Chemicals and regents
Melatonin ELISA kit was purchased from Fine Test (EU0199), Wuhan Fine Biotech Co., Ltd. Wuhan, China. Nicotinamide Adenine Dinucleotide, reduced (NADH), nitro blue tetrazolium (NBT), and glutathione, reduced (GSH) were purchased from SRL, Mumbai, India. 5-methylphenazinium methyl sulphate (PMS), phenylmethylsulfonyl fluoride (PMSF), 1-chloro-2, 4-dinitrobenzen (CDNB), o-phenylenediamine (OPD), trichloro acetic acid (TCA), thiobarbituric acid (TBA), 5, 5’-dithiobis (2-nitrobenzoate) (DTNB), 2’,7’-dichlorofluorescin diacetate (DCFH2DA), Propidium iodide (PI), RNase A, vanadium (III) chloride (VCl3), sulfanilamide, N-(1-napthyl) ethylenediamine dihydrochloride (NEDD) and other chemicals were purchased from Sigma-Aldrich Chemical Co., St. Louis, MO, USA unless otherwise specified.
Collection of fish
To ensure the reliability and consistency of our findings, we gathered adult male catfish Clarias batrachus (males and females were separated on the basis of sexual dimorphic characteristics), weighing about 210 ± 25 g, from the water bodies near the university campus, Purulia, India [23°35' N, 86°33' E], in each month of an annual cycle. The testicular development consists of six distinct phases, phase of slow spermatogenesis (January to February) or Phase-I, early phase of rapid spermatogenesis (March to April) or Phase-II, late phase of rapid spermatogenesis (May to June) or Phase-III, phase of functional maturity (July to mid-August) or Phase-IV, phase of depletion (late-August to mid-October) or Phase-V and phase of relaxation and rehabilitation (mid-October to December) or Phase-VI (Acharyya et al. 2023). Immediately following capture in each month, five adult male catfish (N = 5) were promptly transported to the laboratory and allowed to acclimate for seven days in a glass aquarium under natural photo-thermal conditions. This acclimation period aimed to reduce the stress caused during the collection and transportation of the fish before they were sacrificed. The fish were provided with commercial fish feed pellets, Growfin, from Growel Feeds Pvt. Ltd., (Andhra Pradesh, India), enriched with 40% crude protein along with 12% moisture and crude fat, fibre (6% and 3%, respectively). The Institutional Animal Ethics Committee (IAEC), Department of Zoology (Reg. No. 1973/GO/Re/S/17/CPCSEA), Sidho Kanho Birsha University, reviewed and approved animal care, hygiene, and laboratory procedures in accordance with the guidelines of CPCSEA, New Delhi, Govt. of India.
Collection and preparation of testicular samples
Following the dissection, the testes from each fish were rinsed using phosphate buffer saline (PBS) solution (100 mM; pH 7.4). Subsequently, tissue samples were carefully rid of any additional tissue fragments and placed on blotting paper to remove excess moisture. Each sample was individually weighed to determine the gonadosomatic index (GSI). A portion of each pair of testes was promptly immersed in Bouin’s fixative for histological analysis. Another portion of testicular fragment was minced in sterile PBS (1X, pH 7.4) to obtain a single cell suspension for the evaluation of cell cycle status and reactive oxygen species (ROS) level by flow cytometer. The residual portion of each testis was mixed with Tris-HCl buffer (50 mM, pH 7.4 with 1 mM EDTA, 1 mM PMSF, 100 mM sucrose, 1% leupeptin hemisulfate) and homogenized at 4°C followed by a brief sonication to obtain a 10% tissue homogenate for the measurement of melatonin concentration, levels of malondialdehyde (MDA), total nitrate and the levels of different enzymatic or non-enzymatic antioxidants. The homogenates were centrifuged at 13,000 g for 20 min at a temperature of 4°C. The supernatants were stored at a temperature of − 80°C for subsequent analysis (Hasan et al. 2014).
Histological study of the testes under microscope
Following dehydration of fixed testis tissue fragments, 5 µm thick sections were prepared using an automated microtome (Medimeas MRM-AT). The sections were stained with haematoxylin-eosin (H/E) stain. The relative percentage of different spermatogenic cells was analysed by calculating the count of seminiferous tubules containing a specific germ cell stage divided by the count of total seminiferous tubules, and then multiplying by 100 (Bhattacharya et al. 2007). The slides were visualized using a Leica DM2500 microscope equipped with a photo-micrographic device and image analysis software, LAS v4.9. Spermatogenic cells were examined by evaluating approximately 100 sections of seminiferous tubules from five histological slides per fish (20 sections for each slide), each taken at a scale of 100 µm.
