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.

• Keywords
• Epigenetic inheritance, Epigenetics, Fish sperm, Offspring development, Sperm ageing,
• MeSH
• Embryonic Development * MeSH
• Epigenesis, Genetic MeSH
• Carps * genetics   physiology   embryology   MeSH
• DNA Methylation MeSH
• Multiomics MeSH
• Proteomics MeSH
• Spermatozoa * physiology   metabolism   MeSH
• Transcriptome MeSH
• Semen Preservation * adverse effects   MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

Short-term storage and management of sperm in vitro is an easy and economical process in which suitable extenders can be utilized to extend the storage period and prevent sperm function impairment. Therefore, the current study aimed to evaluate the effect of suitable extenders during the short-term storage of sterlet sperm and determine their fertilizing capacity and hatching success. Three extenders containing a composition of 16, 20, and 24 mM NaCl, 1 mM KCl, 0.1 mM CaCl2, 10 mM Tris, pH 8.0 with osmolarity of 46, 55, and 62 mOsm/kg, were used to dilute the sperm of four sexually mature sterlet males (n = 4). Using a CASA system, the motility and velocity of undiluted and diluted sperm with extenders (E1 - E3) were assessed over 6 days at 0-2 °C. The short-term stored diluted sperm was then used in the fertilization and hatching assay, and undiluted fresh and stored sperm was used as a control. A two-way factorial analysis of variance (ANOVA) model confirmed significant effects on sperm motility, curvilinear velocity (VCL), and straight-line velocity (VSL) (P < 0.001), as well as their interaction with the extender. The model was decomposed into a one-way ANOVA to examine the impacts of extenders and storage time. With increasing storage periods, the sperm motility and velocity gradually decreased for diluted sperm with three extenders (E1-E3) but sharply decreased for undiluted sperm (Control). The motility of undiluted sperm was found 3.77 ± 4.09% at 4 days, whereas sperm diluted with extenders showed 57.57 ± 12.33% (E1), 64.34 ± 11.86% (E2), and 61.40 ± 12.41% (E3) motility at 6 days. This study explored extenders optimized with higher osmolarity (39-62 mOsm/kg) and lower K+ (1 mmol/L) as the most suitable medium for storing sterlet sperm for 6 days. After 6 days post storage, sperm diluted with extenders E1-E3 achieved a fertilization rate of 31.29 ± 14.2%, 31.66 ± 8.84%, and 30.67 ± 10.02%, respectively, and hatching success of 29.58 ± 13.4%, 30.50 ± 7.89%, and 27.95 ± 9.62%, respectively with freshly ovulated eggs.

• Keywords
• CASA, Fertilization rate, Hatching rate, Sperm motility, Sperm short-term storage, Sperm velocity,
• MeSH
• Fertilization * drug effects   MeSH
• Sperm Motility * drug effects   MeSH
• Fishes * physiology   MeSH
• Spermatozoa * physiology   drug effects   MeSH
• Semen Preservation * veterinary   methods   MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

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.

• Keywords
• DNA methylation, common carp, epigenetics, fertilization, fish, milt, sperm aging, sperm quality, sperm storage,
• MeSH
• Carps genetics   growth & development   MeSH
• DNA Methylation genetics   MeSH
• Spermatozoa metabolism   pathology   MeSH
• Aging genetics   pathology   MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

European catfish (Silurus glanis) is a commercially important freshwater fish originating from Eastern Europe. The objective of this study was to examine the short-term storage of its eggs especially in relation to maintaining a low level of malformation in newly hatched fry. The eggs from freshly spawned individuals were stored separately in cell incubators at 17 and 22 °C under aerobic conditions. Changes in fertilization, hatching, and malformation were examined in eggs stored at 1, 3, 5, and 7 h post-stripping. The sperm used for fertilization showed very good motility rates (84-90%) and curvilinear velocity (110-125 μm/s), and straight-line velocity did not drop below 77 μm/s. For all females, a temperature of 17 °C was better than 22 °C for egg storage in vitro. Egg fertilization and total hatching decreased rapidly after 7 h storage at 17 °C. The storage time of eggs in vitro to fertilization should therefore not exceed 5 h at 17 °C. In all females, there was no difference in the total number of eggs hatching between 1 and 3 h of egg storage at 17 °C. The storage time of eggs did not correlate with the level of malformations of the fry. However, the level of hatching and malformations was clearly affected by the storage temperature of eggs when it was > 17 °C. Analysis showed that the storage time of eggs, temperature of storage, and individual females had a significant influence on fertilization and total hatching rates. Regression analysis confirmed a low correlation of fertilization and hatching rates with storage time of eggs.

• Keywords
• Egg, Fertilization, Fish reproduction, Silurus glanis, Spermatozoa,
• MeSH
• Fertilization MeSH
• Sperm Motility MeSH
• Spermatozoa MeSH
• Catfishes * abnormalities   MeSH
• Temperature * MeSH
• Tissue Preservation * MeSH
• Animals MeSH
• Zygote * MeSH
• Check Tag
• Male MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH