Sperm cryopreservation is one of the leading, yet sophisticated techniques to employ both in research purposes and aquaculture production . It has already been reported that sperm cryopreservation may affect the transcriptomic profile of the progeny (Wang et al., 2022) . It is, therefore, justified to hypothesize that by implementation of cryopreservation technology, possibly modifying cell physiology and/or affect structure of the cells retaining fertilizing capacity, a paternal-effect genes – being poorly explored in Teleosts – can be identified. Such strategy has a potential to elucidate paternal contribution to progeny quality what may be used by aquaculturists to fine-tune the selective breeding operations.
This study aims to explore the transcriptomic and phenotypic consequences of progeny obtained following usage for fertilization either cryopreserved or fresh semen in Eurasian perch (Perca fluviatilis), our model of commercial interest, in order to identify paternal-effect genes in this species .
Materials and methods
The semen from wild males (n=6) were stripped, checked for their motility and concentration; divided a portion (~ 1.5 ml) of semen was cryopreserved as described by Judycka et al. (2021), and the second portion (~1.5 ml) was used as fresh semen. Fertilizations were done with e ggs coming from each female (n=3) portioned equally , one for cryopreserved semen (group C) and the other with fresh semen from the same male (group F). Advanced larviculture was carried out as described by Palińska-Żarska et al. (2020) till 16 days post hatch (dph), while noting zootechnical parameters . Larvae at mouth opening (MO) stage and weaning stage were sampled for transcriptomic analysis.
S equencing of RNA of 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 (qRT-PCR) were performed for the differentially expressed genes (DEGs), normalized using 5 housekeeping genes, to verify expression levels of those genes at MO stage and at the weaning stage.
Embryonic survival rate was significantly lower in Group C than in Group F, and the Group C larvae had higher weight wise at their weaning stage. The remaining zootechnical parameters were similar in both groups. Transcriptomic analysis revealed 11 DEGs, out of which 3 genes were further successfully validated by qRT -PCR, namely pde6g , opn1lw1 and rbpl4 (Figure1 ). Interestingly, all the genes are responsible for the development of the eye and had higher expression at MO stage in Group C but later at weaning stage, the expression levels were similar in both groups.
This experiment was done with the intent to not just check the effect of cryopreservation on progeny quality, but also to use the technique as a condition to reveal paternal-effect genes . Phosphodiesterase 6 Gamma (pde6g) is expressed in rod photoreceptors and functions in the phototransduction signaling cascade (Dvir et al., 2010). Long-wave sensitive Opsin (opn1lw1 ) codes for red opsins and are abundant in cone cells (Crespo et al., 2018). Retinol Binding protein 4 (rbp4l ) has a role to play in retina development. Examples of signalling pathways for pde6g and opn1lw1 is G protein mediated phototransduction, for rod and cone cells (Zang n.d.) . Abola et al., (2015) found rbp4l gene being important in pathways like retin oid signalling. Thus we may conclude that development of an eye and potentially other sensory capabilities as well as its functioning durin g the first days post-hatching is under the influence of paternal genome, probably via the methylome status of the sperm (Jiang et al., 2013).
This work was funded by National Science Center of Poland (SONATA BIS project, number UMO-2020/38/E/NZ9/00394).
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