05 µmol/g wet tissue weight) in rainbow trout testis collected from S1 waters and gradually increased in S2 captured fish (0.48 ± 0.07 µmol/g wet tissue weight), which was strongly linked with the severity of heavy metal stress. Both CAT and SOD activity was recorded to be higher in the fish samples captured from S1 waters (0.86 and 0.63 U/mg protein) as compared to S2 fish (0.69 and 0.48 U/mg protein) respectively (Fig. 3c and d). Like the activity of SOD and CAT, the activity of GPx was higher in S1 as compared to S2 fish (Fig. 3e). There was a substantial reduction (P < 0.05) in SOD, CAT (U/mg protein), and GPx (µmole / mg protein) in response to increased heavy metal concentration in S1 waters.

Serum sex steroid levels

S1 and S2 fish have different testosterone and 11-ketotestosterone levels in their serum. Testosterone and 11-ketotestosterone in male rainbow trout followed the same trend as GSI. The amount of testosterone and 11-ketotestosterone is as follows: in S1 fish (0.64 and 0.41 ng/ml) and S2 fish (0.51 and 0.31 ng/ml) respectively (Fig. 3f and g). In male rainbow trout, testosterone and 11-ketotestosterone levels increased substantially in S1 fishes (P < 0.05).

Fatty acid composition

The fatty acid profile of rainbow trout testis is presented in Table 2. The fatty acid content of gonads varied depending on the gonadal stages of maturity. In S1 and S2 collected fishes, saturated fatty acids (SFA) account for 43.67 and 40.48%, respectively. S1 fish gonads had significantly more total monounsaturated fatty acids (MUFA) (P < 0.05). S1 and S2 fishes, respectively, have 11.21 and 8.61% MUFA. Polyunsaturated fatty acids (PUFA) account for most fatty acids in the testes. Docosahexaenoic acid (DHA) is the most abundant PUFA, accounting for about 45% of all PUFAs. Arachidonic acid (20:4, AA) and eicosapentaenoic acid (20:4, EPA) were two others important PUFAs that were higher. S1 and S2 captured fishes have 37.51 and 34.00% PUFAs, respectively.

Discussion

The present research is the first to focus on rainbow trout reproductive disruptions as well as various forms of gonadal changes under the influence of heavy metals. Heavy metal pollution in the environment has been identified as a major contributor to fish poisoning. Pollutants like heavy metals emitted by waste disposal, industries, or mining operations, are responsible for the decline in fisheries resources, and, as is known, these heavy metals cause bioaccumulation and biomagnification in the tissues of the aquatic organisms (Jarup 2003; Bakhshalizadeh et al. 2022). In the present study, the metal levels in testis tissues varied significantly depending on the metal content of the water. When compared to S1 sites, Fe, Zn, Pb, Cu, Cd, and Ar levels in the testis were the highest in S2. According to Varol et al. (2017), Fe has the highest mean content in rainbow trout (Oncorhynchus mykiss) raised in a dam reservoir on the Firat (Euphrates) river. In the samples, Zn was the second-most prevalent metal; these results are in support of the findings of the present study. According to Muinde et al. (2013), the trends for the accumulation of heavy metals in sediment and water samples from Lake Victoria were similar, that is, Zn > Pb > Cr > Cd; however, the trends for tilapia fish were Zn > Pb > Cd > Cr. In the present study, Fe was found to be higher in water samples as well as in fish testis. Heavy metals, such as Cd, accumulate in the gonadal fish tissue if reach up to 1000-fold higher than that in the surrounding water environment and become detrimental to reproduction (Dietrich et al. 2011). Heavy metal accumulation in the gonads is linked to decreased gamete quality, including decreasing sperm (Kocabaşet al. 2022) and ripe oocyte degeneration (Mansour et al. 2018). As a result, many fish reproduction mechanisms, such as seasonal hormonal cycles, spermatogenesis, and testis necrosis are affected (Kime 1999; Dietrich et al. 2010).

The heavy metals accumulated in different tissues affect cellular structure, physiology, and reproduction (Fatima and Usmani 2013). Many studies have shown that pollution, particularly heavy metals, can cause a reduction in the GSI as reported by Gerbron et al. (2014) in Rutilus rutilus which is in the support of the results of the present study. Heavy metals, industrial wastes, pesticides, and agricultural wastes have diverse histopathological effects on the reproductive tissues of fish gonads (Lye et al. 1998; Jaensson et al. 2007). These impacts may disrupt germ cell development and impair the capacity of fish to reproduce (Khillare et al. 2017; Mehanna 2005). Furthermore, spermatogenesis and lobular structures were impaired in the testis of the examined fish obtained from contaminated water, and sperm production was suppressed as reported by Jaensson et al. (2007) and Shobikhuliatul et al. (2013). In the present investigation, structural anomalies of testicular tissues were revealed in rainbow trout testis. The impact of contaminants on fish testes has been reported in previous studies (Sayed and Younes 2017; Heba and Mohamed 2019). The findings of the histological examination of the gonads of Clarias gariepinus also demonstrated that pollution interfered with gonad development (El-Hak et al. 2022). Heavy metal (Cd, Cu, Ar, Hg, and Pb) poisoning causes spermatogenesis inhibition, with large numbers of spermatogonia and spermatocytes but few spermatids and mature sperm, low activity, atrophy or necrosis of the interstitial cells, and changes in Sertoli cell structure (Kime 1995). Furthermore, bivalent ion competition (for example between Zn and Cd) might influence fish reproduction (Favier 1992).

To distinguish viable, early, and dead cells during apoptosis, a microscopic examination of cells was performed. Under a fluorescence microscope, three types of cells were identified: living cells (green), live apoptotic cells (yellow), and dead cells (red), suggesting that heavy metals influenced germ cell death. Heavy metal concentration increased the number of apoptotic cells, which corresponds to the findings of the histopathological alteration.

Reactive oxygen species (ROS) such as superoxide anion, hydrogen peroxide, and hydroxyl radical are known to cause oxidative stress, which is linked to testis damage (Shaikh et al. 1999; Stohs et al. 2000; Murugesan et al. 2005). The findings showed that heavy metal exposure considerably raised the amount of H2O2 in testes indicating that heavy metals such as Cd promoted the formation of reactive oxygen species (ROS). Antioxidant enzyme SOD is the cell’s initial line of defense against ROS, and it can defend against superoxide-induced oxidative damage by accelerating the conversion of the superoxide anion radical to molecular oxygen and hydrogen peroxide (Fridovich 1989). CAT is responsible for decreasing the damaging effects of H2O2 by turning it into water and oxygen. In the present research, S2 caught fish exhibited significantly lower Catalase and SOD activity in testes than S1 captured fish. (P < 0.05). Previous cites on this pathway have shown that excessive production of the superoxide anion radical or the presence of nitrites can inhibit catalase activity (Pandey et al. 2003; Arrillo and Melodia 1991). In addition, S2 fish showed a substantial increase in lipid peroxidation as compared to S1 fish. Similar investigations on fish species were reported from different contaminated areas, such as the Bizerte Lagoon (Ameur et al. 2012) and the Brazilian Pampa Biome (Nunes et al. 2015). The present results are inconsistent with those of Saliu and Bawa-Allah (2012), who found a decrease in SOD and CAT activity in lead-exposed Clarias gariepinus fish. Copper-treated freshwater teleost fish, Esomus danricus, showed decreased antioxidant activities of superoxide dismutase, catalase, and lipid peroxidation (Vutukuru et al. 2006).