Abundance of spermatogenic haploid cells through flow cytometry analysis
Percentage of haploid cells, maturational marker, in testis was determined by flow cytometric analysis in CytoFlex (Beckman Coulter, USA). Testicular fragments were minced in 1 mL sterile PBS (1X, pH 7.4) and filtered through a 40 µm cell strainer for single cell suspension. Following centrifugation at 2,500 g for 5 min at 4°C, cells were fixed in methanol for 15 min at RT and mixed with RNase A (20 mg/mL) after a chill shock at − 20°C for 2 min and kept at 4°C for overnight. Cells were stained with a red-fluorescent DNA dye, propidium iodide (PI) (10 µL of 1 mg/mL stock solution) for 35–40 min. Fluorescence signals from 10000 cells were recorded from the FL2 channel, measured at 565–595 nm. The results were further analysed by using CytExpert v2.4.0.28 software. Forward (FSC) and side scatter (SSC) plots were procured from both unstained and stained samples. A histogram plot, obtained from the ‘events’ within the polygon gate representing sample cells, measures the DNA content. To get the relative percentage of haploid cells, ‘line segment’ gating for respective phases viz. haploid, G0-G1, S and G2-M was further analyzed in different samples (Nóbrega et al. 2015).
Estimation of intra-testicular melatonin concentration
Testicular melatonin concentration was determined by ELISA kit (EU0199) following the instructions provided by the manufacturer (sensitivity: < 4.688 pg/mL, detection range: 7.813 to 500 pg/mL). In brief, 50 µL of test sample was mixed with an equal volume of biotin-labelled anti-melatonin antibody in each well for a period of 45 min at 37°C. 100 µL of HRP-streptavidin conjugated secondary antibody was added. Afterward, 90 µL of 3,3’,5,5’-tetramethylbenzidine (TMB) substrate was introduced. The absorbance was promptly measured at 450 nm after adding the stop solution using a microplate absorbance reader (Bio-Rad, iMark™), and the concentration was determined by interpolating the data with the help of a standard curve (Acharyya et al. 2023).
Estimation of intra-testicular oxidative stress
Generation of reactive oxygen species (ROS)
Prepared testicular single cell suspension was provided with fluorescent probe DCFH2DA (ROS sensitive) and incubated for 15–20 min in dark and examined through a flow cytometer, CytoFlex (Beckman Coulter, USA) to measure the amount of oxidised DCF, indicator of intra-testicular ROS. Data were analysed by using CytExpert v2.4.0.28 software. A histogram plot was taken from the ‘events’ selected in polygon gate, where the X-axis representing the intensity of the probe and Y-axis representing the number of cells (count) containing the probe or DCF+ cells. From the histogram plot, mean fluorescence intensity (MFI) of the probe for each sample was obtained to compare the amount of ROS generated in different samples (Aitken et al. 2013).
Malondialdehyde (MDA) level
Testicular homogenates were centrifuged at 3000 g for 15 min at 4°C (Tarsons, Spinwin, MC-05-R) and the supernatant was mixed with reagent containing TCA (20%), TBA (0.5%), and HCl (2.5 N). The mixture was incubated at boiling water bath for 20 min following centrifugation at 500 g for 15 min at room temperature (RT) and the absorbance of the supernatant was recorded at 532 nm in a microplate absorbance reader (Bio-Rad, iMark™) to measure MDA level, a marker of lipid peroxidation (Mondal et al. 2017).
Estimation of intra-testicular nitrosative stress
Total nitrate level, a marker of nitrosative stress, was measured in testicular sample by incubating with VCl3 and Griess reagent containing sulphanilamide (1%) and NEDD (0.1%) for 30–45 min in dark condition. The absorbance was taken at 540 nm and concentration was determined from a standard curve of sodium nitrate (NaNO3) (Miranda et al. 2001).
Estimation of activity/level of enzymatic and non-enzymatic antioxidants
Enzymatic antioxidants
Superoxide dismutase activity (SOD)
Testicular homogenate (25 µL) was mixed with 200 µL of phosphate buffer (50 mM, pH 7.4) containing reaction mixture 0.1 mM EDTA, 62 µM NBT, 98 µM NADH and 33 µM PMS. The absorbance was recorded at 560 nm in a microplate reader (Moniruzzaman et al. 2016).
