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

Ovulation, fertilization, and embryo development are orchestrated and synchronized processes essential for the optimal health of offspring. Postovulatory aging disrupts this synchronization and impairs oocyte quality. In addition, oocyte aging causes fertilization loss and poor embryo development. This investigation aimed to unravel the endpoint of in vitro oocyte aging in common carp (Cyprinus carpio) to understand the involvement of apoptosis in postovulatory oocyte death. It was observed that the fertilization ability significantly declined (P < 0.001) at 8-h poststripping (HPS), subsequently triggering apoptosis in the advanced stage of oocyte aging, i.e., 48 HPS. This process included an increase in proapoptotic transcripts (fas, bax, cathepsin D, caspase 8, caspase 9, and caspase 3a) (P < 0.05), elevated levels of caspase 3 protein (P < 0.05), and activation of caspase 3 enzyme (P < 0.001), a key player in apoptosis, in aging oocytes. Furthermore, the effects of oocyte aging on the embryonic apoptosis machinery were examined in embryos at 5-h postfertilization (HPF) and 24 HPF derived from fresh and aged oocytes. Expression of apoptotic genes and caspase enzyme activity remained at the basal level in 5 HPF (early blastula embryos) from both fresh and aged oocytes. In contrast, the zymogenic and active forms of caspase 3 increased in 24 HPF embryos from 8-h-aged oocytes (P < 0.01) compared with those from fresh oocytes. Thus, apoptosis intensified in 24 HPF embryos from aged oocytes without affecting the apoptotic machinery of early blastula embryos. Our findings demonstrate that apoptosis initiated by the Fas/FasL system is an important physiological process accompanying oocyte aging in common carp.

The delay in fertilization after ovulation or retention of ovulated oocytes in the fish body causes postovulatory aging. Postovulatory aging leads to time-dependent deterioration of oocyte quality and loss of fertilization capacity. The mechanisms behind losing oocyte quality and developmental capacity due to postovulatory oocyte aging remain elusive. The emerging climate change issues in nature and unfavorable spawning conditions have caused the retention of ovulated oocytes in the female body. Analyzing the apoptotic parameters to understand the fate of these aged oocytes and the consequences of this aging on embryo development was the main objective of this study. The results obtained from this study indicate that aged oocytes die by apoptosis. The embryos from aged oocytes show more apoptosis, stating that oocyte aging affects embryo development by affecting the intensity of apoptosis.

• Keywords
• apoptosis, caspases, early blastula embryos, fish, in-vitro-aged oocytes, spontaneous activation,
• MeSH
• Apoptosis * physiology   MeSH
• Embryo, Nonmammalian physiology   MeSH
• Embryonic Development * physiology   MeSH
• Carps * embryology   physiology   MeSH
• Caspases metabolism   genetics   MeSH
• Oocytes * physiology   MeSH
• Aging * physiology   MeSH
• Gene Expression Regulation, Developmental physiology   MeSH
• Animals MeSH
• Check Tag
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Caspases MeSH

The present study was designed to evaluate sperm phenotypic variables during in vivo and in vitro storage following multiple sperm stripping in common carp (Cyprinus carpio L.). Each male was injected 3 times with carp pituitary 3 days apart. Sperm was stored in vivo in the body cavity for 0.5 days (Fresh sperm) and 3 days (Old sperm) after hormonal stimulation. Then sperm was collected and diluted with a carp extender at a ratio of 1:1, and stored in vitro on ice for 0, 3, and 6 days. The phenotypic parameters, including the number of total motile spermatozoa, number of fast motile spermatozoa, number of motile spermatozoa, percentage of fast motile spermatozoa, and percentage of spermatozoa motility were the major components of principal component analysis (PCA). In general, Fresh sperm from the first stripping showed slightly better quality than Old sperm from the second and third stripping, especially in the phenotypic parameters of a number of total spermatozoa and a number of total motile spermatozoa (P < 0.05). The highest kinetic and quantitative spermatozoa variables were obtained in Fresh and Old sperm just after sperm collection (0-day storage in vitro), and then they were decreased during the period of in vitro storage up to 6 days (P < 0.05). However, the fertilization, hatching, and malformation rates from Fresh sperm were similar compared with the Old sperm. Sperm could be stripped 0.5 days post hormonal treatment and stored in vitro up to 6 days with good fertilization performance (fertility, hatching, and malformation rates were 92.5%, 91.5%, and 1.3%, respectively). Therefore, our results suggested that multiple hormonal treatments with multiple stripping could be used in artificial reproduction in common carp.

• Keywords
• Common carp, In vitro, In vivo, Sperm storage, Spermatozoa aging,
• MeSH
• Carps * MeSH
• Cryopreservation methods   MeSH
• Ice MeSH
• Sperm Motility physiology   MeSH
• Semen physiology   MeSH
• Spermatozoa physiology   MeSH
• Aging MeSH
• Semen Preservation * methods   MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Ice MeSH

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.

• Keywords
• cold-water fish, fatty acid oxidation, glycolysis, maximum critical temperature, oxidative phosphorylation, spawning temperature, sperm motility,
• Publication type
• Journal Article MeSH