Potential roles of sperm-associated tsRNAs in offspring alkaline tolerance in grass carp (Ctenopharyngodon idella).

Transfer RNA-derived small RNAs (tsRNAs) are emerging epigenetic regulators involved in environmental adaptation and paternal effects. Their roles in fish offspring alkaline tolerance, however, remain unclear. In this study, small non-coding RNA (sncRNA) profiles of grass carp (Ctenopharyngodon idella) sperm, oocytes, and embryos were characterized by high-throughput sequencing, and the potential functions of sperm-associated tsRNAs were further investigated. Significant differences in sncRNA expression profiles were observed among sperm, oocytes, and embryos, with tsRNAs representing the most abundant sncRNA class in all groups. Most tsRNAs originated from the nuclear genome and were mainly classified as tiRNA-5, showing biased expression patterns derived from only a few specific tRNAs. A total of 36 tsRNAs were detected exclusively in sperm and embryos but were not detected in oocytes, suggesting potential paternal delivery during fertilization. Integration with alkaline stress-responsive data further identified 18 candidate tsRNAs associated with both alkaline stress response and potential paternal contribution. Predicted target genes were significantly enriched in pathways related to stress response, ion homeostasis, epithelial remodeling, and energy metabolism. Functional microinjection assays in zebrafish and grass carp embryos showed that tRF-33-R9JP9PMNH5SYDQ and tRF-33-KQI34JRX6N380Q significantly enhanced offspring survival under alkaline stress, whereas inhibition of these tsRNAs reduced alkaline tolerance. These findings suggest that sperm-derived tsRNAs may participate in regulating offspring alkaline tolerance in fish and may provide candidate molecular targets for future alkaline-tolerant breeding programs.