# Post-thaw storage of sperm as a tool towards revealing paternal-effect-associated genes in Eurasian perch, Perca fluviatilis

## TL;DR

This study explores how storing thawed sperm affects offspring survival and gene expression in Eurasian perch larvae, revealing paternal-effect-associated genes.

## Contribution

A novel experimental model using post-thaw sperm storage to identify paternal-effect-associated genes and study non-genetic inheritance in fish.

## Key findings

- Post-thaw sperm storage reduced sperm motility but did not affect fertilization success.
- Larvae from stored sperm showed higher mortality and immune-related gene expression differences.
- Transcriptomic analysis identified 41 differentially expressed genes, including several candidate paternal-effect-associated genes.

## Abstract

The influence of paternity on progeny quality, particularly during early developmental stages, has long been underappreciated. However, altered sperm phenotypes are increasingly recognized as effective tools for identifying paternal-effect-associated genes (PEAGs), whose expression in the progeny is influenced by genetic or non-genetic factors carried by the sperm. This study investigated the impact of post-thaw sperm storage (PTS) as a stressor to verify its effect on larval performance in common garden rearing trial and to reveal PEAGs in Eurasian perch (Perca fluviatilis) progeny. In vitro fertilizations were performed using cryopreserved sperm that was either used immediately after thawing (0 min; CON) or after 30 min of post-thaw storage at 4 °C.

Despite a marked decline in sperm motility during PTS, fertilization success remained unaffected, allowing the use of PTS to study its effect on progeny phenotype. Notably, larvae from the PTS group exhibited significantly higher mortality starting from 9 days post hatch, indicating strong paternal influences on early larval viability. Transcriptomic profiling of larvae at the mouth-opening stage, selected to minimize rearing-induced variation, identified 41 differentially expressed genes (DEGs), many linked to immune regulatory pathways. This suggests that paternal inputs may shape larval immune function, potentially contributing to observed mortality differences. Among the DEGs, several genes, mfap4, gimap, hlag, pigr, neo1, and pde6g, emerged as strong candidate PEAGs.

This study shows that even a brief, 30-minute PTS not only reduces sperm motility but also imprints lasting effects on progeny performance and survival. By selectively shaping the pool of functional sperm, PTS acts as an additional layer of selection, enriching for cells with specific traits and offering a powerful, controlled system for studying non-genetic inheritance factors and identifying PEAGs in fish. Transcriptomic analysis uncovered a deeper dimension to this process, revealing that maternal identity can amplify or buffer paternal contributions which serves as evidence of a complex parental interplay that influences early development. In effect, this study provides a robust experimental model based on controlled, paired fertilization design together with transcriptomic profiling of the offspring to identify novel PEAGs and reveal molecular consequences of paternal variation induced by post-thaw storage.

The online version contains supplementary material available at 10.1186/s12864-026-12648-4.