Catalase activity (CAT)
The activity of intra-testicular catalase was measured by spectrophotometric method (Aebi 1984). Briefly, 20 µL of tissue homogenate was mixed with assay buffer, containing 50 mM Tris-Cl (pH ~ 8), 0.25 mM EDTA and 9 mM H2O2 to the final volume of 1 mL in a quartz cuvette and kinetic study was performed at 240 nm in a UV-VIS Spectrophotometer (Thermo Scientific, Genesys, 10S UV-VIS) for 1 min at 15 seconds intervals.
Glutathione peroxidase activity (GPx)
Briefly, 100 µL of testicular homogenate was mixed with OPD (0.4 mg/mL in phosphate citrate buffer, pH ~ 5) in presence of a co-substrate H2O2 (0.013%). After 30 min of incubation at RT, the reaction was terminated with 3N H2SO4 and absorbance was measured at 492 nm in a UV-VIS Spectrophotometer (Thermo Scientific, Genesys, 10S UV-VIS) (Hasan et al. 2014).
Glutathione S transferase activity (GST)
100 µL of sample was mixed with 900 µL of phosphate buffer (50 mM, pH 6.5) containing 100 mM CDNB and 100 mM GSH, and after 5 min of incubation at 30°C absorbance was measured at 340 nm in a UV-VIS Spectrophotometer (Thermo Scientific, Genesys, 10S UV-VIS) at every 60 seconds for 5 min to measure the GST activity (Mondal et al. 2017).
Non-enzymatic antioxidant
Reduced glutathione (GSH)
Testicular samples were mixed with equal volume of perchloric acid (5%) and centrifuged at 800 g for 10 min at 4°C (Tarsons, Spinwin, MC-05-R) followed by an incubation of the supernatant (100 µL) with phosphate buffer (100 mM, pH 8.0) and DTNB (4%) for 3 min at RT for the estimation of GSH level. Absorbance was measured at 412 nm in a UV-VIS Spectrophotometer (Thermo Scientific, Genesys, 10S UV-VIS) at every 60 seconds for 5 min (Hasan et al. 2020).
Statistical analysis
The mean differences were compared by conducting a one-way ANOVA, as all the datasets passed the normality test (p > 0.01) after subjecting the monthly mean data (mean ± S.E.M) of all the variables (GSI, germ cell profiles, percentages of haploid cell population, melatonin concentration, stress levels, enzymatic and non-enzymatic antioxidants) to a Shapiro-Wilks test for assessing normality. During the annual reproductive cycle, the monthly data for each parameter were pooled and expressed as the mean ± S.E.M. of the number of fish samples (N = 5). This pooling was done because no significant differences were observed for each parameter between the consecutive months corresponding to a particular reproductive phase. In cases where the F values indicated statistical significance, a post hoc Duncan’s multiple range test (DMRT) was conducted at a significance level of p < 0.05 to compare the means. The data was analysed using GraphPad Prism v8.4.3, and presented graphically.
Coefficient of correlation and regression (linear and LOESS) analyses
Pearson’s correlation analysis was conducted to explore a potential relationship between melatonin concentration, relative percentage of spermatogenic cells, and biochemical parameters of testis (two variables at a time). Additionally, linear regression analysis was employed to assess the functional relationship between the response variable and independent (predictor) variable, specifically between two variables in isolation. A significance level of p < 0.05 was used as the threshold for determining statistical significance in both the analyses. Furthermore, the LOESS (locally estimated scatterplot smoothing) regression method was employed due to its flexibility in capturing the relationship between two variables without making any prior assumptions. A smoothing parameter of 0.66 was chosen to implement the LOESS regression method.
Principal component analysis (PCA)
Principal Components Analysis (PCA) was conducted to identify underlying patterns and relationships among various seasonal parameters within the dataset and to achieve dimension reduction. This approach was chosen based on the significant correlations observed through correlation and linear regression analyses among the independent variables. To interpret the total multidimensional dataset, coordinate or loading scores (coefficients of correlation), contribution values, and squared cosine values for each variable (representing the studied parameters) or individual (representing each studied sample) were performed. The loading values of each individual or variable for each considered principal component (PC) were also analyzed to extract the key variables that contribute significantly to the dataset interpretation. To assess the quality of representation, contribution of each individual or variable in the principal components (PCs) was determined by dividing 100 by the total number of individuals or variables. Additionally, squared cosine values were calculated to evaluate the quality of representation for each individual or variable across the PCs (Acharyya et al. 2023).