Introduction
The influence of paternal effects on progeny quality, particularly during early developmental stages, has historically been underappreciated. Recently, sperm selection procedures like cryopreservation have been started to be used to identify paternal-effect genes (PEGs; Panda et al., 2024). The expression of these genes in the progeny under the paternal control, meaning their expression rely on paternally-derived factors such as epigenetic status and/or non-genetic inheritance factors (e.g., ncRNAs) contained in the sperm cell. Sperm cryopreservation is a leading and sophisticated process for sperm preservation, keeping the sperm cell viable and functional for artificial reproduction (Cabrita et al., 2010). It is also been reported that sperm cryopreservation may affect the transcriptomic profile of the progeny (Wang et al., 2022). In Eurasian perch (Perca fluviatilis), comparative analysis of transcriptomic profile of freshly hatched larvae obtained following usage of fresh or cryopreserved sperm for fertilization allowed to identify a panel of novel PEGs (Panda et al., 2024). These findings show that exposing sperm to a specific challenge is an excellent approach to identify PEGs.
It is specific feature of cryopreserved E. perch sperm that after thawing its motility decreases over a relatively short period of time, with significant reduction after already 30 min. Thus, post-thaw storage seems to be additional challenging factor for the sperm cells keeping the most robust cells viable. Therefore, the aim of this study was to compare progeny obtained following using freshly thawed (0 min; CON) and stored after thawing (post-thaw stored sperm; PTS) for 30 min through their zootechnical performance and transcriptomic analyses. We hypothesized, that such additional selection pressure will enable to reveal new paternally controlled traits and PEGs.
Methods
The semen from wild males (n=6) were stripped, checked for their motility and concentration; and cryopreserved as described by Judycka et al. (2021). Fertilizations were done with eggs coming from each female (n=3) portioned equally. Cryopreserved sperm straws were thawed at 40°C for 10 seconds in a water bath, and poured into an Eppendorf, stored at 4°C for 30 minutes (PTS), while the same thawing procedure was used for 0 min, CON without providing them a waiting period and taken into use directly. A total of six pairs were created, with two males used to fertilize eggs from single female. Advanced larviculture was carried out as described by Palińska-Żarska et al. (2020) till 27 days post hatch (dph), while noting zootechnical parameters. Larvae at mouth opening (MO) stage, when larvae started weaning and at end (27 DPH) were sampled for transcriptomic and gene expression analysis.
Transcriptomic profile larvae at MO from both groups were compared using RStudio (version 4.1.3) using the package DESeq2 (Love et al., 2014). Differences were considered significant when corrected p-values were inferior to ɑ (ɑ=0.05).
Quantitative real-time PCRs (RT-qPCR) were performed to validate the chosen differentially expressed genes (DEGs), normalized using 5 housekeeping genes, to verify expression levels of those genes at MO stage; weaning stage and end stage if positively validated.
Results
Notably, larvae from the PTS group exhibited higher mortality rates compared to the CON group, with minor or none differences in terms of other zootechnical parameters. Transcriptomic analysis of larvae at the mouth-opening stage revealed 49 DEGs, many of which associated with immune-related functional pathways. Several genes, including mfap, gimap, hlag, pigr, pde6g, and neo1, emerged as promising candidates for PEGs.
Discussion
High mortality in PTS group is a very strong phenotype and directed the study’s focus towards paternal effects. This finding suggests that paternity may compromise immune functions to promote larval survival. Alongside, the gene pde6g, which plays a crucial role in phototransduction, can be confirmed to be a strong candidate as PEG, given its steep downregulation in the PTS group and occurrence in our previous studies identify PEGs.
Overall, this study challenges the long-held view that maternal effects predominantly govern larval development and phenotype. It also represents a significant step towards disentangling individual parental contributions, thereby advancing our understanding of the effect of reproductive techniques in aquaculture which may lead us toward fine-tuning the hatchery protocols. Besides, it broader our knowledge in the field of reproductive and developmental biology bringing us closer to understanding non-genetic inheritance in finfishes.
Acknowledgements
This work was funded by the National Science Center of Poland (SONATA BIS project, number UMO-2020/38/E/NZ9/00394).
References
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