Introduction
Heat stress represents a critical environmental factor that can significantly impair reproductive physiology in aquatic organisms such as Oreochromis niloticus. Chronic exposure to elevated temperatures disrupts spermatogenesis and leads to functional sperm damage. Epigenetic mechanisms, including DNA methylation, histone modifications, and non-coding RNA activity, are known to play essential roles in regulating germ cell development and are sensitive to environmental fluctuations. Despite increasing interest in the relationship between epigenetic mechanisms and male fertility, integrated studies on how chronic heat exposure alters these regulatory layers in Nile tilapia remain limited. The objective of this study was to investigate how long-term thermal stress affects sperm epigenetic signatures and testicular histoarchitecture and to evaluate their association with sperm quality and reproductive success.
Materials and Methods
Experimental setup and animal husbandry: Sexually mature male Nile tilapia was randomly distributed into three groups, each exposed to a specific water temperature: the control group at 28 °C, the moderate stress group at 32 °C, and the high stress group at 36 °C. The fish were reared in aerated tanks under natural photoperiod conditions for a duration of 90 days. At the end of the experimental period, testicular and sperm samples were collected for subsequent analyses.
DNA methylation profiling by whole genome bisulfite sequencing: To evaluate global DNA methylation patterns, genomic DNA was extracted from sperm samples. The DNA was sheared to appropriate fragment sizes and ligated to methylated sequencing adapters. Following bisulfite treatment to convert unmethylated cytosines to uracil, libraries were amplified and sequenced using paired-end next-generation sequencing platforms. Raw sequencing reads were analyzed using the Bismark software pipeline, allowing alignment and methylation calling across CG, CHG, and CHH sequence contexts. Differentially methylated regions were identified between control and heat stress groups.
Expression analysis of small and long non-coding RNAs: Total RNA was isolated from spermatozoa using column-based purification kits. For small RNA analysis, libraries were constructed by ligating 3-prime and 5-prime adapters followed by reverse transcription and PCR amplification. For long non-coding RNA profiling, ribosomal RNA was selectively depleted before sequencing library preparation. High-throughput sequencing was performed on Illumina platforms. Sequencing data were aligned using HISAT2 for long reads and Bowtie2 for small RNAs. Transcripts were assembled using StringTie and differential expression was calculated using DESeq2.
Immunohistochemical detection of epigenetic and stress markers: Testis tissues were fixed in modified Davidson’s solution to ensure optimal nuclear and cytoplasmic preservation. After fixation, tissues were embedded in paraffin, sectioned at five microns, and mounted on positively charged slides. Immunohistochemical staining was performed using primary antibodies specific to 5-methylcytosine as a marker of DNA methylation and histone modifications. Heat shock proteins Hsp70 and Hsp60, as well as the proliferation marker PCNA, were also evaluated. Sections were incubated with corresponding secondary antibodies and visualized using AEC chromogen. Counterstaining was performed with Gill’s hematoxylin and slides were examined microscopically.
Histopathological evaluation of testes: Paraffin-embedded testes fixed in modified Davidson’s fixative were stained with hematoxylin and eosin. Tissue architecture was evaluated under light microscopy, focusing on the organization of seminiferous tubules, thickness of the germinal epithelium, presence of interstitial edema, and vacuolization. Particular emphasis was placed on identifying features associated with feminization such as disrupted spermatogenic layers and disorganized tubular structure.
Results
The 36°C group exhibited significant alterations across all evaluated parameters compared to control. Whole genome bisulfite sequencing analysis revealed widespread hypermethylation, particularly in genes associated with energy metabolism, transcription regulation, and DNA repair pathways. Notably, differentially methylated regions were enriched in fertility-related gene clusters. Small RNA and long non-coding RNA analyses identified numerous differentially expressed small RNAs and novel long non-coding RNAs in heat-stressed sperm. Several downregulated small RNAs were associated with spermatogenic regulation and chromatin remodeling. Long non-coding RNAs were found to co-express with genes involved in oxidative stress response and cellular apoptosis. Immunohistochemical evaluation showed marked increases in nuclear 5-methyl cytosine staining and repressive histone modifications in spermatogenic cells of the 36°C group. PCNA staining was reduced, indicating decreased proliferative capacity, while histone modifications associated with gene activation were also diminished. Increased expression of both Hsp70 and Hsp60 was observed, supporting the presence of a strong cellular stress response. Histopathological analysis revealed degeneration and disorganization of seminiferous tubules, marked reduction in the germinal epithelium, and significant loss of germ cells. In addition, the presence of germ cells resembling oocytes was observed, which was interpreted as a clear indication of partial feminization. Flow cytometric analysis confirmed a substantial decline in sperm viability and mitochondrial function, along with elevated oxidative stress, enhanced lipid peroxidation, and increased rates of apoptosis in the 36°C group. Computer-assisted sperm analysis revealed the lowest motility scores in this group, with sharp reductions in velocity parameters such as straight-line and average path velocities, as well as an increased frequency of morphological abnormalities. Fertilization trials using sperm from heat-stressed males and oocytes from control females demonstrated a marked decrease in fertilization and hatching success in the 36°C group, providing strong evidence that thermal stress leads to epigenetic and cellular disruptions that impair reproductive performance.
Conclusion
This study demonstrates that chronic heat stress induces profound epigenetic and physiological changes in the testes and sperm of Nile tilapia. The observed changes include hypermethylation of key fertility-related genes, altered non-coding RNA expression, increased histone repressive marks, and clear histological damage. These molecular and cellular disruptions were strongly associated with decreased sperm function and reduced fertilization success. Collectively, these findings suggest that specific epigenetic markers could serve as predictive biomarkers for thermal stress-induced subfertility and may aid in the development of thermotolerant Nile tilapia lines for sustainable aquaculture.