The purpose of the current study was to analyze phenotypic and functional characteristics of common carp (Cyprinus carpio) spermatozoa during in vitro aging and to investigate whether global DNA methylation is affected by sperm aging. Milt was collected from five individual males, stored in vitro on ice in a refrigerator for up to 96 h post stripping (HPS) and used to fertilize eggs with intervals of 1, 24 and 96 h. Computer-assisted sperm analysis and a S3e Cell Sorter was employed to determine the spermatozoa phenotypic characteristics (motility, velocity, concentration and viability). In addition, pH and osmolality of the seminal fluid and the capacity of the spermatozoa to fertilize, hatching rate and health of the resulting embryos were examined at different aging times. Whole-genome bisulfite sequencing was used to compare the global and gene-specific DNA methylation in fresh and aged spermatozoa. The results demonstrated that spermatozoa aging in common carp significantly affects their performance and thus the success of artificial fertilization. The methylation level at the cytosine-phosphate-guanine (CpG) sites increased significantly with 24 HPS spermatozoa compared to the fresh group at 1 HPS and then decreased significantly at 96 HPS. A more detailed investigation of gene specific differences in the DNA methylation was hindered by incomplete annotation of the C. carpio genome in the public databases.

Regarding the sperm of cold-water fish, the contributions of different bioenergetic pathways, including mitochondrial respiration, to energy production at the spawning temperature and its adaptation at the maximum critical temperature (CTmax) are unclear. The roles of glycolysis, fatty acid oxidation, oxidative phosphorylation (OXPHOS) at 4 °C, and OXPHOS at 15 °C for energy production in burbot (Lota lota) spermatozoa were studied by motility and the oxygen consumption rate (OCR) (with and without pathway inhibitors and the OXPHOS uncoupler). At both temperatures, the effects of the inhibitors and the uncoupler on the motility duration, curvilinear velocity, and track linearity were insignificant; in addition, the OCRs in activation and non-activation media differed insignificantly and were not enhanced after uncoupler treatment. After inhibitor treatment in both media, OXPHOS was insignificantly different at the 2, 30, and 60 s time points at 4 °C but was reduced significantly at the 30 and 60 s time points after treatment with sodium azide at 15 °C. In conclusion, for burbot sperm at both the spawning temperature and the CTmax, the energy synthesized via OXPHOS during motility was insufficient. Therefore, the majority of the energy required to sustain motility was derived from pre-accumulated energy produced and stored during the quiescent state of the spermatozoa.

The sensitivity to stress and its impact on immunity are supposedly related to a fish's personality. In the present study, European perch (Perca fluviatilis) were exposed to an open-field and a novel-object test to identify distinctive shy and bold individuals. This series of cognitive tests revealed clear differences between proactive individuals with pronounced exploration behavior (bold personality) and reactive individuals that took a freeze-hide position (shy personality). A cohort of shy and bold perch was then exposed to elevated stocking density. Frozen activity and lower explorative behavior were related to higher basal and stocking-induced cortisol levels compared to proactive individuals. Since cortisol is a well-known modulator of immune-gene expression, we used multiplex real-time PCR to profile the differential immune responses to the intraperitoneal injection of Aeromonas hydrophila in the head kidney and peritoneal cells of bold and shy perch individuals. These expression differences between stimulated bold and shy perch were generally modest, except for the genes encoding the complement component c3 and the matrix metallopeptidase mmp9. The strong differential expression of these two bactericidal and inflammatory genes in the context of the modestly regulated features suggests that a fish's personality is linked to a particular immune-defense strategy. In conclusion, our approach, based on behavioral video observations, phagocytosis and enzyme assays, immunogene-expression profiling, and quantification of stress-relevant metabolites, revealed indications for divergent coping styles in cohorts of bold or shy European perch. This divergence could be exploited in future selective breeding programs.

Background: Recent studies have demonstrated that prolonged sperm storage adversely affects offspring through epigenetics, yet its broader effects on other molecular levels such as transcription and proteomics in progeny have been rarely explored. Results: We employed comprehensive multi-omics approaches to uncover storage-induced epigenetic changes in sperm and their effects on embryonic development and offspring health. Sperm from common carp (Cyprinus carpio) was stored in vitro in artificial seminal plasma for 14 days, and the impacts of storage on functional properties of sperm and progeny development were investigated. We combined DNA methylome, transcriptomic and proteomic data to elucidate the potential mechanisms by which sperm storage influences progeny development. Prolonged in vitro storage significantly reduced sperm motility and fertilising ability which coincided with changes in the DNA methylation pattern. Integrated analyses of the offspring DNA methylome, comparative transcriptomics and cardiac performance measurements revealed storage-induced alterations of genes associated with nervous system development, myocardial morphogenesis and cellular responses to stimuli. Proteomic analyses showed that in addition to visual perception and nervous system function, pathways of the immunity system were also enriched. Results provide strong evidence of the epigenetic inheritance of the offspring’s performances when short-term stored sperm was used for fertilisation. Conclusions: Short-term sperm storage induces heritable molecular and phenotypic changes in offspring, raising concerns over the potential intergenerational consequences of assisted reproductive practices in aquaculture and possibly other vertebrates.