In nature, trout usually consumes invertebrates from both terrestrial and aquatic communities (Fochetti et al. 2003). Dietary habits have a significant impact on lipid and protein levels (Oz, 2016). As a result, fatty acid content can vary greatly. In the present study, saturated and polyunsaturated fatty acids (PUFAs) made up most of the fatty acid reservoir in rainbow trout testis, followed by monounsaturated fatty acids (MUFAs). The main testicular fatty acids were 16:0, 18:0, 16:1, 18:1, 20:4 (AA), 20:5 (EPA), 22:5, and 22:6 (DHA), and all the fatty acids got decreased from S1 to S2 captured fish. The three main types of fatty acids found in Rhamdia quelen that were collected from the Rio Uruguay River were saturated (SFA = 35.5%), (MUFA = 28.1%), and (PUFA = 33.5%). DHA, palmitic acid, and AA were all found in large amounts (Anido et al. 2015). These results are in support to the findings of the present study. Fish exposed to Ni, Hg, or Ni plus Hg had relatively low levels of SFAs in their muscles (Senthamilselvan et al. 2016). In the present study, we also reported a decrease in SFAs in S2 captured fishes. Some desaturase enzymes may have been inhibited, resulting in a decrease in the amount of saturated fatty acids. This observation is supported by the fact that cadmium exposure reduced the activity of microsomal Δ9 desaturase as well as hepatic stearoyl-CoA desaturase (SCD) (Alvarez et al. 2007). PUFAs are regarded as essential for human and animal nutrition (Innis 2004). Saito et al. (1997) have reported that the fatty acid content of fish varies depending on the fishing site and is impacted by environmental factors and geographical conditions. Konar et al. (2010) also reported that the content of fatty acids like palmitoleic acid, linoleic acid, arachidonic acid, eicosapentaenoic acid, and docosapentaenoic acid (DPA) was less in cadmium-exposed muscle tissues as compared to without cadmium groups. The reduction in polyunsaturated fatty acids may be due to heavy metals stimulating the prostaglandin synthesis pathway. The alteration of metabolic pathways may be a major factor in the reduction of polyunsaturated fatty acid levels (Choi et al. 2002). The fatty acid composition and lipid levels of fish were impacted by the species, sex, degree of pollution, nutritional state, and seasonal fluctuation (Kitts et al. 2004).

In the present study, the concentrations of Testosterone and 11-ketotestosterone hormones in rainbow trout collected from S1 and S2 water were found to be significantly higher (P < 0.05) in fish captured in S1 waters (Fig. 3f and g). Hecker et al. (2002) reported that fish exposed to heavy metals had endocrine disruptor effects, resulting in a reduction in sex steroid hormone levels. Heavy metals, according to Salim (2015), can affect hormone production and function by inhibiting hormone synthesis, imitating natural hormones, and supplying receptors that limit hormone synthesis in cells. Especially, Cd ions have been shown to influence numerous mechanisms involved in fish reproduction, including seasonal hormonal cycles, spermatogenesis, and testis necrosis (Kime 1999; Mousa and Mousa 1999). Cadmium has also been shown to stimulate the production of gonad inhibitory hormone in the crab Uca pugilator (Rodriguez et al. 2000). Similarly, in male and female Japanese medaka (Oryzias latipes) exposed to Cd, alterations in circulating sex hormones have been observed (Foran et al. 2002; Tilton et al. 2003).

Conclusion

Heavy metal contamination not only increased the incidence of testicular deformities and apoptosis but also disrupted the normal steroidogenesis pattern in fish, resulting in impaired hormone production in male fish. The present study also found that the fish inhabiting the S2 site had higher levels of oxidative stress as measured by lipid peroxidation, as well as weaker antioxidant defense as measured by SOD, CAT, and GPx. Heavy metals have also been shown to affect the fatty acid components of the testis. As a result, this study indicated that the S2 was subjected to severe pollution, which had a significant impact on the fish population. In the context of biomonitoring, our findings might be used as a reference for fish ecotoxicological research in the polluted region in the future. The rainbow trout is a suitable bio-indicator of environmental pollution with heavy metals.